Volumetric absorption microsampling device and method of use thereof

Abstract

It is an object of the present invention to provide a microsampling device that allows for the analysis of proteins in a biological sample, such as a whole blood sample, and to provide methods for using the same.

Description

[Technical Field]

[0001] The present invention relates to a microsampling device and analytical method for detecting target components in a biological sample, such as a blood sample.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application Nos. 63 / 299,281, filed January 13, 2022, and 63 / 392,336, filed July 26, 2022, the entire contents of each of which are incorporated herein by reference. [Background technology]

[0003] Over the past two decades, the concept of precision medicine—preventive and therapeutic strategies that take individual differences into account—has emerged as a way to transform disease prediction, prognosis, and empower individuals to manage their own health (Collins and Varmus, N Engl J Med. 2015;372(9):793-5; Denny et al., N Engl J Med. 2019;381(7):668-76; Franks et al., J Intern Med. 2021; Snyderman, Biotechnol J. 2012;7(8):973-9). Driven by increasingly affordable access to genomic technologies, precision medicine genetic analysis is a game changer in medicine.

[0004] Blood microsampling, which allows patients to provide longitudinal samples without visiting a hospital or pathology laboratory, is rapidly developing and is a key innovation linking precision medicine with the growing trend of telemedicine (Hollander and Sites, NEJM Catalyst. 2020). Blood volumes of typically less than 100 μL are collected in small devices, providing sufficient material for numerous molecular detection techniques (Lei and Prow, Biomed Microdevices. 2019;21(4):81).

[0005] Microsampling of patient blood promises several advantages for facilitating precision medicine research compared to conventional phlebotomy practice and overcomes the need for a licensed phlebotomist, allowing for frequent on-site collection, home collection, and remote sampling, which facilitates compatibility with remote medical monitoring, minimal post-collection processing, and unrefrigerated transport and storage.

[0006] Microsampling also increases patient accessibility to testing and overcomes reluctance among patients who find venipuncture stressful (Chapman et al. Bioanalysis. 2014;6(22):2965-8.). A concomitant of collecting small blood volumes is the direct analysis of preselected targets from dried blood spots (DBS), as exemplified by newborn screening of small molecule metabolites for inherited metabolic diseases (Moat et al., Int J Neonatal Screen. 2020;6(2):26).

[0007] The use of DBS for bottom-up, untargeted discovery proteomic analysis of whole blood (WB), which would support precision medicine research, has not been thoroughly investigated. However, for proteomic biomarker studies, mass spectrometry of WB has generally been avoided in favor of plasma or serum obtained via venipuncture.

[0008] While conventional DBS involves the application of blood to paper followed by drying, newer approaches are being developed. One such approach involves a microsampling device known as volumetric absorptive microsampling (VAMS) (Kok and Fillet, J Pharm Biomed Anal. 2018;147:288-96). This device allows for precise volumes of blood to be collected to account for variations in hematocrit, an important variable in diseased individuals. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Collins and Varmus, N Engl J Med. 2015;372(9):793-5 [Non-patent document 2] Denny et al., N Engl J Med. 2019;381(7):668-76 [Non-patent document 3] Franks et al., J Intern Med. 2021 [Non-patent document 4] Snyderman, Biotechnol J. 2012;7(8):973-9 [Non-patent document 5] Hollander and Sites, NEJM Catalyst. 2020 [Non-patent document 6] Lei and Prow, Biomed Microdevices. 2019;21(4):81 [Non-Patent Document 7] Chapman et al. Bioanalysis. 2014;6(22):2965-8. [Non-patent document 8] Moat et al., Int J Neonatal Screen. 2020;6(2):26 [Non-Patent Document 9] Kok and Fillet, J Pharm Biomed Anal. 2018;147:288-96 Summary of the Invention [Problem to be solved by the invention]

[0010] Under conventional blood separation methods, protein detection is hindered by the high relative concentrations of hemoglobin, other structural components of red blood cell membranes, and the high concentrations of many plasma proteins. The present disclosure provides methods for analyzing proteins and cells in whole blood that would otherwise not be detected by conventional blood separation methods due to dynamic range limitations. The present disclosure also provides devices and methods for utilizing the devices for analyzing proteins and cells in whole blood that would otherwise not be detected by conventional blood separation methods due to dynamic range limitations. [Means for solving the problem]

[0011] In one aspect, provided herein is a method of fractionating a sample, comprising: (a) introducing a sample into a porous material; (b) optionally centrifuging the porous material containing the sample; (c) drying the sample in the porous material; (d) extracting a first set of proteins from the dried sample-containing porous material, comprising (1) incubating the dried sample-containing porous material in a first extraction solution; and (2) separating the first extraction solution from the first incubated porous material; and (e) detecting the first set of proteins in the separated extraction solution.

[0012] In one aspect, provided herein is a method of fractionating a sample, comprising: (a) introducing a sample into a porous material; (b) optionally centrifuging the porous material containing the sample; (c) drying the sample in the porous material; (d) extracting a first set of proteins from the dried sample-containing porous material, comprising (1) incubating the dried sample-containing porous material in a first extraction solution; (2) separating the first extraction solution from the first incubated porous material; and (3) optionally washing the separated first extracted porous material; (e) extracting a second set of proteins from the first extracted porous material, comprising (1) incubating the first extracted porous material in a second extraction solution; (2) separating the second extraction solution from the second incubated porous material; and (f) detecting the first or second set of proteins from the separated first or second extraction solution, respectively.

[0013] In one aspect, provided herein is a method further comprising: (g) optionally washing the separated second extracted porous material; (h) extracting a third set of proteins from the second extracted porous material, comprising: (1) incubating the second extracted porous material in a third extraction solution; and (2) separating the third extraction solution from the third incubated porous material; and (i) detecting the third set of proteins in the separated third extraction solution.

[0014] In some embodiments, the method further comprises sequentially extracting one or more additional sets of proteins from the third extracted porous material, comprising repeating steps (g)-(i) with one or more additional extraction solutions.

[0015] In one aspect, provided herein is a method of fractionating a sample, comprising: (a) introducing a sample into a porous material; (b) optionally centrifuging the porous material containing the sample; (c) drying the sample in the porous material; (d) extracting a first set of proteins from the dried sample-containing porous material, comprising (1) incubating the dried sample-containing porous material in a first extraction solution; (2) separating the first extraction solution from the first incubated porous material; and (3) optionally washing the separated first extracted porous material; (e) digesting proteins remaining in the first extracted porous material, comprising (1) incubating the first extracted porous material in a digestion solution; and (2) separating the digestion solution from the digest-incubated porous material; and (f) detecting one or more proteins in the separated digestion solution.

[0016] In some embodiments, prior to the digestion step, the method further comprises: (A) extracting a second set of proteins from the first extracted porous material, comprising: (1) incubating the first extracted porous material in a second extraction solution; (2) separating the second extraction solution from the second incubated porous material; and (g) optionally washing the separated second extracted porous material; and (B) optionally detecting the second set of proteins in the separated second extraction solution.

[0017] In some embodiments, prior to the digestion step, the method further comprises sequentially extracting one or more additional sets of proteins from the second extracted porous material, comprising repeating steps (A)-(B) with one or more additional extraction solutions.

[0018] In one aspect, provided herein is a method of fractionating a sample, comprising: (a) introducing a sample into a porous material; (b) optionally centrifuging the porous material containing the sample; (c) drying the sample in the porous material; (d) extracting a first set of molecules from the dried sample-containing porous material, comprising (1) incubating the dried sample-containing porous material in a first extraction solution; (2) separating the first extraction solution from the porous material; and (3) optionally washing the porous material; (e) digesting proteins remaining in the porous material, comprising (1) incubating the porous material in a digestion solution; and (2) separating the digestion solution from the porous material; and (f) optionally detecting one or more molecules in the separated first extraction solution; and (g) detecting one or more peptides or proteins in the digestion solution.

[0019] In one aspect, provided herein is a method further comprising, following extraction of the first set of molecules and prior to the digestion step, (A) extracting a second set of molecules from the first extracted porous material, comprising: (1) incubating the first extracted porous material in a second extraction solution; (2) separating the second extraction solution from the second incubated porous material; and (h) optionally washing the separated second extracted porous material; and (B) optionally detecting the second set of molecules in the separated second extraction solution.

[0020] In some embodiments, the method further comprises, prior to the digestion step, sequentially extracting one or more additional sets of molecules from the second extracted porous material, comprising repeating steps (A)-(B) with one or more additional extraction solutions.

[0021] In some embodiments, the set of molecules is selected from the group consisting of proteins, nucleic acids, and glycans, hi another embodiment, the proteins are phosphoproteins.

[0022] In some embodiments, the sample is or comprises a bodily fluid sample. In some embodiments, the sample comprises cells or tissue. In some embodiments, the sample comprises cells suspended in a liquid. In some embodiments, the sample comprises cultured cells suspended in a culture medium. In some embodiments, the sample is or comprises blood, a blood fraction, plasma, a nucleic acid-containing stabilized sample, a stabilized blood sample (e.g., blood in a Streck tube sample), urine, tears, wound fluid, CSF, bronchoalveolar lavage fluid, or ascites. In some embodiments, the sample is a blood sample. In some embodiments, the sample is or comprises a nucleic acid-containing stabilized sample or a stabilized blood sample, such as a nucleic acid-containing stabilized blood sample, e.g., a blood sample in a Streck tube. In some embodiments, the sample has a volume in the range of 100 μL to 2 μL, 100 μL to 5 μL, 100 μL to 10 μL, 99 μL to 2 μL, 90 μL to 2 μL, 80 μL to 2 μL, 70 μL to 2 μL, 60 μL to 2 μL, 50 μL to 2 μL, 40 μL to 2 μL, 30 μL to 2 μL, 20 μL to 2 μL, 10 μL to 2 μL, or 5 μL to 2 μL. In some embodiments, the sample has a volume of less than 100 μL, less than 50 μL, less than 30 μL, less than 10 μL, or less than 5 μL. In some embodiments, the sample has a volume of at least 2 μL.

[0023] In some embodiments, the method includes centrifuging the porous material containing the sample prior to the drying step.

[0024] In some embodiments, the drying, extraction, and digestion steps are performed with the sample-containing porous material in the tube.

[0025] In some embodiments, the drying step comprises air-drying the sample within the porous material, after which the sample is dried within the porous material. In some embodiments, the drying step comprises centrifuging the sample within the porous material, after which the sample is dried within the porous material. In some embodiments, the drying step comprises drying for a period of time to allow the sample to adhere to the porous material. In some embodiments, the drying step comprises drying for a period of time to allow the sample to adhere to the porous material, after which the sample-containing porous material is stored for a period of time prior to the first extraction step. In some embodiments, the sample-containing porous material is frozen prior to the first extraction step. In some embodiments, the drying step comprises air-drying the sample within the porous material. In some embodiments, the drying step takes less than 30 minutes. In some embodiments, the drying step takes less than 20 minutes. In some embodiments, the drying step takes less than 10 minutes. In some embodiments, the drying step takes less than 5 minutes. In some embodiments, the drying step takes less than 5 minutes. In some embodiments, the air-drying step is for at least 30 minutes, 1, 2, 4, 6, 8, 10, 12, 16, 20, or 24 hours. In some embodiments, the drying step comprises centrifuging the sample within the porous material. In some embodiments, the sample is frozen immediately after the sample is collected in the porous material, or optionally, the sample is air-dried for less than 30 minutes before being frozen.

[0026] In some embodiments, the method comprises storing the sample-containing porous material for a period of time prior to the first extraction step, hi some embodiments, the method comprises storing the sample-containing porous material immediately after the drying step.

[0027] In some embodiments, the sample-containing porous material is stored frozen. In some embodiments, the sample-containing porous material is stored frozen at -20°C. In some embodiments, the sample-containing porous material is stored frozen at -80°C. In some embodiments, the sample-containing porous material is thawed and refrozen prior to the first extraction step. In some embodiments, the sample-containing porous material is stored at room temperature.

[0028] In some embodiments, the first, second, third, and further extraction solutions comprise an extraction agent comprising a salt, a weak detergent, a strong detergent, a chaotropic agent, a reducing agent, a thiol-containing reducing agent, or an alkylating agent, an acid, an organic solvent, or an enzyme, or a combination thereof.

[0029] In some embodiments, the first extraction solution is different from the second extraction solution. In some embodiments, the first, second, and third extraction solutions are different. In some embodiments, the first, second, third, and further extraction solutions are different. In some embodiments, the first, second, third, and further extraction solutions are selected to fractionate proteins contained in the porous material through differential solubility. In some embodiments, each successive extraction solution selected is characterized as having a relatively large solubility of proteins remaining in the porous material. In some embodiments, the first, second, third, and further extraction solutions contain increasing concentrations of the same extractant as one extraction proceeds to the next.

[0030] In some embodiments, the separated first, second, third, or further extraction solution comprises one or more proteins. In some embodiments, the separated first extraction solution comprises a first set of proteins. In some embodiments, the separated second extraction solution comprises a second set of proteins. In some embodiments, the separated third extraction solution comprises a third set of proteins. In some embodiments, the separated further extraction solution comprises a further set of proteins. In some embodiments, the first, second, third, or further extraction solution has a volume of less than 100 μL. In some embodiments, the first, second, third, or further extraction solution has a volume of 1 to 5 times the volume of the porous material.

[0031] In some embodiments, the incubating step in the first, second, third, or additional extraction solution is carried out for a time sufficient to solubilize one or more proteins contained within or attached to the porous material. In some embodiments, the incubating step in the first, second, third, or additional extraction solution is carried out for a time sufficient to extract one or more proteins contained within or attached to the porous material. In some embodiments, the incubating step is carried out for a time period ranging from 1 minute to 48 hours. In some embodiments, the incubating step is carried out at ambient temperature. In some embodiments, the incubating step is carried out at an elevated temperature above ambient temperature. In some embodiments, the incubating step is carried out at an elevated temperature below ambient temperature. In some embodiments, the incubating step is carried out with agitation.

[0032] In some embodiments, the step of incubating in the digestion solution is carried out for a time sufficient to digest one or more proteins contained within or adhered to the porous material. In some embodiments, the incubating step is carried out for a time period between 1 minute and 48 hours. In some embodiments, the incubating step is carried out for a time period between 1 minute and 24 hours. In some embodiments, the incubating step is carried out for a time period between 24 hours and 48 hours. In some embodiments, the incubating step is carried out at ambient temperature. In some embodiments, the incubating step is carried out at an elevated temperature above ambient temperature. In some embodiments, the incubating step is carried out at an elevated temperature below ambient temperature. In some embodiments, the incubating step is carried out with agitation.

[0033] In some embodiments, the separating step comprises removing the first, second, third, or additional incubated porous material from the first, second, third, or additional extraction solution, respectively. In some embodiments, the separating step comprises removing the first, second, third, or additional extraction solution from the first, second, third, or additional incubated porous material, respectively. In some embodiments, the separating step of the first, second, third, or additional extraction solution from the first, second, third, or additional incubated porous material, respectively, is via centrifugation, filtration, or a combination of centrifugation and filtration. In some embodiments, the separating step is performed via centrifugation. In some embodiments, the separating step is performed via filtration. In some embodiments, the separating step is performed via a combination of centrifugation and filtration.

[0034] In some embodiments, the separating step comprises removing the digestion-incubated porous material from the digestion solution. In some embodiments, the separating step comprises removing the digestion solution from the digestion-incubated porous material. In some embodiments, the separating step of the digestion solution from the digestion-incubated porous material is via centrifugation, filtration, or a combination of centrifugation and filtration. In some embodiments, the separating step is via centrifugation. In some embodiments, the separating step is via filtration. In some embodiments, the separating step is via a combination of centrifugation and filtration.

[0035] In some embodiments, the method includes washing the separated first, second, third, or additional extracted porous material. In some embodiments, the separated first, second, third, or additional extracted porous material is washed with a wash volume of the first, second, third, or additional extraction solution, respectively. In some embodiments, the separated first extracted porous material is washed with an extraction solution different from the first extraction solution. In some embodiments, the selection of the different extraction solution is limited to solutions based on proteins remaining in the separated first extracted porous material that have the same or lower solubility in the different extraction solution than the first extraction solution. In some embodiments, the selection of the different extraction solution is based on proteins remaining in the separated first extracted porous material that have the same or lower solubility in the different extraction solution than the first extraction solution.

[0036] In some embodiments, the wash volume of the first, second, third, or further extraction solution is 50 μL or more. In some embodiments, the wash volume of the first, second, third, or further extraction solution is 100 μL or more. In some embodiments, the wash volume of the first, second, third, or further extraction solution is between 5 times the volume of the porous material and 3 mL. In some embodiments, the washing step of the separated first, second, third, or further extracted porous material is repeated one, two, or three times.

[0037] In some embodiments, the digestion solution comprises a reducing agent, an alkylating agent, a buffering agent, a surfactant, or a combination thereof. In some embodiments, the digestion solution further comprises a salt, a weak surfactant, a strong surfactant, a chaotropic agent, or a thiol-containing reducing agent, or a combination thereof. In some embodiments, the digestion solution further comprises a trypsin digestion solution. In some embodiments, the digestion solution comprises a protease or a combination of proteases. In some embodiments, the protease is trypsin, or the combination of proteases comprises trypsin.

[0038] In some embodiments, the detecting step comprises detecting proteins in the separated first extraction solution. In some embodiments, the detecting step comprises detecting proteins in the separated second extraction solution. In some embodiments, the detecting step comprises detecting proteins in the separated third extraction solution. In some embodiments, the detecting step comprises detecting proteins in a further extraction solution. In some embodiments, detecting the first, second, third, or further set of proteins from the first, second, third, or further extraction solution, respectively, is via immunoassay. In some embodiments, detecting the first, second, third, or further set of proteins from the first, second, third, or further extraction solution, respectively, is via mass spectrometry (MS). In some embodiments, detecting via MS comprises subjecting the first, second, third, or further extraction solution to digestion prior to said detecting via mass spectrometry (MS). In some embodiments, prior to detecting the first, second, third, or further set of proteins in the first, second, third, or further extraction solution, respectively, the method further comprises incubating said first, second, third, or further extraction solution in a digestion solution.

[0039] In some embodiments, the porous material is a three-dimensional porous material. In some embodiments, the three-dimensional porous material is the tip of a volumetric absorption microsampling (VAMS) device. In some embodiments, the porous material is a non-preloaded porous material. In some embodiments, the porous material is not preloaded with a protease inhibitor. In some embodiments, the porous material is a preloaded porous material. In some embodiments, the porous material is preloaded with a protease inhibitor. In some embodiments, the porous material is preloaded with an anticoagulant. In some embodiments, the porous material is preloaded with an enzyme. In some embodiments, the porous material is preloaded with a surfactant.

[0040] In some embodiments, the method further comprises placing a sample-containing porous material in a test tube. In some embodiments, the method further comprises placing a dried sample-containing porous material in a test tube. In some embodiments, the method uses a plurality of porous materials. In some embodiments, the sample is introduced into a plurality of porous materials. In some embodiments, the plurality of porous materials comprises two or more porous materials.

[0041] In one aspect, provided herein is a method of generating a protein profile comprising the use of any one of the methods disclosed herein.

[0042] In one aspect, provided herein is a method of isolating one or more proteins comprising the use of any one of the methods disclosed herein.

[0043] In one aspect, provided herein is a method of resolving proteins in a sample by differential solubility, comprising the use of any one of the methods disclosed herein.

[0044] In one aspect, provided herein is a method for preparing sequential samples using an absorption device, comprising the use of any one of the methods disclosed herein.

[0045] In one aspect, provided herein is a microsampling device comprising: (a) a first porous material; (b) a second porous material; and (c) a test tube.

[0046] In one aspect, provided herein is a microsampling device comprising: (a) a first porous material; (b) a second porous material; (c) a third porous material; and (d) a test tube.

[0047] In some embodiments, the test tube is an Eppendorf tube.

[0048] In one aspect, provided herein is a microsampling device comprising: (a) a first porous material; (b) a second porous material; and (c) a shaft; the first porous material is located at one end of the shaft and the second porous material is disposed at a position along the shaft, such that the first porous material and the second porous material are separated and do not physically contact each other.

[0049] In one aspect, provided herein is a microsampling device comprising: (a) a first porous material; (b) a second porous material; (c) a third porous material; and (d) a shaft; the first porous material is located at one end of the shaft, and the second porous material and the third porous material are disposed at positions on the shaft, such that the first porous material, the second porous material, and the third porous material are separated and do not physically contact each other.

[0050] In some embodiments, the microsampling device is a volumetric absorption microsampling (VAMS) device.

[0051] In some embodiments, the first porous material and the second porous material are located on the shaft and separated from each other by a spacer, such that the first porous material and the second porous material are separated and do not physically contact each other.

[0052] In some embodiments, the first porous material, the second porous material, and the third porous material are located on the shaft and separated from each other by spacers, such that the first porous material, the second porous material, and the third porous material are separated and do not physically contact each other.

[0053] In some embodiments, the shaft is a threaded shaft. In some embodiments, the shaft is a non-linear shaft. In some embodiments, the shaft is a curved shaft. In some embodiments, the shaft has a length of 10 mm to 50 mm. In some embodiments, the shaft has a diameter of 0.5 mm to 3 mm. In some embodiments, the shaft fits into a test tube. In some embodiments, the shaft fits into a test tube having a volume of 5 mL, 2 mL, 1.5 mL, or 1 mL. In some embodiments, the shaft fits into an Eppendorf tube. In some embodiments, the shaft fits into a 96-well plate.

[0054] In some embodiments, the first porous material and the second porous material are preloaded with an anticoagulant. In some embodiments, the first porous material has an absorbent capacity in the range of 2.5 μL to 50 μL. In some embodiments, the first porous material and the second porous material have the same absorbent capacity. In some embodiments, the first porous material and the second porous material have different absorbent capacities. In some embodiments, the first porous material, the second porous material, and the third porous material have the same absorbent capacity. In some embodiments, the first porous material, the second porous material, and the third porous material have different absorbent capacities. In some embodiments, the first porous material and the second porous material have the same absorbent capacity, and the third porous material has a different absorbent capacity compared to the first and second porous materials.

[0055] In one aspect, provided herein is a method of using any one of the devices disclosed herein according to any one of the methods disclosed herein.

[0056] In one aspect, a method of generating a protein profile is provided, comprising: (a) obtaining at least one protein profile generated according to any one of the methods disclosed herein from a sample obtained from a subject having a disease or disorder; (b) obtaining at least one protein profile generated according to any one of the methods disclosed herein from a sample obtained from at least one subject not having the disease or disorder; (c) comparing the protein profile of the subject having the disease or disorder with the protein profile of at least one subject not having the disease or disorder; and (d) generating a disease protein profile from the comparison, wherein said generated disease protein profile comprises one or more proteins that have a different presence or level in the protein profile from the subject having the disease or disorder as compared to the protein profile of the at least one subject not having the disease or disorder.

[0057] In some embodiments, the protein profile is obtained from proteins obtained from the extraction solution and from one or more proteins obtained from the digestion solution. [Brief explanation of the drawings]

[0058] [Figure 1] Figure 1A. VAMS DBS washing procedure. Figure 1A. The VAMS DBS chip was removed from its spindle and placed in extraction solution for 24 hours. Figure 1B. The VAMS chip was then pulsed three times from 0 to 10,000 g. Figure 1C. The VAMS chip was then placed in a custom column insert in a waste collection microcentrifuge tube and centrifuged at 3,000 g for 3 minutes. Steps B and C were repeated three times. Figure 1D. After the third wash, the extraction solution was removed and the dried chip was placed in a new microcentrifuge tube for trypsin digestion. [Figure 2] FIG. 1 shows an UpSet plot showing proteins identified from whole blood (WB) and plasma using the VAMS device. [Figure 3] Figure 3 shows various DBS washing conditions. Figure 3A. Upset plot showing the effect of different DBS washing conditions on protein detection. Figure 3B. Principal component analysis (PC1, PC2) demonstrating that urea washing of DBS yields a distinct subset of proteins. [Figure 4] Figure 4 shows LC-MS analysis of proteins recovered from VAMS. Figure 4A. Upset plot showing proteins detected from whole blood using paper DBS (PBS paper and LiCl paper) or VAMS devices loaded fresh or frozen (LiCl fresh and LiCl frozen). Figure 4B. Principal component analysis (PC1, PC2) demonstrating differences in protein recovery with PBS-washed DBS on paper. [Figure 5] Volcano plot identifying differentially abundant proteins in frozen blood cell pellets from a group of patients with the same disease. FC>2, p<0.05. [Figure 6-1] Figure 6 shows cellular components of plasma and white blood cell (WBC) fractions. Figure 6A shows a bar graph depicting three types of cellular components, including cellular anatomical entities, intracellular proteins, and protein-containing complexes. Figure 6B shows a bar graph further classifying categories of non-membrane-associated protein complexes. Figure 6C shows a bar graph depicting categories of membrane-associated protein complexes. [Figure 6-2] Figure 6 shows cellular components of plasma and white blood cell (WBC) fractions. Figure 6A shows a bar graph depicting three types of cellular components, including cellular anatomical entities, intracellular proteins, and protein-containing complexes. Figure 6B shows a bar graph further classifying categories of non-membrane-associated protein complexes. Figure 6C shows a bar graph depicting categories of membrane-associated protein complexes. [Figure 7-1]Figure 7 shows the cellular components of RBC fractions. Figure 7A shows a bar graph depicting three types of cellular components, including cellular anatomical entities, intracellular proteins, and protein-containing complexes. Figure 7B shows a bar graph further classifying categories of non-membrane-associated protein complexes. Figure 7C shows a bar graph depicting categories of membrane-associated protein complexes. [Figure 7-2] Figure 7 shows the cellular components of RBC fractions. Figure 7A shows a bar graph depicting three types of cellular components, including cellular anatomical entities, intracellular proteins, and protein-containing complexes. Figure 7B shows a bar graph further classifying categories of non-membrane-associated protein complexes. Figure 7C shows a bar graph depicting categories of membrane-associated protein complexes. [Figure 8] 8A-8D illustrate a 96-well expansion of the device provided herein. FIG. 8A shows a Neoteryx tip containing a blood sample absorbed onto the tip placed in extraction solution in a 96-well plate. FIG. 8B shows the tip being removed from the extraction solution. FIG. 8C shows a new 96-well plate with holes in the bottom placed on top of the original 96-well plate. FIG. 8D shows the tip being lowered into the new 96-well plate. FIG. 8E shows the tip remover plate being placed on top of the 96-well plate. FIG. 8F shows the tip being removed from the shaft by sliding the tip remover plate sideways and lifting the tip shaft. FIG. 8G shows the shaft and tip remover plate being removed. FIG. 8H shows the empty tip being retained in the upper plate and the extract being retained in the lower 96-well plate. [Figure 9] FIG. 10 shows an UpSet plot depicting that washing a VAM device three times with 1 mL of 0.1 M Tris-HCl containing 0.5 M NaCl resulted in the identification of 1465 proteins common to frozen whole blood (WB), WB and white blood cells (WBC), and the subsequent identification of 1233 proteins unique to WBC. [Figure 10]FIG. 10 shows an UpSet plot demonstrating that washing the Neoteryx chip twice with 1 mL of 2 M urea results in the identification of a distinct protein profile containing approximately 550 fewer identified proteins compared to FIG. 9. [Figure 11] FIG. 1 shows an UpSet plot depicting that washing Neoteryx tips or filter papers with LiCl or NaCl identifies 1321 proteins present in filter papers washed with LiCl, filter papers washed with NaCl, Neoteryx tips washed with LiCl, and Neoteryx tips washed with NaCl. [Figure 12] Figure 1 shows a clustergram depicting sample or detergent types that produce different protein sets. The x-axis depicts different sample types or detergents. The y-axis depicts proteome groupings identified by the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The legend indicates statistical significance by gray scale, with darker gray reflecting lower p-values ​​and therefore more significant values. 2M urea produced significantly different protein profiles compared to the other detergents. [Figure 13] Figure 1 shows an example of two small pieces of Mitra® 3-D porous material from Neoteryx inside a plastic test tube, the bottom of which was sealed to prevent sample loss. [Figure 14] FIG. 1 shows a test tube containing two small pieces of Mitra® 3-D porous material from Neoteryx after addition of a whole blood sample (approximately 100 μL) and fractionation by centrifugation (1500 g, 5 min). [Figure 15]Figure 1 shows two pieces of Mitra® 3-D porous material from Neoteryx after application of a whole blood sample and fractionation by centrifugation (1500 g, 5 minutes). The two pieces of Mitra® 3-D porous material were removed from the test tube and placed back on their handles to allow for storage and drying. The left piece of Mitra® 3-D porous material is the upper (plasma and leukocyte-rich) portion sample, and the right piece of Mitra® 3-D porous material is the lower (RBC-rich) portion sample. [Figure 16] FIG. 1 shows a filtered centrifuge tube containing the upper and lower portions of the test tube in which the extract was stored frozen at −20° C. until required for analysis. [Figure 17] FIG. 1 shows an experimental flow chart of a method using the devices provided herein. [Figure 18] FIG. 1 shows a Venn diagram depicting the number of identified proteins unique to the plasma fraction (246 proteins) and the RBC-rich fraction (255 proteins), as well as the number of proteins identified in both fractions (206 proteins). [Figure 19] FIG. 1 shows a Venn diagram depicting the number of identified proteins unique to the plasma fraction (3 proteins) and the RBC-rich fraction (300 proteins), as well as the number of proteins identified in both fractions (51 proteins). [Figure 20] FIG. 20A shows an example of two small pieces of Mitra® 3-D porous material from Neoteryx mounted on a metal shaft, the individual portions of which are identified schematically in FIG. 20B. [Figure 21] 21A shows the device (ie, the device from FIG. 20) and in FIG. 21B after a whole blood sample has been added and fractionated by centrifugation (1500 g, 5 min). [Figure 22]FIG. 1 shows a Venn diagram depicting the number of identified proteins unique to the plasma fraction (1017 proteins) and the RBC-rich fraction (80 proteins), as well as the number of proteins identified in both fractions (262 proteins). [Figure 23] FIG. 1 shows a Venn diagram depicting the number of identified proteins unique to the plasma / WBC fraction (791 proteins) and the RBC-rich fraction (29 proteins), as well as the number of proteins identified in both fractions (40 proteins). [Figure 24]

[0023] Figure 1 shows an UpSet plot depicting the presence of various proteins in frozen whole blood cell pellets from five healthy donors and five stage III cancer donors. Healthy and cancer samples were matched for both age and sex. Healthy samples are represented by odd numbers 1, 3, 5, 7, and 9, while cancer samples are represented by even numbers 2, 4, 6, 8, and 10. [Figure 25] FIG. 25 shows a box plot depicting the middle 50 percent of data values, also known as the interquartile range, or IQR. The median value is depicted as a line dividing the box in half. The IQR illustrates variability within a set of values. A large IQR indicates a large spread of values, while a smaller IQR indicates that the majority of values ​​fall near the center. The box plot also illustrates the minimum and maximum data values ​​through the whiskers extending from the box, and optionally outliers as points extending beyond the whiskers. FIG. 25A shows unfiltered label-free quantification (LFQ). FIG. 25B shows filtered LFQ with shifted zeros. FIG. 25C shows filtered normalized median. [Figure 26]FIG. 26 shows a box plot depicting the middle 50 percent of data values, also known as the interquartile range, or IQR. The median value is depicted as a line dividing the box in half. The IQR illustrates variability within a set of values. A large IQR indicates a large spread of values, while a smaller IQR indicates that the majority of values ​​fall near the center. The box plot also illustrates the minimum and maximum data values ​​through the whiskers extending from the box, and optionally outliers as points extending beyond the whiskers. FIG. 26A shows imputed missing values ​​for each sample. FIG. 26B shows the filtered normalized median. FIG. 26C shows the normalized median LFQ with zeros imputed. [Figure 27] Figure 27 shows Volcano plots depicting differentially expressed (DE) proteins / genes that were significantly different between cancer and controls. Figure 27A shows a simple t-test. Figure 27B shows a Limma adjusted t-test. [Figure 28] FIG. 1 shows a heatmap depicting the clear separation of differentially expressed (DE) proteins / genes between cancer and controls. [Figure 29] FIG. 1 shows a clustergram depicting enriched intracellular proteins between cancer (frozen samples) and freshly prepared and frozen samples from healthy individuals. [Figure 30] 30A shows a volumetric absorption microsampling device. Figure 30A shows a test tube containing two "plugs" of sponge-like absorbent material that capture a defined volume of fluid. The absorbent plugs may also contain an anticoagulant substance (wet or dry) to prevent blood clotting during separation. Figure 30B shows the introduction of blood into the top of the test tube. Figure 30C shows the test tube after centrifugation. RBCs, WBCs, and platelets are removed from the plasma and contained within absorbent plug 1. Absorbent plug 2 contains the plasma. [Figure 31]31A shows a volumetric absorption microsampling device. Figure 31A shows a test tube containing two "plugs" of sponge-like absorbent material connected to each other by a shaft. Figure 31B shows the introduction of blood into the top of the test tube. Figure 31C shows the test tube after centrifugation. RBCs, WBCs, and platelets are removed from the plasma and contained within absorbent plug 1. Absorbent plug 2 contains the plasma. Figure 31D shows the removal of the absorbent plug after centrifugation. [Figure 32] 32A shows a volumetric absorption microsampling device. Figure 32A shows a test tube provided with a lid to prevent leakage and suppress aerosol release during centrifugation. Figure 32B shows the introduction of blood into the top of the test tube. Figure 32C shows the test tube after centrifugation. RBCs, WBCs, and platelets are removed from the plasma and contained within absorbent plug 1. Absorbent plug 2 contains the plasma. Figure 32D shows the removal of the absorbent plug after centrifugation. [Figure 33] 33A shows a volumetric absorption microsampling device. Figure 33A shows a test tube that can have a relatively wide opening at the top to facilitate blood collection by wiping or scraping a drop of blood from a fingertip. Figure 33B shows a long, narrow test tube with variable spacing between absorbent plugs to ensure that the plasma sample is free of cells or platelets. [Figure 34] FIG. 10 shows the squeeze tube shaft as indicated by the arrow to create suction and aspirate the blood droplet. [Figure 35] Figure 1 shows the wire used to create the shaft (handle), which was shaped to separate the absorbent plug. The blood sample was venous blood anticoagulated with EDTA. The fractionation device shown was placed in an empty 1,500 μL Eppendorf tube. Blood was added using a pipette until the upper absorbent plug was covered. The tube was closed and centrifuged at 1,500 g for 4 minutes. The wire shaft was used to remove the device from the tube, and the absorbent plug was allowed to air dry. Cellular components, particularly RBCs, were substantially removed from the upper absorbent plug by visual assessment. [Figure 36] Figure 1 shows a 2 mm threaded bolt used to create the shaft (handle). The threads on the shaft prevented the upper (plasma) absorbent plug from falling off during centrifugation. The blood sample was finger-prick capillary blood. The absorbent plug was moistened with 10x EDTA anticoagulant. The fractionation device shown was placed into an empty 500 μL Eppendorf tube. Blood was collected from a finger-prick using a disposable pipette, and approximately 50 μL was added. The tube was closed and centrifuged for 2 minutes using a modified battery-powered fan. The shaft containing the absorbent plug was removed using tweezers, and the absorbent plug was allowed to air dry. Cellular components, particularly RBCs, were substantially removed from the upper absorbent plug by visual assessment. [Figure 37] Figure 1 shows the experimental workflow of a method for sequential sample preparation using an absorbent device. In this basic workflow, a low-volume extraction is performed using PBS, followed by a high-volume wash. These steps are repeated prior to trypsin digestion of the chip. [Figure 38] Figure 1 shows the experimental workflow of a method for sequential sample preparation using an absorbent device. In this workflow, two extractions and two analyses are performed. The first extraction is a low-volume extraction using PBS or Tween (or PBS and Tween), followed by a high-volume wash using NaCl or LiCl. The wash can be repeated one, two, or three times prior to trypsin digestion of the chip. [Figure 39] This figure shows the experimental workflow of a method for sequential sample preparation using an absorbent device. This workflow involves two rounds of extraction and analysis, including a stringent wash to remove non-membrane proteins. This workflow improves on the basic workflow. First, a low-volume extraction is performed using PBS or Tween (or PBS and Tween), followed by a high-volume wash using urea, urea / thiourea, or a surfactant. The washes are repeated one, two, or three times prior to trypsin digestion of the chip. [Figure 40]Figure 1 shows the experimental workflow of a method for sequential sample preparation using an absorbent device. This workflow involves three rounds of extraction and analysis, including a stringent extraction as an intermediate step. First, a low-volume extraction using PBS or Tween (or PBS and Tween) is performed, followed by a low-volume extraction using urea, urea / thiourea, or a surfactant, followed by trypsin digestion of the chip. [Figure 41] Figure 1 shows the experimental workflow of a method for sequential sample preparation using an absorbent device. In this workflow, two porous materials were used in the device. The device was centrifuged, and two extractions and two analyses were performed. A low-volume extraction was performed using Tween or PBS (or PBS and Tween), followed by one, two, or three high-volume extractions (1 mL) using NaCl or LiCl, followed by trypsin digestion of the chip. [Figure 42] FIG. 1 shows the total peptide yield recovered according to Example V. [Figure 43] FIG. 1 shows the number of protein and peptide identifications (IDs) observed according to Example V. [Figure 44] FIG. 1 shows the percentage of deletion truncations after trypsin digestion according to Example V. [Figure 45] FIG. 1 shows the total protein yield recovered according to Example VI. [Figure 46] FIG. 1 shows the number of protein and peptide identifications (IDs) observed according to Example VI. [Figure 47] FIG. 1 shows the percentage of deletion cleavage products after trypsin digestion according to Example VI and in Hela cells (Hela standard) as a control. [Figure 48] FIG. 1 shows the percentage of alkylated peptides (containing carbamidomethyl modifications (CAM)) according to Example VI. [Figure 49]FIG. 1 shows a Venn diagram depicting the number of identified proteins unique to peripheral blood mononuclear cell (PBMC) and whole blood (WB) samples, as well as the number of proteins identified in both fractions, according to Example VI. [Figure 50] FIG. 1 shows protein classes of proteins found exclusively in PBMCs according to Example VI. [Figure 51] FIG. 1 shows protein classes of proteins found exclusively in WB according to Example VI. [Figure 52] FIG. 1 shows the total protein yield recovered according to Example VII. [Figure 53] FIG. 1 shows the number of protein IDs observed according to Example VII. [Figure 54] FIG. 1 shows the number of peptide IDs observed according to Example VII. [Figure 55] FIG. 1 shows the percentage of deletion truncations after trypsin digestion according to Example VII. [Figure 56] FIG. 10 shows a Venn diagram depicting the number of identified proteins unique to the WB sample and the WB Streck tube sample, as well as the number of proteins identified in both fractions, according to Example VII. [Figure 57] FIG. 10 shows a Venn diagram depicting the number of identified proteins unique to PBMC and WB samples, as well as the number of proteins identified in both fractions, according to Example VII. [Figure 58] FIG. 10 shows a Venn diagram depicting the number of identified proteins unique to the PBMC sample, the WB sample, the WB Streck tube sample, as well as the number of identified proteins in all three fractions according to Example VII. [Figure 59] FIG. 1 shows protein classes of proteins found exclusively in PBMC samples according to Example VII. [Figure 60]FIG. 10 shows protein classes of proteins found exclusively in WB and WB Streck tube samples according to Example VII. [Figure 61] FIG. 10 shows the protein yield recovered according to Example VIII. [Figure 62] FIG. 10 shows the number of protein IDs observed according to Example VIII. [Figure 63] FIG. 10 shows the number of peptide IDs observed according to Example VIII. [Figure 64] FIG. 10 shows the percentage of deletion truncations after trypsin digestion according to Example VIII. [Figure 65] FIG. 8 shows a Venn diagram depicting the number of identified proteins unique to buffy coat samples washed with LiCl (BC LiCl), buffy coat samples washed with urea / thiourea (BC Urea / Thiourea), and the number of proteins identified in both fractions according to Example VIII. [Figure 66] FIG. 8 shows a Venn diagram depicting the number of identified proteins unique to WB samples washed with LiCl (WB LiCl), WB samples washed with urea / thiourea (WB Urea / Thiourea), and the number of proteins identified in both fractions according to Example VIII. [Figure 67] FIG. 10 shows the top protein IDs observed from buffy coat samples with LiCl washes according to Example VIII. [Figure 68] FIG. 10 shows the top protein IDs observed from buffy coat samples with urea / thiourea washes according to Example VIII. [Figure 69] FIG. 8 shows a Venn diagram depicting the number of identified proteins unique to WB samples washed with LiCl (WB LiCl) and buffy coat samples (BC LiCl), respectively, as well as the number of proteins identified in both fractions, according to Example VIII. [Figure 70]FIG. 8 shows a Venn diagram depicting the number of identified proteins unique to PBMC and buffy coat samples, and the number of proteins identified in both fractions, according to Example VIII. [Figure 71] FIG. 10 shows the total protein yield recovered according to Example IX. [Figure 72] FIG. 10 shows the number of protein IDs observed according to Example IX. [Figure 73] FIG. 10 shows a Venn diagram depicting protein ID overlap for buffy coat (BC) chips washed with LiCl; urea / thiourea (UT); urea / thiourea / SDC; urea / thiourea / SDC / TBP according to Example IX. [Figure 74] FIG. 10 shows protein classes of proteins found exclusively in TBP according to Example IX. [Figure 75] FIG. 10 shows protein classes of proteins found exclusively in LiCl according to Example IX. [Figure 76] FIG. 10 shows overlap of protein IDs for RBCs, RBC pellets and supernatants according to Example IX. [Figure 77] FIG. 10 shows proteins found exclusively in RBC pellets according to Example IX. [Figure 78] FIG. 10 shows proteins found exclusively in RBC supernatants according to Example IX. [Figure 79] FIG. 10 shows a Venn diagram showing protein overlap for PBMCs and whole blood according to Example IX. [Figure 80] FIG. 1 shows the top abundant proteins in whole blood according to Example IX. [Figure 81] FIG. 10 shows the top abundant proteins in PBMCs according to Example IX. [Figure 82] FIG. 1 shows the total protein yield recovered according to Example X. [Figure 83] FIG. 1 shows the number of protein IDs observed according to Example X. [Figure 84] FIG. 10 shows protein and peptide IDs from DIA-NN for PBMCs digested directly in solution or in-chip followed by sequential extraction according to Example X. [Figure 85] FIG. 1 shows a summary of down-regulated proteins from PBMCs in VAMS according to Example X. [Figure 86] FIG. 1 shows a summary of upregulated proteins from PBMCs in VAMS according to Example X. [Figure 87] FIG. 10 shows protein and peptide IDs from DIA-NN for samples subjected to different washing conditions according to Example X. [Figure 88] FIG. 1 shows a Venn diagram displaying the overlap of upregulated proteins in TBP and CLA according to Example X. [Figure 89] FIG. 10 shows a Venn diagram displaying the overlap of down-regulated proteins in TBP and CLA, respectively, according to Example X. [Figure 90] FIG. 10 shows protein quantification plots for various samples according to Example XI. [Figure 91] FIG. 1 shows a Volcano plot depicting differential expression for various samples according to Example XI. [Figure 92] FIG. 10 shows a heatmap of 13 selected differentially expressed proteins that clustered well with each participant group according to Example XI. [Figure 93] FIG. 10 shows protein yields for individual chip replicates according to Example XII. [Figure 94] FIG. 10 shows the overall yield from batch to batch according to Example XII. [Figure 95] FIG. 11 shows total protein and peptide IDs from DIA-NN according to Example XIII. [Figure 96]FIG. 10 shows the average defect cutoff for individual chip replicates according to Example XI. [Figure 97] FIG. 11 shows the number of proteins with %CV less than 20% (blue) and %CV greater than 20% (orange) from analytical replicates using the same chip (intra-chip MS1), chips from each batch (inter-chip batch 1 or 2), chips from batch 1 reanalyzed when batch 2 was analyzed (intra-chip MS2), all chips on each instrument (inter-instrument), and finally all chips together (inter-chip) according to Example XII. [Figure 98] FIG. 11 shows box plots of %CV from analytical replicates using the same chip (intra-chip MS1), chips from each batch (inter-chip batch 1 or 2), chips from batch 1 reanalyzed when batch 2 was analyzed (intra-chip MS2), all chips on each instrument (inter-instrument), and finally all chips together (inter-chip), according to Example XII. [Figure 99] From left to right, violin plots of %CV for each batch (B1 and B2) and for both batches together (intra-batch) are shown. The dots indicate the median %CV according to Example XII. [Figure 100] FIG. 10 shows a PCA plot of chip and batch reproducibility according to Example XII. [Figure 101] FIG. 10 shows the total yield from DBS dried at room temperature for 1 week (dry) and from DBS prevented from drying by freezing and cycling through 3× freeze-thaw, according to Example XIII. [Figure 102] FIG. 10 shows protein IDs from DIA-NN for dried and frozen DBS according to Example XIII. [Figure 103] FIG. 10 shows a Venn diagram showing overlap of protein IDs from dried and frozen VAMS according to Example XIII. [Figure 104] FIG. 10 shows the %CV of replicates for dried and frozen DBS according to Example XIII. [Figure 105-1]FIG. 1 shows the most abundant proteins in dried (A) and frozen (B) samples according to Example XII. [Figure 105-2] FIG. 1 shows the most abundant proteins in dried (A) and frozen (B) samples according to Example XII. [Figure 106-1] FIG. 10 shows the most abundant blood proteins in dried and frozen samples according to Example XIII. [Figure 106-2] FIG. 10 shows the most abundant blood proteins in dried and frozen samples according to Example XIII. [Figure 106-3] FIG. 10 shows the most abundant blood proteins in dried and frozen samples according to Example XIII. [Figure 107] FIG. 10 shows the dynamic range, defined as the area difference between the highest and lowest peaks from both room temperature dried and frozen DBS, according to Example XIII. [Figure 108] FIG. 10 shows total protein yield for whole blood DBS after storage at room temperature (dry), 37° C. (dry 37° C.), freezing at −80° C. (freezing), or thawing and then freezing at −80° C. (thaw-freezing) according to Example XIV. [Figure 109] FIG. 10 shows total protein yield for plasma DBS after storage at room temperature (dry) or frozen at −80° C. (frozen) according to Example XIV. [Figure 110] FIG. 10 shows a Venn diagram illustrating the overlap of dried and frozen whole blood DBS extracted with DUTRA detergent according to Example XIV. [Figure 111] FIG. 10 shows proteins found exclusively in dried samples according to Example XIV. [Figure 112] FIG. 10 shows proteins found exclusively in frozen samples according to Example XIV. [Figure 113]FIG. 10 shows a Venn diagram showing the overlap of up- and down-regulated proteins in LiCl and DUTRA extractions according to Example XIV. [Figure 114] FIG. 10 shows the dynamic range, defined as the area difference between the highest and lowest peaks from both room temperature dried and frozen DBS followed by DUTRA extraction, according to Example XIV. [Figure 115] FIG. 10 shows overlap of down- and up-regulated protein IDs from VAMS stored at room temperature (RT), 37° C. (37) or frozen at −80° C. (Fz) according to Example XIV. [Figure 116] Figure 1 shows the overlap of proteins identified in whole blood (unlysed), chips (unlysed frozen), and whole blood that was lysed prior to application to the chips (lysed frozen). Both sets of chips were frozen at -80°C according to Example XIV. [Figure 117] FIG. 10 shows overlap of protein IDs from whole blood and plasma VAMS that were dried at room temperature or otherwise immediately frozen according to Example XIV. [Figure 118] FIG. 10 shows protein classes upregulated in plasma VAMS dried at room temperature or frozen at −80° C. according to Example XIV. [Figure 119] FIG. 10 shows protein classes down-regulated in plasma VAMS dried at room temperature or frozen at −80° C., respectively, according to Example XIV. [Figure 120] FIG. 10 shows the total protein yield from all samples according to Example XV. [Figure 121] FIG. 14 shows the number of protein IDs from various processing methods according to Example XV. [Figure 122] FIG. 10 shows a Venn diagram showing overlap of protein IDs from VAMS dried and stored at various temperatures: room temperature (control), 37° C. (37), or 99° C. (100), according to Example XV. [Figure 123] FIG. 10 shows a Venn diagram showing overlap of protein IDs from venous and finger prick blood according to Example XV. [Figure 124] FIG. 10 shows protein classes unique to venous blood samples according to Example XV. [Figure 125] FIG. 10 shows protein classes unique to finger-prick blood samples according to Example XV. [Figure 126] FIG. 10 shows a Venn diagram showing overlap of protein IDs from samples processed using standard methods and samples extracted first for DNA and subsequently for protein, according to Example XV. [Figure 127] FIG. 10 shows a Venn diagram showing overlap of protein IDs after using various wash buffers for extraction according to Example XV. [Figure 128] FIG. 10 shows an inverted upset plot of protein ID overlap from various cleaning and extraction methods, including sonication and benzonase during extraction, according to Example XV. [Figure 129] FIG. 10 shows a PCA plot comparing each of the washing and drying methods for whole blood VAMS according to Example XV. [Figure 130] FIG. 10 shows a summary of protein peak areas for the most abundant proteins (most abundant proteins ranked 1-20) using various washing methods according to Example XV. [Figure 131] FIG. 10 shows a summary of protein peak areas for the most abundant proteins (most abundant proteins at positions 21-30) using various washing methods according to Example XV. [Figure 132] FIG. 10 shows a Venn diagram showing the overlap in protein IDs for samples treated with Benzonase at different stages of extraction compared to untreated controls according to Example XV. [Figure 133] FIG. 10 shows the total protein yield from all digested samples according to Example XVI. [Figure 134]FIG. 10 shows protein IDs from liquid samples (digests) or in-chip processed samples (chips) for MBCC and yeast cells according to Example XVI. [Figure 135] FIG. 10 shows protein ID overlap between liquid samples (digests) and in-chip samples for MBCC and yeast cells according to Example XVI. [Figure 136] FIG. 10 shows proteins found exclusively in MBCC fluid samples according to Example XVI. [Figure 137] FIG. 10 shows proteins found exclusively in samples within the MBCC chip according to Example XVI. [Figure 138] FIG. 10 shows proteins found exclusively in yeast liquid samples according to Example XVI. [Figure 139] FIG. 10 shows proteins found exclusively in yeast in-chip samples according to Example XVI. [Figure 140] FIG. 10 shows highly abundant mouse proteins across serially diluted MBCCs in whole blood according to Example XVI. [Figure 141] FIG. 10 shows highly abundant human proteins across serially diluted MBCCs in whole blood according to Example XVI. [Figure 142] FIG. 10 shows protein IDs from liquid sample chip processed samples subjected to various washing, digestion, or processing methods according to Example XVII. [Figure 143] FIG. 10 shows protein IDs from liquid sample chip processed samples subjected to various washing, digestion, or processing methods according to Example XVII. [Figure 144] FIG. 10 shows a PCA plot comparing each of the washing methods of sample collection in whole blood VAMS and relatively low volume VAMS and collection into a PVDF membrane according to Example XVII. [Figure 145]FIG. 10 shows protein ID overlap from whole blood VAMS samples extracted according to standard methods (LiCl) or with PBS for DNA extraction or immunoassay analysis prior to mass spectrometry extraction and analysis according to Example XVII. [Figure 146] FIG. 14 shows the relative abundance of the top abundant proteins for whole blood VAMS extracted using standard method (WB control-LiCl), DTT, 2M urea & CHAPS, citric acid, or a combined wash including benzonase, LiCl, CHAPS and sonication according to Example XVII. [Figure 147] FIG. 10 shows protein classes found exclusively in 2M urea & 2% CHAPS extractions according to Example XVII. [Figure 148] FIG. 10 shows protein classes found exclusively with DTT reduction during extraction according to Example XVII. [Figure 149] FIG. 10 shows protein classes found exclusively using a combination wash including benzonase, LiCl, CHAPS, and sonication according to Example XVII. [Figure 150] FIG. 1 shows overlap of (A) peptides and (B) proteins from whole blood samples digested with trypsin, Asp-N, or Glu-C according to Example XVII. [Figure 151] FIG. 10 shows a duplicate of proteins extracted from 30 μL and 10 μL VAMS using the same protocol according to Example XVII. [Figure 152] FIG. 1 shows the relative abundance of the top abundant proteins for whole blood samples collected either in 30 μL VAMS (Mitra®) or on a PVDF membrane according to Example XVII. [Figure 153] FIG. 10 shows the relative abundance of the top abundant proteins for plasma samples collected onto VAMS preloaded with no preload (control), BSA, or SDS according to Example XVII. [Fig. 154]FIG. 1 shows overlap of phosphopeptides identified across three analytical platforms: Fraggler, DIANN, and Max Quant, according to Example XVII. [Figure 155] FIG. 10 shows overlap of protein specificity from various biological fluids including whole blood, plasma, and saliva according to Example XVII. [Figure 156] FIG. 10 shows protein ID from whole blood VAMS or filter paper samples subjected to various washing methods according to Example XVIII. [Figure 157] Figure 1 shows a Venn diagram comparison of protein profiles from a single chip extracted three times sequentially (A: first extraction; B: second extraction; and C: third extraction). Mass spectrometry was performed according to Example XVIII on all three extractions according to Example XVIII. [Figure 158] FIG. 10 shows a heat map and Venn diagram comparison of protein profiles from samples washed with 250 mM, 500 mM, or 1 M LiCl according to Example XVIII. [Figure 159] FIG. 10 shows a Venn diagram of protein IDs from VAMS washed with CaCl or LiCl according to Example XVIII. [Figure 160] FIG. 10 shows Venn diagrams of protein IDs from VAMS (M) and filter paper (FP) for (A) collected and frozen or (B) collected and dried samples according to Example XVIII. [Figure 161] FIG. 10 shows a PCA plot of the differences between whole blood samples collected onto filter paper or VAMS (Mitra®) and samples that were immediately frozen or dried overnight according to Example XVIII. [Figure 162] FIG. 1 shows (A) protein and (B) peptide IDs from samples digested with trypsin overnight (Tryp control), trypsin overnight followed by an additional 2 hours (Tryp-Tryp), or trypsin overnight followed by 2 hours of digestion with Glu-C (Tryp-GluC) according to Example XVIII. [Figure 163] FIG. 10 shows total protein yield from whole blood or plasma VAMS subjected to various sample processing and washing methods according to Example XIX. [Fig. 164] FIG. 10 shows a Venn diagram comparison of protein profiles from (A) plasma samples or (B) whole blood samples that were either untreated (control), adjusted to pH 8 (TEAB), or adjusted to pH 6 (citrate) according to Example XIX. [Figure 165-1] 1 shows protein class assessment of unique IDs from whole blood samples adjusted to (A) pH 8 or (B) pH 6. Each colored bar represents a different protein class according to Example XIX. [Figure 165-2] 1 shows protein class assessment of unique IDs from whole blood samples adjusted to (A) pH 8 or (B) pH 6. Each colored bar represents a different protein class according to Example XIX. [Figure 166] FIG. 10 shows a Venn diagram comparison of protein profiles from (A) plasma samples or (B) whole blood samples spiked into standard VAMS (control) or long shelf-life VAMS (NM) according to Example XIX. [Figure 167] FIG. 10 shows a Venn diagram comparison of protein IDs from whole blood samples washed with LiCl at 500 mM, 125 mM, or 50 mM, or with CaCl at 50 mM, according to Example XIX. [Figure 168] Venn diagram comparison of protein IDs from (A) plasma or (B) whole blood samples extracted sequentially for glycan analysis and subsequent proteome analysis. According to Example XX, samples were either prepared according to standard methods (control), washed and then prepared for glycomics (SB161B and SB159B glyco), prepared directly for glycomics (SB162 glyco and SB160 glyco), or the reduced / alkylated fraction prior to glycomics digestion was analyzed (R / A). [Figure 169] Figure 2 shows PCA plots of protein IDs from (A) plasma or (B) whole blood samples extracted sequentially for glycan analysis and subsequent proteome analysis. According to Example XX, samples were either prepared according to standard methods (Control), prepared for glycomics (Glyco), or the reduced / alkylated fraction prior to glycomics digestion was analyzed (Red / Alk). [Figure 170] FIG. 10 shows a Venn diagram comparison of protein IDs from whole blood samples extracted with 100 mM Tris, H2O, or 500 mM LiCl+100 mM Tris (control) according to Example XXI. [Figure 171] FIG. 2 shows total protein yield from bovine liver tissue samples digested either in VAMS (chip) or in vitro as a liquid digest (digest) after solubilization with either SDC or PBS according to Example XXII. [Fig. 172] FIG. 2 shows protein IDs from bovine liver tissue samples digested either in VAMS (chip) or in vitro as a liquid digest (digest) after solubilization with either SDC or PBS according to Example XXII. [Figure 173] FIG. 2 shows a Venn diagram comparison of bovine liver tissue samples digested either in VAMS (chip) or in vitro as a liquid digest (digest) after solubilization with either SDC or PBS according to Example XXII. [Fig. 174]

[0023] Figure 1 shows the experimental workflow of a method for sequential sample preparation using an absorbent device. In this workflow, three or optionally four extractions and analyses are performed. First, a low-volume extraction is performed using PBS or Tween (or PBS and Tween), followed by a series of washes to target specific protein groups. The porous material is then treated to remove glycans, followed by trypsinization of the porous material. Analysis can be performed on the first PBS-based extraction, optionally with the collected detergent (which can be considered a second extraction), the removed glycans, and a tryptic digest of the porous material. DETAILED DESCRIPTION OF THE INVENTION

[0059] As used herein, the terms "Mitra®," "Neoteryx tip," and "absorbable plug" are used interchangeably and refer to the porous materials disclosed herein.

[0060] As used herein, the terms "VAMS" and "microsampling device" are used interchangeably.

[0061] Provided herein is a volume absorption microsampling (VAMS) device for use in precision medicine genetic analysis.Specifically, provided herein is a VAMS device comprising a first porous material, a second porous material, and a shaft.Provided herein is a VAMS device comprising a first porous material, a second porous material, and a test tube.Provided herein is a VAMS device comprising a first porous material, a second porous material, a third porous material, and a shaft.Provided herein is a VAMS device comprising a first porous material, a second porous material, a third porous material, and a test tube.Also provided herein is a method of using a VAMS device to generate protein profiles, fractionate blood samples, or isolate blood cells.

[0062] Cost-effective single nucleotide polymorphism (SNP) arrays containing approximately 800,000 variants allow for the rapid assessment of a wide range of polymorphisms. In recent years, the cost of SNP arrays has decreased significantly, driven by the very large sample sizes required to perform genome-wide association studies (GWAS) (Suratannon N et al., Frontiers in Immunology. 2020;11(614)). Various genome sequencing modalities, including whole-genome sequencing, whole-exome sequencing, targeted gene panels, and transcriptomics, have reduced costs and are increasingly being applied in clinical settings (Pleasance et al., Nature Cancer. 2020;1(4):452-68; Lievre et al., J Clin Oncol. 2008;26(3):374-9; Mosele et al., Ann Oncol. 2020;31(11):1491-505).

[0063] At least in principle, further levels of personalization can be achieved by using other omics technologies to obtain quantitative readouts of functional molecules such as proteins and metabolites (Geyer et al., Cell Syst. 2016;2(3):185-95). In practice, proteomics technologies have not kept pace with genomics, especially in quantitative longitudinal analysis of patient blood. While thousands of proteins can be quantified from highly fractionated plasma using long LC-MS run times, such workflows are impractical for routine precision medicine applications, reducing the number of studies reported for demonstration projects (Dey et al., Clinical Proteomics. 2019;16(1):16; Wewer et al., Cell Syst. 2018;7(6):601-12.e3). Specifically, the issue is that the time required to fractionate a plasma sample by multidimensional chromatography (multiple chromatography steps and / or types, such as reversed-phase and size exclusion) is typically several hours for each sample. Therefore, implementing this strategy for hundreds or thousands of samples is completely impractical. Additionally, performing proteomics on liquid components (plasma or serum) does not allow for the identification of intracellular proteins, membrane proteins, or other cell-associated proteins (e.g., membrane-bound).

[0064] For blood proteomics to provide significant value to precision medicine research, repeated longitudinal sample collection using systems simple enough to generate use by the majority of participants is required. Furthermore, simplified analytical workflows compatible with high-throughput measurement techniques are needed to quantify protein abundance in these blood samples.

[0065] method In one aspect, provided herein is a method of fractionating a sample, comprising: (a) introducing a sample into a porous material; (b) optionally centrifuging the porous material containing the sample; (c) drying the sample in the porous material; (d) extracting a first set of proteins from the dried sample-containing porous material, comprising (1) incubating the dried sample-containing porous material in a first extraction solution; and (2) separating the first extraction solution from the first incubated porous material; and (e) detecting the first set of proteins in the separated extraction solution.

[0066] In one aspect, provided herein is a method of fractionating a sample, comprising: (a) introducing a sample into a porous material; (b) optionally centrifuging the porous material containing the sample; (c) drying the sample in the porous material; (d) extracting a first set of proteins from the dried sample-containing porous material, comprising (1) incubating the dried sample-containing porous material in a first extraction solution; (2) separating the first extraction solution from the first incubated porous material; and (3) optionally washing the separated first extracted porous material; (e) extracting a second set of proteins from the first extracted porous material, comprising (1) incubating the first extracted porous material in a second extraction solution; (2) separating the second extraction solution from the second incubated porous material; and (f) detecting the first or second set of proteins from the separated first or second extraction solution, respectively.

[0067] In one aspect, provided herein is a method further comprising: (g) optionally washing the separated second extracted porous material; (h) extracting a third set of proteins from the second extracted porous material, comprising: (1) incubating the second extracted porous material in a third extraction solution; and (2) separating the third extraction solution from the third incubated porous material; and (i) detecting the third set of proteins in the separated third extraction solution.

[0068] In some embodiments, the method further comprises sequentially extracting one or more additional sets of proteins from the third extracted porous material, comprising repeating steps (g)-(i) with one or more additional extraction solutions.

[0069] In one aspect, provided herein is a method of fractionating a sample, comprising: (a) introducing a sample into a porous material; (b) optionally centrifuging the porous material containing the sample; (c) drying the sample in the porous material; (d) extracting a first set of molecules from the dried sample-containing porous material, comprising: (1) incubating the dried sample-containing porous material in a first extraction solution; (2) separating the first extraction solution from the first incubated porous material; and (3) optionally washing the separated first extracted porous material; (e) digesting proteins remaining in the first extracted porous material, comprising: (1) incubating the first extracted porous material in a digestion solution; and (2) separating the digestion solution from the digest-incubated porous material; and (f) detecting one or more proteins in the separated digestion solution; and (g) optionally detecting one or more molecules in the first set of molecules.

[0070] In some embodiments, prior to the digestion step, the method further comprises: (A) extracting a second set of molecules from the first extracted porous material, comprising: (1) incubating the first extracted porous material in a second extraction solution; (2) separating the second extraction solution from the second incubated porous material; and (g) optionally washing the separated second extracted porous material; and (B) optionally detecting the second set of molecules in the separated second extraction solution.

[0071] In some embodiments, prior to the digestion step, the method further comprises sequentially extracting one or more additional sets of molecules from the second (and subsequent) extracted porous material, comprising repeating steps (A)-(B) with one or more additional extraction solutions.

[0072] In some embodiments, the molecule is selected from the group consisting of a protein, a nucleic acid, and a glycan. In some embodiments, the protein is a phosphorylated protein.

[0073] In one aspect, the method includes: (a) introducing a sample into a porous material; (b) optionally centrifuging the sample-containing porous material; (c) drying the sample in the porous material; (d) (1) incubating the dried sample-containing porous material in a first extraction solution; (2) separating the first extraction solution from the first incubated porous material; and (3) optionally washing the separated first extracted porous material; and (4) optionally detecting one or more molecules in the first set of molecules; (e) (1) incubating the first extracted porous material in a second extraction solution; (2) separating the second extraction solution from the second incubated porous material; and (3) optionally washing the separated second extracted porous material; and (4) Provided herein are methods for fractionating a sample, comprising: extracting a second set of molecules from the first extracted porous material, optionally including detecting one or more molecules in the second set of molecules in a separated second extraction solution; and (f) digesting proteins remaining in the first extracted porous material, including (1) incubating the separated second extracted porous material in a digestion solution; and (2) separating the digestion solution from the digestion-incubated porous material; and (3) detecting one or more proteins in the separated digestion solution.

[0074] In some embodiments, the first set of molecules is or comprises a protein. In some embodiments, the first set of molecules is or comprises a nucleic acid. In some embodiments, the first set of molecules is or comprises a glycan. In some embodiments, the first set of molecules is a nucleic acid, and the second first set of molecules is or comprises a protein. In some embodiments, the first set of molecules is or comprises a protein, and the second set of molecules is or comprises a glycan. In some embodiments, the protein is a phosphorylated protein.

[0075] In some embodiments, the proteins in the first, second, or one or more additional sets of molecules and / or one or more peptides or proteins in the digestion solution are phosphoproteins or phosphopeptides, respectively. In some embodiments, the method further comprises performing an enrichment step to enrich for phosphorylated proteins and / or peptides in the separated first, second, or one or more additional extraction solutions and / or digestion solutions. In some embodiments, the method comprises pooling two or more separated first extraction solutions, two or more separated second extraction solutions, two or more of the separated one or more additional extraction solutions, and / or pooling two or more digestion solutions obtained from a fractionation method as described herein performed on one or more replicate samples or one or more separate samples. In some embodiments, the enrichment step comprises an affinity-based technique, an immunoprecipitation technique, or a chemical modification technique. In one embodiment, the enrichment step comprises an immobilized metal affinity chromatography (IMAC) and / or a metal oxide affinity chromatography (MOAC) enrichment step. In another embodiment, the enrichment step utilizes zirconium-based IMAC particulates.

[0076] In one aspect, provided herein is a method of generating a protein profile comprising utilizing a method of fractionating a sample as disclosed herein. In some embodiments, the protein profile is generated by an immunoassay workflow. In some embodiments, the protein profile is generated by a proteomics workflow. In some embodiments, the protein profile is generated by a combination of an immunoassay workflow and a proteomics workflow. In some embodiments, the method generates a protein profile of non-membrane / soluble protein complexes. In some embodiments, the method generates a protein profile of cell membrane-associated proteins. In some embodiments, the method generates a protein profile of phosphorylated proteins.

[0077] In one aspect, provided herein is a method of isolating one or more proteins comprising utilizing a method of fractionating a sample as disclosed herein.

[0078] In one aspect, provided herein is a method of resolving proteins in a sample by differential solubility, comprising utilizing a method of fractionating the sample as disclosed herein.

[0079] In one aspect, provided herein is a method of preparing sequential samples using an absorbent device, comprising utilizing a method of fractionating a sample as disclosed herein.

[0080] In one aspect, provided herein is a method of generating a protein profile comprising: (a) introducing a blood sample into a porous material; (b) drying the blood sample in the porous material; (c) detecting a first set of extracted proteins from the dried sample-containing porous material using a first extraction solution in a container; and (d) detecting digested cell membrane-associated proteins from the first extracted sample-containing porous material using a digestion solution in a container; wherein the protein profile generated from the extracted first set of proteins and the digested cell membrane-associated proteins comprises one or more additional proteins compared to a protein profile generated from detecting proteins in plasma from said blood sample.

[0081] In one aspect, provided herein is a method of fractionating a blood sample, comprising: (a) introducing a blood sample into a porous material; (b) drying the blood sample in the porous material; (c) detecting a first set of extracted proteins from the dried sample-containing porous material using a first extraction solution in a container; and (d) detecting digested cell membrane-associated proteins from the first extracted sample-containing porous material using a digestion solution in a container; wherein a protein profile generated from the extracted first set of proteins and the digested cell membrane-associated proteins comprises one or more additional proteins compared to a protein profile generated from detecting proteins in plasma from said blood sample.

[0082] In one aspect, provided herein is a method for isolating blood cells, comprising: (a) introducing a blood sample into a porous material; (b) drying the blood sample in the porous material; (c) detecting a first set of extracted proteins from the dried sample-containing porous material using a first extraction solution in a container; and (d) detecting digested cell membrane-associated proteins from the first extracted sample-containing porous material using a digestion solution in a container; wherein a protein profile generated from the extracted first set of proteins and the digested cell membrane-associated proteins comprises one or more additional proteins compared to a protein profile generated from detecting proteins in plasma from said blood sample.

[0083] In one aspect, the method includes the steps of: (a) introducing a sample into a porous material; (b) drying the sample in the porous material; (c) (i) incubating the first mixture of dried sample-containing porous material in a first extraction solution in a vessel; (ii) centrifuging the first mixture in the vessel; (iii) separating the first extraction solution from the centrifuged first mixture in the vessel, wherein the separated first extraction solution comprises a first set of proteins; and (iv) optionally collecting the first set of proteins from the dried sample-containing porous material, the step comprising: repeating steps (i)-(iii) of step (c) one or more times with the separated first extracted sample-containing porous material in the vessel and the first extraction solution; (d) (i) incubating the digestion mixture of the separated first extracted sample-containing porous material from step (c) in a digestion solution in the vessel; (ii) centrifuging the digestion mixture in the vessel; and (iii) Provided herein is a method for generating a protein profile, comprising: (a) collecting cell membrane-associated proteins from the separated first extracted sample-containing porous material, comprising the steps of separating a digestion solution from the centrifuged digestion mixture in the vessel, wherein the separated digestion solution comprises cell membrane-associated proteins; and (e) detecting the first set of proteins and the cell membrane-associated proteins.

[0084] In one aspect, the method includes the steps of: (a) introducing a sample into a porous material; (b) drying the sample in the porous material; (c) (i) incubating the dried sample-containing porous material first mixture in a first extraction solution in a vessel; (ii) centrifuging the first mixture in the vessel; (iii) separating the first extraction solution from the centrifuged first mixture in the vessel, wherein the separated first extraction solution comprises a first set of proteins; and (iv) optionally collecting the first set of proteins from the dried sample-containing porous material, the step comprising: repeating steps (i)-(iii) of step (c) one or more times with the separated first extracted sample-containing porous material in the vessel and the first extraction solution; (d) (i) incubating the digestion mixture of the separated sample-containing porous material from step (c) in a digestion solution in the vessel; (ii) centrifuging the digestion mixture in the vessel; and (iii) Provided herein is a method for fractionating a sample, comprising: (a) collecting cell membrane-associated proteins from the separated first extracted sample-containing porous material, comprising the steps of separating a digestion solution from the centrifuged digestion mixture in the container, wherein the separated digestion solution comprises cell membrane-associated proteins; and (b) detecting the first set of proteins and the cell membrane-associated proteins.

[0085] In one aspect, the method includes the steps of: (a) introducing a sample into a porous material; (b) drying the sample in the porous material; (c) (i) incubating the dried sample-containing porous material first mixture in a first extraction solution in a vessel; (ii) centrifuging the first mixture in the vessel; (iii) separating the first extraction solution from the centrifuged first mixture in the vessel, wherein the separated first extraction solution comprises a first set of proteins; and (iv) optionally collecting the first set of proteins from the dried sample-containing porous material, the step comprising: repeating steps (i)-(iii) of step (c) one or more times with the separated sample-containing porous material in the vessel and the first extraction solution; (d) (i) incubating the digestion mixture of the separated sample-containing porous material from step (c) in a digestion solution in the vessel; (ii) centrifuging the digestion mixture in the vessel; and (iii) Provided herein is a method for isolating blood cells, comprising: (a) collecting a set of cell membrane-associated proteins from a first extracted sample-containing porous material, the step comprising separating a digestion solution from the centrifuged digestion mixture in the container, wherein the separated digestion solution comprises cell membrane-associated proteins; and (b) detecting the first set of proteins and the cell membrane-associated proteins.

[0086] In one aspect, provided herein is a method of generating a protein profile comprising: (a) introducing a sample into a porous material; (b) drying the sample in the porous material; (c) detecting a first set of proteins extracted from the dried sample-containing porous material using a first extraction solution in a vessel; and (d) detecting digested cell membrane-associated proteins from the first extracted sample-containing porous material using a digestion solution in a vessel; wherein the protein profile generated from the extracted first set of proteins and the digested cell membrane-associated proteins comprises one or more additional proteins compared to the protein profile generated from the detection of proteins from the sample.

[0087] In one aspect, provided herein is a method of fractionating a sample, comprising: (a) introducing a sample into a porous material; (b) drying the sample in the porous material; (c) detecting a first set of extracted proteins from the dried sample-containing porous material using a first extraction solution in a container; and (d) detecting digested cell membrane-associated proteins from the first extracted sample-containing porous material using a digestion solution in a container; wherein a protein profile generated from the extracted first set of proteins and the digested cell membrane-associated proteins comprises one or more additional proteins compared to a protein profile generated from detecting proteins from the sample.

[0088] Table 1 shows a list of membrane-associated and non-membrane-associated protein complexes in Figures 6A-6C.

[0089] Table 2 shows a list of membrane-associated and non-membrane-associated protein complexes in Figures 7A-7C.

[0090] [Table 1] TIFF2025504594000002.tif63170

[0091] [Table 2]

[0092] In some embodiments, the method further comprises the steps of: (i) incubating a second mixture of the separated first extracted sample-containing porous material in a second extraction solution in a container; (ii) centrifuging the second mixture in the container; (iii) separating the second extraction solution from the centrifuged second mixture in the container, wherein the separated second extraction solution comprises the second set of proteins; and (iv) optionally collecting the second set of proteins from the separated first extracted sample-containing porous material from step (c), comprising repeating steps (i)-(iii) one or more times using the separated second extracted sample-containing porous material in the container and the second extraction solution.

[0093] In some embodiments, collecting the cell membrane-associated proteins comprises collecting the cell membrane-associated proteins from the separated second extracted sample-containing porous material, comprising: (i) incubating the digestion mixture of the separated second extracted sample-containing porous material in a digestion solution in the vessel; (ii) centrifuging the digestion mixture in the vessel; and (iii) separating the digestion solution from the centrifuged digestion mixture in the vessel, wherein the separated digestion solution comprises the cell membrane-associated proteins.

[0094] In some embodiments, the detecting step comprises detecting said first set of proteins, said second set of proteins, and said cell membrane-associated protein.

[0095] In some embodiments, the method further comprises the steps of: (i) incubating a third mixture of the separated second extracted sample-containing porous material in a third extraction solution in a container; (ii) centrifuging the third mixture in the container; (iii) separating the third extraction solution from the centrifuged second mixture in the container, wherein the separated third extraction solution comprises a third set of proteins; and (iv) optionally collecting the third set of proteins from the separated second extracted sample-containing porous material, comprising repeating steps (i)-(iii) one or more times using the separated third extracted sample-containing porous material in the container and the third extraction solution.

[0096] In some embodiments, collecting the cell membrane-associated proteins comprises collecting the cell membrane-associated proteins from the separated third extracted sample-containing porous material, comprising: (i) incubating the digestion mixture of the separated third extracted sample-containing porous material in a digestion solution in the container; (ii) centrifuging the digestion mixture in the container; and (iii) separating the digestion solution from the centrifuged digestion mixture in the container, wherein the separated digestion solution comprises the cell membrane-associated proteins.

[0097] In some embodiments, the detecting step comprises detecting said first set of proteins, said second set of proteins, said third set of proteins, and said cell membrane-associated proteins.

[0098] In some embodiments, the proteins in the first set of proteins, the second set of proteins, the third set of proteins, and the cell membrane-associated proteins are phosphoproteins or phosphopeptides. In some embodiments, the method further comprises performing an enrichment step to enrich for phosphorylated proteins and / or peptides in the separated first, second, or third or one or more additional extraction and / or digestion solutions. In some embodiments, the method comprises pooling two or more separated first extraction solutions, two or more separated second extraction solutions, or two or more of the separated one or more additional extraction solutions, and / or pooling two or more digestion solutions obtained from a fractionation method as described herein performed on one or more replicate samples or one or more separate samples. In some embodiments, the enrichment step comprises an affinity-based technique, an immunoprecipitation technique, or a chemical modification technique. In one embodiment, the enrichment step comprises an immobilized metal affinity chromatography (IMAC) and / or a metal oxide affinity chromatography (MOAC) enrichment step. In another embodiment, the enrichment step utilizes zirconium-based IMAC particulates.

[0099] In some embodiments, the sample is or comprises a bodily fluid. In some embodiments, the sample comprises cells or tissue. In some embodiments, the sample comprises cells suspended in a liquid. In some embodiments, the sample comprises cultured cells suspended in a culture medium. In some embodiments, the bodily fluid is or comprises blood, a blood fraction, plasma, a nucleic acid-containing stabilized sample, a stabilized blood sample (e.g., blood in a Streck tube sample), urine, tears, wound fluid, CSF, bronchoalveolar lavage fluid, or ascites. In some embodiments, the sample is or comprises plasma. In some embodiments, the sample is or comprises a blood sample. In some embodiments, the blood sample is a whole blood (WB) sample. In some embodiments, the blood sample is a red blood cell (RBC) sample. In some embodiments, the blood sample is a white blood cell (WBC) sample. In some embodiments, the blood sample is a frozen blood sample. In some embodiments, the blood sample is fresh. As used herein, the term "fresh" refers to a sample that is not frozen. In some embodiments, the blood sample is frozen. In some embodiments, the sample is or comprises a nucleic acid-containing stabilized sample or stabilized blood sample, such as a nucleic acid-containing stabilized sample or a stabilized blood sample, such as a blood sample in a Streck tube. In some embodiments, the bodily fluid comprises blood or a blood fraction. In some embodiments, the bodily fluid comprises frozen blood or a frozen blood fraction. In some embodiments, the blood fraction comprises WBCs, platelets, or RBCs.

[0100] In other embodiments, the sample is or comprises a blood sample. In some embodiments, the sample is or comprises a whole blood sample. In some embodiments, the sample is a blood sample, and the protein profile generated from the extracted at least a first set of proteins and the digested cell membrane-associated proteins comprises one or more additional proteins compared to a protein profile generated from detecting proteins in plasma from said blood sample. In some embodiments, the blood sample is a whole blood (WB) sample. In some embodiments, the blood sample is a red blood cell sample (RBC). In some embodiments, the blood sample is a fractionated blood sample. In some embodiments, the fractionated blood sample comprises white blood cells (WBC), platelets and / or RBCs. In some embodiments, the blood sample is a frozen blood sample. In some embodiments, the blood sample is a fresh blood sample.

[0101] In some embodiments, the blood sample has a volume in the range of 100 μL to 2 μL, 100 μL to 5 μL, 100 μL to 10 μL, 99 μL to 2 μL, 90 μL to 2 μL, 80 μL to 2 μL, 70 μL to 2 μL, 60 μL to 2 μL, 50 μL to 2 μL, 40 μL to 2 μL, 30 μL to 2 μL, 20 μL to 2 μL, 10 μL to 2 μL, or 5 μL to 2 μL. In some embodiments, the blood sample has a volume of less than 100 μL, less than 50 μL, less than 30 μL, less than 10 μL, or less than 5 μL. In some embodiments, the blood sample has a volume of at least 2 μL.

[0102] In some embodiments, the introducing step comprises absorbing the sample into the porous material. In some embodiments, the introducing step comprises absorbing the sample into the porous material via a finger prick. In some embodiments, the introducing step comprises immersing the porous material into the sample. In some embodiments, the introducing step comprises pipetting a known volume of the sample into the porous material.

[0103] In some embodiments, the method further comprises collecting the sample. In some embodiments, the method further comprises collecting the sample using a microsampling device. In some embodiments, introducing the sample comprises absorbing the sample into a porous material.

[0104] In some embodiments, the blood sample is divided. In some embodiments, the dividing step comprises centrifugation. In some embodiments, the method comprises centrifuging the porous material containing the sample prior to the drying step. In some embodiments, the centrifugation is performed at a speed of 1,500 g for 5 minutes. In some embodiments, the centrifugation is performed at 500 g to 10,000 g. In some embodiments, the centrifugation is performed at 500 g to 3,000 g. In some embodiments, the centrifugation is performed for at least 1-15 minutes. In some embodiments, the centrifugation is performed for 15 minutes or less. In other embodiments, the blood sample is divided via filtration.

[0105] In some embodiments, the method comprises sequential extraction. In some embodiments, the sequential extraction is solubility-based protein fractionation. In some embodiments, the method comprises a single or non-sequential extraction.

[0106] In some embodiments, the method recovers 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, or 2000 or more proteins compared to conventional microsampling. In some embodiments, the method recovers a fraction of non-membrane / soluble protein complexes from a red blood cell (RBC) sample that are not obtained by conventional microsampling. In some embodiments, the method recovers a fraction of non-membrane / soluble protein complexes from a plasma sample not obtained by conventional microsampling. In some embodiments, the method recovers a fraction of non-membrane / soluble protein complexes from a whole blood sample not obtained by conventional microsampling. In some embodiments, the method recovers a fraction of membrane protein complexes from a red blood cell (RBC) sample not obtained by conventional microsampling. In some embodiments, the method recovers a fraction of membrane protein complexes from a plasma sample not obtained by conventional microsampling. In some embodiments, the method recovers a fraction of membrane protein complexes from a whole blood sample not obtained by conventional microsampling.

[0107] In some embodiments, the drying, extraction, and digestion steps are performed in the test tube with the sample-containing porous material.

[0108] In some embodiments, the drying step comprises air-drying the sample within the porous material, after which the sample is dried within the porous material. In some embodiments, the drying step comprises centrifuging the sample within the porous material, after which the sample is dried within the porous material. In some embodiments, the drying step comprises drying for a period of time to allow the sample to adhere to the porous material. In some embodiments, the drying step comprises drying for a period of time to allow the sample to adhere to the porous material, after which the sample-containing porous material is stored for a period of time prior to the first extraction step. In some embodiments, the sample-containing porous material is frozen prior to the first extraction step. In some embodiments, the drying step comprises air-drying. In some embodiments, the drying step lasts for less than 30 minutes. In some embodiments, the drying step lasts for less than 20 minutes. In some embodiments, the drying step lasts for less than 10 minutes. In some embodiments, the drying step lasts for 5 minutes. In some embodiments, the drying step lasts for less than 5 minutes. In some embodiments, the air-drying step is for at least 30 minutes, 1, 2, 4, 6, 8, 10, 12, 16, 20, or 24 hours. In some embodiments, the air-drying step is for 24 hours. In some embodiments, the air-drying step is for at least 1 day. In some embodiments, the drying step comprises a vacuum drying step. In some embodiments, the drying step comprises drawing a vacuum on the sample within the porous material. In some embodiments, the drying step is performed immediately after blood collection on the porous material. In some embodiments, drying the porous material immediately after sample collection comprises an air-drying step. In some embodiments, the drying step is performed after extraction and digestion. In some embodiments, drying the porous material immediately after blood collection comprises centrifugation. In some embodiments, the drying step comprises centrifuging the sample within the porous material. In some embodiments, the centrifugation step is at a speed of 500 g to 10,000 g. In some embodiments, the centrifugation step is for at least 1 to 15 minutes.In some embodiments, the centrifugation step lasts for 15 minutes or less. In some embodiments, the sample is frozen immediately after it is collected in the porous material, or optionally, the sample is air-dried for less than 30 minutes before being frozen.

[0109] In some embodiments, the first, second, third, and further extraction solutions comprise an extractant comprising a salt, a weak detergent, a strong detergent, a chaotropic agent, a reducing agent, a thiol-containing reducing agent, an alkylating agent, an acid, an organic solvent, or an enzyme, or a combination thereof. In some embodiments, the first extraction solution comprises an extractant comprising a salt, a weak detergent, a strong detergent, a chaotropic agent, a reducing agent, a thiol-containing reducing agent, an alkylating agent, an acid, an organic solvent, or an enzyme, or a combination thereof. In some embodiments, the second extraction solution comprises an extractant comprising a salt, a weak detergent, a strong detergent, a chaotropic agent, a reducing agent, a thiol-containing reducing agent, an alkylating agent, an acid, an organic solvent, or an enzyme, or a combination thereof. In some embodiments, the third extraction solution comprises an extractant comprising a salt, a weak detergent, a strong detergent, a chaotropic agent, a reducing agent, a thiol-containing reducing agent, an alkylating agent, an acid, an organic solvent, or an enzyme, or a combination thereof. In some embodiments, the additional extraction solution comprises an extractant comprising a salt, a weak detergent, a strong detergent, a chaotropic agent, a reducing agent, a thiol-containing reducing agent, an alkylating agent, an acid, an organic solvent, or an enzyme, or a combination thereof.

[0110] In some embodiments, the first, second, third, and further extraction solutions comprise a salt. In some embodiments, the first, second, third, and further extraction solutions comprise a weak detergent. In some embodiments, the first, second, third, and further extraction solutions comprise a strong detergent. In some embodiments, the first, second, third, and further extraction solutions comprise a chaotropic agent. In some embodiments, the first, second, third, and further extraction solutions comprise a reducing agent. In some embodiments, the first, second, third, and further extraction solutions comprise a thiol-containing reducing agent. In some embodiments, the first, second, third, and further extraction solutions comprise an alkylating agent. In some embodiments, the first, second, third, and further extraction solutions comprise an acid. In some embodiments, the first, second, third, and further extraction solutions comprise an organic solvent. In some embodiments, the first, second, third, and additional extraction solutions contain enzymes. In some embodiments, the salt is or includes NaCl, LiCl, or Tris-HCl. In some embodiments, the weak detergent is a non-ionic detergent such as PBS or Tween. In some embodiments, the weak detergent is an ionic detergent such as sodium deoxycholate. In some embodiments, the weak detergent is a zwitterionic detergent such as sulfobetaine or amidosulfobetaine. In some embodiments, the weak detergent is or includes PBS, Tween, or sodium deoxycholate. In some embodiments, the weak detergent is or includes Tween or sodium deoxycholate. In some embodiments, the strong detergent is or includes cetyltrimethylammonium bromide (CTAB), 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonic acid (CHAPS), or sodium dodecyl sulfate (SDS). In some embodiments, the chaotropic agent is or includes urea, thiourea, or guanidine. In some embodiments, the reducing agent is or comprises a phosphine.In some embodiments, the phosphine is or comprises tributyl phosphate (TBP) or tris(2-carboxyethyl)phosphine (TCEP). In some embodiments, the thiol-containing reducing agent is or comprises β-mercaptoethanol or dithiothreitol. In some embodiments, the alkylating agent is or comprises iodoacetamide or acrylamide. In some embodiments, the acid is or comprises citric acid or trifluoroacetic acid. In some embodiments, the organic solvent is methanol. In some embodiments, the enzyme is or comprises benzonase.

[0111] In some embodiments, the first extraction solution is a salt solution, a weak detergent-containing solution, or a salt and a weak detergent-containing solution. In some embodiments, the first extraction solution is a salt solution, a chaotropic agent-containing solution, or a salt and a chaotropic agent-containing solution. In some embodiments, the first extraction solution comprises NaCl, LiCl, Tris-HCl, PBS, or Tween, or a combination thereof. In some embodiments, the first extraction solution comprises NaCl, LiCl, Tris-HCl, PBS, Tween, sodium deoxycholate, CTAB, CHAPS, SDS, urea, thiourea, or guanidine, or a combination thereof. In some embodiments, the first extraction solution comprises NaCl, LiCl, Tris-HCl, PBS, Tween, sodium deoxycholate, CHAPS, SDS, urea, thiourea, guanidine, TBP, TCEP, β-mercaptoethanol, dithiothreitol, iodoacetamide, or acrylamide, or a combination thereof.

[0112] In some embodiments, when glycans present in a sample are to be evaluated, the first, second, third, or one or more additional extraction solutions comprise an endoglycosidase, in some embodiments, the endoglycosidase is peptide-N-glycosidase F (PNGase F) and / or an O-glycosidase.

[0113] In some embodiments, the first extraction solution is different from the second extraction solution. In some embodiments, the first, second, and third extraction solutions are different. In some embodiments, the first, second, third, and further extraction solutions are different. In some embodiments, the first, second, third, and further extraction solutions are selected to fractionate proteins contained in the porous material through differential solubility. In some embodiments, each successive extraction solution selected is characterized as having a relatively large solubility of proteins remaining in the porous material. In some embodiments, the first, second, third, and further extraction solutions contain increasing concentrations of the same extractant as one extraction proceeds to the next.

[0114] In some embodiments, the selection of the different extraction solution is limited to solutions based on proteins remaining in the separated first extracted porous material that have the same or lower solubility in the different extraction solution than in the first extraction solution. In some embodiments, the selection of the different extraction solution is based on proteins remaining in the separated first extracted porous material that have the same or lower solubility in the different extraction solution than in said first extraction solution.

[0115] In some embodiments, the method includes extracting or washing the dried sample-containing porous material one or more times with a first extraction solution. In some embodiments, the dried sample-containing porous material is extracted or washed one, two, or three times with the first extraction solution. In some embodiments, the dried sample-containing porous material is extracted or washed one time with the first extraction solution. In some embodiments, the dried sample-containing porous material is extracted or washed two times with the first extraction solution. In some embodiments, the dried sample-containing porous material is extracted or washed three times with the first extraction solution.

[0116] In some embodiments, the concentration of salt in the first, second, third, or additional extraction solution is 0.01-1M. In some embodiments, the concentration of salt in the first, second, third, or additional extraction solution is 0.5M. In some embodiments, the concentration of salt in the first, second, third, or additional extraction solution is 0.1M. In some embodiments, the salt is NaCl, LiCl, or Tris-HCl. In some embodiments, the concentration of NaCl in the first, second, third, or additional extraction solution is 0.5M. In some embodiments, the concentration of LiCl in the first, second, third, or additional extraction solution is 0.5M. In some embodiments, the concentration of Tris-HCl in the first, second, third, or additional extraction solution is 0.1M. In some embodiments, the concentration of the chaotropic agent in the first, second, third, or additional extraction solution is 0.01-8 M. In some embodiments, the concentration of the chaotropic agent in the first, second, third, or additional extraction solution is 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8 M. In some embodiments, the concentration of the chaotropic agent in the first, second, third, or additional extraction solution is 0.01-8 M. In some embodiments, the chaotropic agent is urea, thiourea, or guanidine. In some embodiments, the first, second, third, or additional extraction solution comprises urea, thiourea, guanidine, or a combination thereof. In some embodiments, the first, second, third, or additional extraction solution comprises urea at a concentration of 2 M. In some embodiments, the first, second, third, or additional extraction solution comprises urea at a concentration of 5 M and thiourea at a concentration of 1 M. In some embodiments, the first, second, third, or additional extraction solution comprises urea at a concentration of 6 M and thiourea at a concentration of 1.5 M. In some embodiments, the first, second, third, or additional extraction solution comprises urea at a concentration of 7 M and thiourea at a concentration of 2 M.

[0117] In some embodiments, the first extraction solution is a salt solution or a urea-containing solution. In some embodiments, the salt solution is a Tris-HCl solution containing NaCl or LiCl. In some embodiments, the urea-containing solution is a Tris-HCl solution containing urea. In some embodiments, the first extraction solution is a salt solution, a urea-containing solution, or a salt and urea-containing solution. In some embodiments, the first extraction solution comprises Tris-Cl, NaCl, LiCl, or urea, or a combination thereof. In some embodiments, the first extraction solution comprises Tris-HCl. In some embodiments, the first extraction solution comprises NaCl. In some embodiments, the first extraction solution comprises LiCl. In some embodiments, the first extraction solution comprises urea. In some embodiments, Tris-HCl is present in the first extraction solution at a concentration of 0.1 M. In some embodiments, NaCl is present in the first extraction solution at a concentration of 0.5 M. In some embodiments, LiCl is present in the first extraction solution at a concentration of 0.5 M. In some embodiments, urea is present in the first extraction solution at a concentration of 2M. In some embodiments, the first extraction solution and the second extraction solution are different. In some embodiments, the first extraction solution and the second extraction solution are salt solutions or urea-containing solutions. In some embodiments, the first extraction solution and the second extraction solution are different. In some embodiments, the second extraction solution comprises Tris-HCl, NaCl, LiCl, or urea, or a combination thereof. In some embodiments, the first extraction solution is a salt solution, and the second extraction solution is a salt solution different from the first extraction solution or a urea-containing solution. In some embodiments, the first extraction solution comprises Tris-HCl. In some embodiments, the first extraction solution comprises NaCl. In some embodiments, the first extraction solution comprises LiCl. In some embodiments, the first extraction solution comprises urea. In some embodiments, the second extraction solution comprises Tris-HCl. In some embodiments, the second extraction solution comprises NaCl. In some embodiments, the second extraction solution comprises LiCl. In some embodiments, the second extraction solution comprises urea. In some embodiments, Tris-HCl is present in the extraction solution at a concentration of 0.1 M.In some embodiments, NaCl is present in the extraction solution at a concentration of 0.5 M. In some embodiments, LiCl is present in the extraction solution at a concentration of 0.5 M. In some embodiments, urea is present in the extraction solution at a concentration of 2 M.

[0118] In some embodiments, the separated first, second, third, or further extraction solution comprises one or more proteins. In some embodiments, the separated first extraction solution comprises a first set of proteins. In some embodiments, the separated first extraction solution comprises albumin, hemoglobin, IgG, or one or more additional proteins compared to conventionally isolated serum or plasma. In some embodiments, the separated second extraction solution comprises a second set of proteins. In some embodiments, the separated third extraction solution comprises a third set of proteins. In some embodiments, the separated further extraction solution comprises a further set of proteins.

[0119] In some embodiments, the first, second, third, or additional extraction solution has a volume of less than 100 μL. In some embodiments, the first, second, third, or additional extraction solution has a volume of 95 μL or less. In some embodiments, the first, second, third, or additional extraction solution has a volume of 90 μL or less. In some embodiments, the first, second, third, or additional extraction solution has a volume of 1 to 5 times the volume of the porous material. In some embodiments, the first, second, third, or additional extraction solution has a volume of 1, 2, 3, 4, or 5 times the volume of the porous material. In some embodiments, the first, second, third, or additional extraction solution has a volume of 3 times the volume of the porous material.

[0120] In some embodiments, the incubation step in the first, second, third, or additional extraction solution is carried out for a time sufficient to solubilize one or more proteins contained within or attached to the porous material. In some embodiments, the incubation step in the first, second, third, or additional extraction solution is carried out for a time sufficient to extract one or more proteins contained within or attached to the porous material. In some embodiments, the incubation step is carried out for 1 minute to 48 hours. In some embodiments, the incubation step is carried out for 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes. In some embodiments, the incubation step is carried out for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes. In some embodiments, the incubating step is carried out for 1 to 48 hours, 1 to 40 hours, 1 to 30 hours, 1 to 20 hours, 1 to 10 hours, 1 to 5 hours, or 1 to 40 hours. In some embodiments, the incubating step is carried out at ambient temperature. In some embodiments, the incubating step is carried out at an elevated temperature above ambient temperature. In some embodiments, the incubating step is carried out at an elevated temperature below ambient temperature. In some embodiments, the incubating step is carried out with agitation.

[0121] In some embodiments, the separating step comprises removing the first, second, third, or additional incubated porous material from the first, second, third, or additional extraction solution, respectively. In some embodiments, the separating step comprises removing the first, second, third, or additional extraction solution from the first, second, third, or additional incubated porous material, respectively. In some embodiments, the separating step of the first, second, third, or additional extraction solution from the first, second, third, or additional incubated porous material, respectively, is via centrifugation, filtration, or a combination of centrifugation and filtration. In some embodiments, the separating step is via centrifugation. In some embodiments, the separating step is via filtration. In some embodiments, the separating step is via a combination of centrifugation and filtration. In some embodiments, the centrifugation step is at a speed between 500 g and 10,000 g. In some embodiments, the centrifugation step is for at least 1 minute. In some embodiments, the centrifugation step is for 1 to 15 minutes. In some embodiments, the centrifugation step lasts for 15 minutes or less.

[0122] In some embodiments, the digestion solution comprises a reducing agent, an alkylating agent, a buffer, a detergent, or a combination thereof. In some embodiments, the reducing agent is or comprises a phosphine. In some embodiments, the phosphine is or comprises TBP or TCEP. In some embodiments, the alkylating agent is or comprises iodoacetamide or acrylamide. In some embodiments, the digestion solution further comprises a salt, a weak detergent, a strong detergent, a chaotropic agent, or a thiol-containing reducing agent, or a combination thereof. In some embodiments, the salt is or comprises NaCl, LiCl, or Tris-HCl. In some embodiments, the weak detergent is a non-ionic detergent. In some embodiments, the weak detergent is or comprises PBS or Tween. In some embodiments, the weak detergent is an ionic detergent such as sodium deoxycholate. In some embodiments, the weak detergent is a zwitterionic detergent such as sulfobetaine or amidosulfobetaine. In some embodiments, the weak detergent is or comprises PBS, Tween, or sodium deoxycholate. In some embodiments, the weak detergent is or comprises Tween or sodium deoxycholate. In some embodiments, the strong detergent is or comprises cetyltrimethylammonium bromide (CTAB), 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonic acid (CHAPS), or sodium dodecyl sulfate (SDS). In some embodiments, the chaotropic agent is or comprises urea, thiourea, or guanidine. In some embodiments, the thiol-containing reducing agent is or comprises β-mercaptoethanol or dithiothreitol. In some embodiments, the digestion solution further comprises a trypsin digestion solution. In some embodiments, the trypsin digestion solution comprises triethylammonium bicarbonate, SDC, TCEP, chloroacetamide, or a combination thereof.In some embodiments, the digestion solution comprises triethylammonium bicarbonate, sodium deoxycholate (SDC), TCEP, or chloroacetamide, or a combination thereof. In some embodiments, the digestion solution comprises triethylammonium bicarbonate. In some embodiments, triethylammonium bicarbonate is present in the digestion solution at a concentration of 0.1 M. In some embodiments, the digestion solution comprises sodium deoxycholate (SDC). In some embodiments, sodium deoxycholate (SDC) is present in the digestion solution at a concentration of 0.05% to 10% (w / v). In some embodiments, the digestion solution comprises TCEP. In some embodiments, TCEP is present in the digestion solution at a concentration of 1 mM to 100 mM. In some embodiments, the digestion solution comprises chloroacetamide. In some embodiments, chloroacetamide is present in the digestion solution at a concentration of 5 mM to 100 mM. In some embodiments, the digestion solution comprises a protease or a combination of proteases. In some embodiments, the protease is trypsin, or the combination of proteases includes trypsin.

[0123] In some embodiments, the digestion solution comprises triethylammonium bicarbonate, sodium deoxycholate (SDC), TCEP, or chloroacetamide, or a combination thereof. In some embodiments, the digestion solution comprises PBS. In some embodiments, the digestion solution comprises triethylammonium bicarbonate. In some embodiments, the digestion solution comprises sodium deoxycholate (SDC). In some embodiments, the digestion solution comprises TCEP. In some embodiments, the digestion solution comprises chloroacetamide. In some embodiments, the digestion solution comprises a protease or a combination of proteases. In some embodiments, the protease is trypsin, or the combination of proteases includes trypsin. In some embodiments, PBS is present in the digestion solution at a concentration of 0.01 M. In some embodiments, PBS can further comprise potassium chloride and sodium chloride. In some embodiments, the potassium chloride is at a concentration of 0.0027 M. In some embodiments, the sodium chloride is at a concentration of 0.137 M. In some embodiments, any PBS solution known in the art can be used. In some embodiments, triethylammonium bicarbonate is present in the digestion solution at a concentration of 0.1 M. In some embodiments, sodium deoxycholate (SDC) is present in the digestion solution at a concentration of 1% (w / v). In some embodiments, TCEP is present in the digestion solution at a concentration of 10 mM. In some embodiments, chloroacetamide is present in the digestion solution at a concentration of 40 mM.

[0124] In some embodiments, the digestion solution comprises a protease or a combination of proteases. In some embodiments, the digestion solution comprises a protease or a combination of proteases and further comprises triethylammonium bicarbonate, sodium deoxycholate (SDC), TCEP, or chloroacetamide, or a combination thereof. In some embodiments, the digestion solution comprises trypsin.

[0125] In some embodiments, the step of incubating in the digestion solution is carried out for a time sufficient to digest one or more proteins contained within or adhered to the porous material. In some embodiments, the incubation step is carried out for 1 minute to 48 hours. In some embodiments, the incubation step is carried out for 1 minute to 24 hours. In some embodiments, the incubation step is carried out for 24 hours to 48 hours. In some embodiments, the incubation step is carried out for 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes. In some embodiments, the incubation step is carried out for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. In some embodiments, the incubation step is carried out at ambient temperature. In some embodiments, the incubation step is carried out at an elevated temperature above ambient temperature. In some embodiments, the incubating step is carried out at an elevated temperature below ambient temperature. In some embodiments, the incubating step is carried out with agitation.

[0126] In some embodiments, the separating step comprises removing the digestion-incubated porous material from the digestion solution. In some embodiments, the separating step comprises removing the digestion solution from the digestion-incubated porous material. In some embodiments, the separating step of the digestion solution from the digestion-incubated porous material is via centrifugation, filtration, or a combination of centrifugation and filtration. In some embodiments, the separating step is via centrifugation. In some embodiments, the separating step is via filtration. In some embodiments, the separating step is via a combination of centrifugation and filtration. In some embodiments, the centrifugation step is at a speed of 500 g to 10,000 g. In some embodiments, the centrifugation step is for at least 1 minute. In some embodiments, the centrifugation step is for 1 to 15 minutes. In some embodiments, the centrifugation step is for 15 minutes or less.

[0127] In some embodiments, the method includes washing the separated first, second, third, or additional extracted porous material. In some embodiments, the method includes washing the separated first extracted porous material. In some embodiments, the method includes washing the separated second extracted porous material. In some embodiments, the method includes washing the separated third extracted porous material. In some embodiments, the method includes washing the separated additional extracted porous material. In some embodiments, the separated first, second, third, or additional extracted porous material is washed with a wash volume of the first, second, third, or additional extraction solution, respectively. In some embodiments, the separated first extracted porous material is washed with a wash volume of the first extraction solution. In some embodiments, the separated second extracted porous material is washed with a wash volume of the first, second, or a combination comprising the first and second extraction solutions. In some embodiments, the separated third extracted porous material is washed with a wash volume of the first, second, third, or a combination comprising the first, second, or third extraction solution. In some embodiments, the separated further extracted porous material is washed with a wash volume of the first, second, third, or a combination comprising the first, second, third, or further extraction solution. In some embodiments, the separated first extracted porous material is washed with an extraction solution different from the first extraction solution.

[0128] In some embodiments, the wash volume of the first, second, third, or additional extraction solution is 50 μL or more. In some embodiments, the wash volume of the first, second, third, or additional extraction solution is 100 μL or more. In some embodiments, the wash volume of the first, second, third, or additional extraction solution is between 5 times the volume of the porous material and 3 mL. In some embodiments, the wash volume of the first, second, third, or additional extraction solution is 0.05 to 3 mL. In some embodiments, the wash volume of the first, second, third, or additional extraction solution is 0.1 to 3 mL. In some embodiments, the wash volume of the first, second, third, or additional extraction solution is 0.5 to 3 mL. In some embodiments, the wash volume of the first, second, third, or additional extraction solution is 0.5, 1, 1.5, 2, 2.5, or 3 mL. In some embodiments, the washing step of the separated first, second, third, or further extracted porous material is repeated one, two, or three times.

[0129] In some embodiments, the porous material is washed one or more times with Tris-HCl. In some embodiments, the porous material is washed once with Tris-HCl. In some embodiments, the porous material is washed twice with Tris-HCl. In some embodiments, the porous material is washed three times with Tris-HCl. In some embodiments, the Tris-HCl is at a concentration of 0.1 M.

[0130] In some embodiments, the porous material is washed one or more times with Tris-HCl and NaCl. In some embodiments, the porous material is washed once with Tris-HCl and NaCl. In some embodiments, the porous material is washed twice with Tris-HCl and NaCl. In some embodiments, the porous material is washed three times with Tris-HCl and NaCl. In some embodiments, the Tris-HCl is at a concentration of 0.1 M. In some embodiments, the NaCl is at a concentration of 0.5 M.

[0131] In some embodiments, the porous material is washed one or more times with Tris-HCl and LiCl. In some embodiments, the porous material is washed once with Tris-HCl and LiCl. In some embodiments, the porous material is washed twice with Tris-HCl and LiCl. In some embodiments, the porous material is washed three times with Tris-HCl and LiCl. In some embodiments, the Tris-HCl is at a concentration of 0.1 M. In some embodiments, the LiCl is at a concentration of 0.5 M.

[0132] In some embodiments, the porous material is washed one or more times with Tris-HCl and urea. In some embodiments, the porous material is washed once with Tris-HCl and urea. In some embodiments, the porous material is washed twice with Tris-HCl and urea. In some embodiments, the porous material is washed three times with Tris-HCl and urea. In some embodiments, the Tris-HCl is at a concentration of 0.1 M. In some embodiments, the urea is at a concentration of 2 M.

[0133] In some embodiments, the second extraction solution is a salt solution or a urea-containing solution. In some embodiments, the first extraction solution and the second extraction solution are different. In some embodiments, the second extraction solution comprises Tris-HCl, NaCl, LiCl, or urea, or a combination thereof. In some embodiments, the first extraction solution is a salt solution, and the second extraction solution is a salt solution different from the first extraction solution or a urea-containing solution. In some embodiments, the third extraction solution is a salt solution or a urea-containing solution. In some embodiments, the third extraction solution comprises Tris-HCl, NaCl, LiCl, or urea, or a combination thereof. In some embodiments, the third extraction solution comprises Tris-HCl. In some embodiments, the third extraction solution comprises NaCl. In some embodiments, the third extraction solution comprises LiCl. In some embodiments, the third extraction solution comprises urea. In some embodiments, Tris-HCl is present in the third extraction solution at a concentration of 0.1 M. In some embodiments, NaCl is present in the third extraction solution at a concentration of 0.5 M. In some embodiments, LiCl is present in the third extraction solution at a concentration of 0.5 M. In some embodiments, urea is present in the third extraction solution at a concentration of 2 M.

[0134] In some embodiments, the first extraction solution, the second extraction solution, and the third extraction solution are different. In some embodiments, the first extraction solution is a salt solution, the second extraction solution is a different salt solution from the first extraction solution, and the third extraction solution is a different salt solution from the first extraction solution and the second extraction solution or a urea-containing solution. In some embodiments, the second extraction solution comprises Tris-HCl. In some embodiments, the second extraction solution comprises NaCl. In some embodiments, the second extraction solution comprises LiCl. In some embodiments, the second extraction solution comprises urea. In some embodiments, Tris-HCl is present in the second extraction solution at a concentration of 0.1 M. In some embodiments, NaCl is present in the second extraction solution at a concentration of 0.5 M. In some embodiments, LiCl is present in the second extraction solution at a concentration of 0.5 M. In some embodiments, urea is present in the second extraction solution at a concentration of 2 M.

[0135] In some embodiments, the separated sample mixture is extracted with at least a second extraction solution.

[0136] In some embodiments, the first extraction solution or the second extraction solution comprises Tris-HCl. In some embodiments, the Tris-HCl is at a concentration of 0.1 M. In some embodiments, the first extraction solution or the second extraction solution comprises Tris-HCl and NaCl. In some embodiments, the Tris-HCl is at a concentration of 0.1 M. In some embodiments, the NaCl is at a concentration of 0.5 M. In some embodiments, the first extraction solution or the second extraction solution comprises Tris-HCl and LiCl. In some embodiments, the Tris-HCl is at a concentration of 0.1 M. In some embodiments, the LiCl is at a concentration of 0.5 M. In some embodiments, the first extraction solution or the second extraction solution comprises Tris-HCl and urea. In some embodiments, the Tris-HCl is at a concentration of 0.1 M. In some embodiments, the urea is at a concentration of 2 M.

[0137] In some embodiments, the detecting step comprises detecting proteins in the separated first extraction solution. In some embodiments, the detecting step comprises detecting proteins in the separated second extraction solution. In some embodiments, the detecting step comprises detecting proteins in the separated third extraction solution. In some embodiments, the detecting step comprises detecting proteins in a further extraction solution. In some embodiments, the detecting step of the first, second, third, or further set of proteins from the first, second, third, or further extraction solution, respectively, is via immunoassay. In some embodiments, the detecting step of the first, second, third, or further set of proteins from the first, second, third, or further extraction solution, respectively, is via mass spectrometry (MS).

[0138] In some embodiments, detecting via MS comprises subjecting the first, second, third, or further extraction solution to digestion prior to said detecting via mass spectrometry (MS). In some embodiments, prior to detecting the first, second, third, or further set of proteins in the first, second, third, or further extraction solution, respectively, the method further comprises incubating the first, second, third, or further extraction solution in a digestion solution.

[0139] In some embodiments, any one of the methods described herein further comprises generating a protein profile of a first set of proteins. In some embodiments, the first protein profile is generated from the collected first set of proteins. In some embodiments, the first protein profile is generated by a proteomics workflow. In some embodiments, the first protein profile is generated by an immunoassay workflow.

[0140] In some embodiments, the detecting step comprises an immunoassay workflow. In some embodiments, the immunoassay is or comprises an ELISA. In some embodiments, the immunoassay is or comprises a Western blot. In some embodiments, the immunoassay comprises a Luminex and proximity extension assay. In some embodiments, the Luminex and proximity extension assay further comprise a Slow Offrate Modified Aptamer (SOMAmer) reagent.

[0141] In some embodiments, the detecting step comprises a proteomics workflow. In some embodiments, the proteomics workflow comprises mass spectrometry (MS). In some embodiments, the MS is LC-MS. In some embodiments, the MS is selected reaction monitoring mass spectrometry (SRM-MS). In some embodiments, the MS is data-dependent acquisition MS (DDA-MS). In some embodiments, the MS is data-independent acquisition MS (DIA-MS). In some embodiments, the MS is matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) MS; MALDI-TOF post-source decay (PSD); MALDI-TOF / TOF; surface-enhanced laser desorption / ionization time-of-flight mass spectrometry (SELDI-TOF) MS; electrospray ionization mass spectrometry (ESI-MS); ESI-MS / MS; ESI-MS / (MS)n (n is an integer greater than zero); ESI Selected from the group consisting of 3D or linear (2D) ion trap MS; ESI triple quadrupole MS; ESI quadrupole orthogonal time-of-flight (Q-TOF); ESI Fourier transform MS system; desorption / ionization on silicon (DIOS); secondary ion mass spectrometry (SIMS); atmospheric pressure chemical ionization mass spectrometry (APCI-MS); APCI-MS; APCI-(MS)n; ion mobility spectrometry (IMS); inductively coupled plasma mass spectrometry (ICP-MS), atmospheric pressure photoionization mass spectrometry (APPI-MS), APPI-MS / MS; and APPI-(MS)n.

[0142] In some embodiments, any one of the methods described herein further comprises generating a protein profile of the cell membrane-associated protein. In some embodiments, the cell membrane-associated protein profile is generated from a collected set of cell membrane-associated proteins. In some embodiments, the cell membrane-associated protein profile is generated by LC-MS. In some embodiments, the cell membrane-associated protein profile is generated by immunoassay. In some embodiments, the cell membrane-associated protein is or comprises a cell membrane-bound protein or a cell membrane-integral protein. In some embodiments, the cell membrane-associated protein comprises one or more membrane protein complexes.

[0143] In some embodiments, the porous material is a three-dimensional porous material. In some embodiments, the three-dimensional porous material comprises a plastic. In some embodiments, the three-dimensional porous material comprises a sponge. In some embodiments, the three-dimensional porous material is the tip of a volumetric absorption microsampling (VAMS) device. In some embodiments, the porous material is a non-preloaded porous material. In some embodiments, the porous material is not preloaded with a protease inhibitor. In some embodiments, the porous material is a preloaded porous material. In some embodiments, the porous material is preloaded with a protease inhibitor. In some embodiments, the porous material is preloaded with an anticoagulant. In some embodiments, the anticoagulant is EDTA. In some embodiments, the anticoagulant is heparin.

[0144] In some embodiments, the method further comprises placing the sample-containing porous material in a test tube. In some embodiments, the method further comprises placing the dried sample-containing porous material in a test tube. In some embodiments, the test tube is an Eppendorf tube.

[0145] In some embodiments, the method uses a plurality of porous materials. In some embodiments, the sample is introduced into the plurality of porous materials. In some embodiments, the plurality of porous materials comprises two or more porous materials. In some embodiments, the plurality of porous materials comprises two porous materials. In some embodiments, the plurality of porous materials is three porous materials.

[0146] In some embodiments, one or more proteins detected in the first, second, third, or additional set of proteins are or comprise non-membrane / soluble protein complexes. In some embodiments, one or more proteins detected in the separated digestion solution are or comprise non-membrane / soluble protein complexes. In some embodiments, one or more proteins detected in the first, second, third, or additional extraction solution after incubating in the digestion solution are or comprise non-membrane / soluble protein complexes. In some embodiments, one or more proteins detected in the first, second, third, or additional set of proteins are or comprise cell membrane-associated proteins. In some embodiments, one or more proteins detected in the separated digestion solution are or comprise cell membrane-associated proteins. In some embodiments, one or more proteins detected in the first, second, third, or additional extraction solution after incubating in the digestion solution are or comprise cell membrane-associated proteins. In some embodiments, the plasma membrane-associated protein is a plasma membrane-bound protein or an integral plasma membrane protein. In some embodiments, the plasma membrane-associated protein is a membrane protein complex.

[0147] In some embodiments, when glycans present in a sample are of interest, the method includes a multiglycomics workflow, in which different glycans can be released sequentially prior to the digestion step. Glycosaminoglycans (GAGs), glycosphingolipids (GSLs), and N-glycans can all be released sequentially through enzymatic means while preserving the peptide backbone. In some embodiments, the method further includes, prior to the digestion step, dividing the porous material and analyzing a portion of the porous material for remaining glycans. In one embodiment, the remaining glycans are those remaining after enzymatic treatment to release the glycans. In one embodiment, the remaining glycans are analyzed after release of O-glycans via reductive β-elimination.

[0148] device Dried blood spots (DBS) can be used as a sample collection system. However, while DBS provides a simple and reliable means for collecting blood samples without the need for medical intervention, it does have some drawbacks. For example, one drawback is the lack of separation in DBS, which means that the entire blood sample (plasma and cells) is collected in a single spot. This can be a concern when an analyte is present in multiple compartments, such as plasma and cells, and DBS extracts give different results than plasma separated from venous blood.

[0149] Provided herein is a device for obtaining a defined volume fraction from a small blood volume.

[0150] In one aspect, provided herein is a microsampling device comprising: (a) a first porous material; (b) a second porous material; and (c) a test tube.

[0151] In one aspect, provided herein is a microsampling device comprising: (a) a first porous material; (b) a second porous material; (c) a third porous material; and (d) a test tube.

[0152] In one aspect, provided herein is a microsampling device comprising: (a) a first porous material; (b) a second porous material; and (c) a shaft; the first porous material is located at one end of the shaft and the second porous material is disposed at a position along the shaft, such that the first porous material and the second porous material are separated and do not physically contact each other.

[0153] In one aspect, provided herein is a microsampling device comprising: (a) a first porous material; (b) a second porous material; (c) a third porous material; and (d) a shaft; the first porous material is located at one end of the shaft, and the second porous material and the third porous material are disposed at positions on the shaft, such that the first porous material, the second porous material, and the third porous material are separated and do not physically contact each other.

[0154] In some embodiments, the microsampling device is a volumetric absorption microsampling (VAMS) device.

[0155] In some embodiments, the test tube is an Eppendorf tube.

[0156] In some embodiments, a test tube containing two absorbent materials (sometimes referred to as absorbent "plugs" of sponge-like absorbent material) can capture a defined volume of fluid. In some embodiments, the absorbent plugs can also contain an anticoagulant substance (wet or dry) to prevent blood clotting during separation. In some embodiments, the device comprises the device of FIG.

[0157] In some embodiments, blood is introduced into the top of the test tube. In some embodiments, blood is collected from a finger prick. In some embodiments, the test tube comprises any one of the test tubes of Figures 30-33. In some embodiments, after centrifugation, cells (RBCs, WBCs, and platelets) are removed from the plasma, with the plasma remaining in absorbent plug 2 and the RBCs and other cells remaining in absorbent plug 1.

[0158] In some embodiments, the devices provided herein allow small volumes of blood, including those from finger prick samples, to be readily processed to obtain plasma and cellular fractions. The absorbent material used in the devices disclosed herein can be a sponge-like material. In some embodiments, the absorbent material can be prepared from or is a Neoteryx tip.

[0159] In one embodiment, the two absorbable plugs are connected by a shaft that also functions as a handle for removing the absorbable plugs (see Figure 31).

[0160] In some embodiments, the test tube can include a lid to prevent leakage and suppress the release of aerosols during centrifugation (see FIG. 32). In some embodiments, the test tube can have a relatively wide opening at the top to facilitate blood collection by wiping or scraping a drop of blood from a fingertip (see FIG. 33). In some embodiments, the test tube can also be elongated with variable spacing between absorbent plugs to ensure that the plasma sample is free of cells or platelets. In some embodiments, the test tube can also be elongated with flexible walls between the absorbent plugs that allow blood drops to be aspirated by squeezing and releasing the test tube (see FIG. 34).

[0161] In some embodiments, the device can use a wire to create a shaft (handle), which is shaped to keep the absorbable plugs apart (see Figure 35).

[0162] The device can use a 2 mm threaded bolt to create the shaft (handle). The threads on the shaft prevented the upper (plasma) absorbent plug from falling out during centrifugation (see Figure 36).

[0163] In some embodiments, the porous material is a preloaded porous material. In some embodiments, the porous material is preloaded with a protease inhibitor. In some embodiments, the porous material is preloaded with an anticoagulant. In some embodiments, the porous material is preloaded with an enzyme. In some embodiments, the porous material is preloaded with a surfactant. In some embodiments, the anticoagulant is EDTA. In some embodiments, the anticoagulant is heparin. In some embodiments, the enzyme is benzonase. In some embodiments, the surfactant is sodium dodecyl sulfate. In some embodiments, the porous material is a non-preloaded porous material. In some embodiments, the porous material is not preloaded with a protease inhibitor. In some embodiments, the first porous material and the second porous material are preloaded with an anticoagulant.

[0164] In some embodiments, the first porous material and the second porous material are equally spaced. In some embodiments, the first porous material, the second porous material, and the third porous material are equally spaced.

[0165] In some embodiments, the first porous material and the second porous material are air-dried. In some embodiments, the first porous material and the second porous material are air-dried for at least 1, 2, 4, 6, 8, 10, 12, 16, 20, or 24 hours. In some embodiments, the first porous material and the second porous material are air-dried for 24 hours. In some embodiments, the first porous material and the second porous material are air-dried for at least 1 day. In some embodiments, the first porous material and the second porous material are air-dried for a time period sufficient to allow the sample to adhere to the porous material. In some embodiments, the first porous material and the second porous material are vacuum-dried.

[0166] In some embodiments, the first porous material has an absorption capacity in the range of 2.5 μL to 50 μL. In some embodiments, the first porous material has an absorption capacity of 2.5 μL, 5 μL, 10 μL, 20 μL, 30 μL, 40 μL, or 50 μL. In some embodiments, the second porous material has an absorption capacity in the range of 2.5 μL to 50 μL. In some embodiments, the second porous material has an absorption capacity in the range of 2.5 μL, 5 μL, 10 μL, 20 μL, 30 μL, 40 μL, or 50 μL. In some embodiments, the third porous material has an absorption capacity in the range of 2.5 μL to 50 μL. In some embodiments, the third porous material has an absorption capacity in the range of 2.5 μL, 5 μL, 10 μL, 20 μL, 30 μL, 40 μL, or 50 μL.

[0167] In some embodiments, the first porous material has an absorbent capacity of 5 μL. In some embodiments, the first porous material has an absorbent capacity of 10 μL. In some embodiments, the first porous material has an absorbent capacity of 20 μL. In some embodiments, the first porous material has an absorbent capacity of 30 μL. In some embodiments, the second porous material has an absorbent capacity of 5 μL. In some embodiments, the second porous material has an absorbent capacity of 10 μL. In some embodiments, the second porous material has an absorbent capacity of 20 μL. In some embodiments, the second porous material has an absorbent capacity of 30 μL.

[0168] In some embodiments, the first porous material and the second porous material have the same absorbent capacity. In some embodiments, the first porous material, the second porous material, and the third porous material have the same absorbent capacity. In some embodiments, the first porous material and the second porous material have different absorbent capacities. In some embodiments, the first porous material and the second porous material have different absorbent capacities. In some embodiments, the first porous material and the second porous material have the same absorbent capacity, and the third porous material has a different absorbent capacity compared to the first and second porous materials. In some embodiments, the first porous material has an absorbent capacity of 5 μL and the second porous material has an absorbent capacity of 5 μL. In some embodiments, the first porous material has an absorbent capacity of 10 μL and the second porous material has an absorbent capacity of 10 μL. In some embodiments, the first porous material has an absorbent capacity of 20 μL and the second porous material has an absorbent capacity of 20 μL. In some embodiments, the first porous material has an absorbent capacity of 30 μL and the second porous material has an absorbent capacity of 30 μL. In some embodiments, the first porous material has an absorbent capacity of 30 μL and the second porous material has an absorbent capacity of 20 μL. In some embodiments, the first porous material has an absorbent capacity of 30 μL and the second porous material has an absorbent capacity of 10 μL. In some embodiments, the first porous material has an absorbent capacity of 30 μL and the second porous material has an absorbent capacity of 5 μL. In some embodiments, the first porous material has an absorbent capacity of 5 μL and the second porous material has an absorbent capacity of 30 μL. In some embodiments, the first porous material has an absorbent capacity of 10 μL and the second porous material has an absorbent capacity of 30 μL. In some embodiments, the first porous material has an absorbent capacity of 20 μL and the second porous material has an absorbent capacity of 30 μL.

[0169] In some embodiments, the first porous material, the second porous material, and the third porous material have the same absorbent capacity. In some embodiments, the first porous material, the second porous material, and the third porous material have different absorbent capacities. In some embodiments, the first porous material and the second porous material have the same absorbent capacity, and the third porous material has a different absorbent capacity compared to the first and second porous materials.

[0170] In some embodiments, the microsampling device further comprises a shaft. In some embodiments, the shaft is a threaded shaft. In some embodiments, the shaft is a non-linear shaft. In some embodiments, the shaft is a curved shaft. In some embodiments, the shaft has a length of 10 mm to 50 mm. In some embodiments, the shaft has a diameter of 0.5 mm to 3 mm. In some embodiments, the shaft has a diameter of 0.5 mm to 5 mm. In some embodiments, the shaft can be a hollow tube to allow airglow and prevent airlock after the sample is added. In some embodiments, the shaft fits into a test tube. In some embodiments, the shaft fits into a test tube having a volume of 5 mL, 2 mL, 1.5 mL, or 1 mL. In some embodiments, the shaft fits into an Eppendorf tube. In some embodiments, the shaft fits into an Eppendorf tube having a volume of 5 mL, 2 mL, 1.5 mL, or 1 mL. In some embodiments, the shaft fits into an Eppendorf tube having a volume of 5 mL. In some embodiments, the shaft fits into an Eppendorf tube having a volume of 2 mL. In some embodiments, the shaft fits into an Eppendorf tube having a volume of 1.5 mL. In some embodiments, the shaft fits into an Eppendorf tube having a volume of 1 mL. In some embodiments, the shaft fits into a 96-well plate.

[0171] In some embodiments, the first porous material and the second porous material are located on the shaft and separated from one another by a spacer, whereby the first porous material and the second porous material are separated and do not physically contact one another. In some embodiments, the first porous material, the second porous material, and the third porous material are located on the shaft and separated from one another by a spacer, whereby the first porous material, the second porous material, and the third porous material are separated and do not physically contact one another.

[0172] In some embodiments, any one of the methods provided herein can be performed using any one of the devices provided herein. In some embodiments, the methods and devices include the method and device of Figure 37. In some embodiments, the methods and devices include the method and device of Figure 38. In some embodiments, the methods and devices include the method and device of Figure 39. In some embodiments, the methods and devices include the method and device of Figure 40. In some embodiments, the methods and devices include the method and device of Figure 41. In some embodiments, the methods and devices include the method and device of Figure 174.

[0173] In some embodiments, provided herein is a kit comprising a microsampling device provided herein. In some embodiments, the kit further provides instructions for using the microsampling device. [Example]

[0174] The following example evaluates the use of a volumetric absorption microsampling (VAMS) device as a substrate for whole blood samples to enable untargeted proteomic analysis of dried whole blood. In particular, the purpose of the following example was to evaluate the protein specificity of VAMS compared to traditional DBS methods.

[0175] The use of VAMS resulted in the detection and robust quantification of up to 1,600 proteins from a single shotgun LC-MS analysis of dried whole blood, greatly enhancing proteomic depth compared to conventional single-shot LC-MS analysis of undepleted plasma. Several proteins not previously reported in blood were detected using this approach. Various wash reagents were used to demonstrate that proteins can be preferentially removed from the VAMS device prior to downstream analysis. Proof-of-concept experiments also demonstrate that storage of frozen blood cell pellets stored under long-term storage (exceeding 5 years) is also compatible with VAMS, enabling the quantification of potential biomarker proteins from biobank repertoires for use in biomarker research. These demonstrations are an important step in establishing a viable analytical workflow to support large-scale precision medicine research.

[0176] VAMS has been demonstrated to be an ideal substrate for the depletion of the most abundant blood proteins through washing, enabling the detection of over 1,600 proteins from a single run of dried whole blood, including plasma proteins typically detected in LC-MS analysis of undepleted plasma.

[0177] Example I: Proteomic analysis of whole blood and plasma using a volumetric absorption microsampling (VAM) device Blood Collection and DBS Matrix Application. Blood was collected from healthy volunteers with written consent under a study approved by the Northern Sydney Local Health District Ethics Committee (2020 / ETH01974). DBS samples were prepared using a 30 μL Mitra® VAMS device (Neoteryx, Torrance, CA) or Whatman FTA-DMPK C filter paper where specified.

[0178] Venous blood was collected from participants by venipuncture into an EDTA vacutainer (BD, North Ryde, Australia). Whole blood was added directly to the VAMS tip until completely filled. In some cases, frozen whole blood was compared with fresh whole blood. For this, aliquots of EDTA blood were immediately frozen at -80°C for 24 hours. The blood was thawed and added to the VAMS tip or filter paper. For plasma sample preparation, whole blood was centrifuged (10 minutes, 1000g, 50% brake), and the resulting plasma was collected and added to the VAMS tip. Samples were dried at room temperature for 5 minutes and then transferred to foil Ziploc bags containing desiccant. These samples were stored at 4°C for a minimum of 24 hours before extraction. Whole blood was added to Whatman FTA-DMPK C filter paper using a pipette, dried at room temperature for 30 minutes, and stored at 4°C for a minimum of 24 hours before extraction.

[0179] Frozen whole blood cell pellets from cancer patients. Frozen whole blood cell pellets (plasma removed) from five age- and sex-matched healthy donors and five stage III cancer donors were purchased from PrecisionMed (Solana Beach, CA, USA). Samples were collected between 2014 and 2018. The supplier indicated that samples from healthy participants were stored at -20°C for 35.6 to 263.0 weeks, and samples from cancer patients were stored for 43.0 to 165.4 weeks. Upon receipt at our laboratory, samples were stored at -80°C for 101 weeks prior to the analysis reported herein. Samples were thawed on ice and added to the microsampling device until completely filled. Samples were then allowed to dry at room temperature for 5 minutes before being transferred to a foil ziplock bag containing desiccant. All samples were stored at 4°C for a minimum of 24 hours before extraction.

[0180] DBS Washing and Extraction. VAMS DBS chips were removed from their spindles, suspended in 1 mL of extraction solution in 1.5 mL Eppendorf tubes, and incubated at room temperature with gentle shaking (100 rpm) for 24 h. The extraction solution was 0.1 M Tris-HCl containing either 0.5 M NaCl, 0.5 M LiCl, or 2 M urea, as indicated. After incubation, VAMS chips were washed three times in fresh extraction solution. This was performed by pulse centrifugation of the chips in the extraction solution (3× pulse, 0–10,000 g), followed by centrifuging the extraction solution out of the chips using a custom column insert device (Micro BioSpin P-30 gel column with column material and base filter removed, BioRad, USA) and resuspending them in fresh extraction solution (3000 g, 3 min, full brake). After the final wash, the dried chips, from which the extraction solution had been removed, were placed in a new Eppendorf tube for trypsin digestion. An overview of the method is shown in Figure 1.

[0181] The paper DBS discs were washed in 1 mL of PBS or 0.5 M LiCl for 24 hours. The discs were then placed in the custom column insert device described above inside a waste collection microcentrifuge tube and centrifuged at 3000 g for 3 minutes. The wash solution was removed, and the filter paper discs were placed in a new microcentrifuge tube for trypsin digestion.

[0182] Trypsin digestion. Washed DBS chips / papers were incubated with 90 μL digestion solution (0.1 M triethylammonium bicarbonate, 1% (w / v) sodium deoxycholate (SDC), 10 mM TCEP, 40 mM chloroacetamide). The samples were then heated at 95°C for 10 minutes. 1 μg trypsin was added and incubated at 37°C for 16 hours. After incubation, the chips were removed from the solution, and the remaining liquid was centrifuged out of the chip as described above and combined. Formic acid was added to quench the reaction and precipitate SDC. Peptides were desalted using a Stage-tip as previously described (Rappsilber et al., Anal Chem. 2003;75(3):663-70).

[0183] Mass Spectrometry Acquisition. Peptides were loaded onto a 75 μm × 40 cm 1.9 μm C18 (ReproSil-Pur C18-AQ) nanoLC column in 0.1% formic acid and eluted over a 90-minute gradient up to 30% solvent B (80% ACN, 0.1% FA).

[0184] Peptides were detected using an HF-X orbitrap mass spectrometer (Thermo, CA, USA) with the following instrument settings when operated in DDA mode: MS1 - AGC 3e6, resolution 60K, scan range 300–1650 m / z. MS2 - AGC 1e5, resolution 15K, loop count 15.

[0185] When run in DIA mode, the following instrument parameters were used: a full MS scan with a resolution of 60K, an AGC of 3e6, a maximum injection time of 20 ms, and a scan range of 350-1300 m / z, followed by two DIA events with a total of 117 x 6 m / z mass windows to scan from 400-1218 m / z using settings of 15K resolution, an AGC of 3e6, and a maximum injection time of 32 ms. The normalized collision energy was set to 30, and the default charge state was 2.

[0186] Protein Identification. For DDA experiments, raw files were searched by MaxQuant V1.6.5 against the UniProt human proteome database (UP000005640, 20,350 screened protein entries) using the following settings: 1% FDR PSM and reverse target-decoy strategy to generate proteins, a minimum of 2 peptides, an MSMS mass tolerance of 20 ppm, deletion truncations set to a maximum of 2, variable oxidation of methionine and fixed carbamidomethyl modification of Cys, and an effective LFQ requiring 2 peptides using default settings.

[0187] For DIA experiments, analysis was performed using Spectronaut Pulsar X v12.0.2 (Biognosys, Switzerland) with a custom library generated from a DDA experiment of the same specimen. A mutant decoy search method was applied with 1% precursor and protein FDR.

[0188] Mass spectrometry proteomic data have been deposited in the ProteomeXchange consortium via the PRIDE partner repository with the dataset identifier PXD028605.

[0189] Quantification and statistical analysis. For DDA experiments, Perseus software was used to filter out reverse hits and peptide matches to single peptides (two peptides was the minimum required for protein quantification). Data were log2 transformed and median normalization was applied. Missing measurement values ​​were replaced by imputation with Perseus using default settings: values ​​sampled from a Gaussian distribution downshifted by 1.8 standard deviations with a width of 0.3. For DIA experiments, protein quantification was performed for peptides quantified by a minimum of three fragments, with the top n = 5 peptides. Data were then exported as a "Q-value Sparse" pivoted report matrix containing normalized protein areas, with missing values ​​represented as "NaN." Upset plots and principal component analysis plots were generated using R. Statistical analysis of sample group protein specificity differences was performed by ANOVA with Tukey post-hoc analysis using Q < 0.05. Gene ontology enrichment was performed using the R package PloGO2 (Wu et al., Journal of Proteome Research. 2020;19(7):2898-906) using a default set of 42 GO categories. To determine GO category enrichment, the number of proteins in each category was compared to a baseline set of all proteins identified in the experiment using Fisher's exact test; counts, percentages, and fdr-corrected Fisher's exact test p-values ​​were generated and summarized. Differentially expressed proteins were identified using Limma's adjusted T-test, with FC>2 and P<0.05 considered significant.

[0190] LC-MS of dried whole blood and dried plasma added to a VAMS device. Capillary blood microsampling and deposition as dried blood spots (DBS) has important practical advantages over traditional venous blood phlebotomy. However, to date, proteomic biomarker discovery studies have typically avoided the use of DBS in favor of shotgun LC / MS analysis of plasma or serum, as this avoids the highly abundant red blood cell proteins that would otherwise dominate DDA-MS studies and significantly limit proteomic depth. Nevertheless, it is widely accepted that LC-MS analysis of plasma will not solve these problems due to the presence of highly abundant protein species, including immunoglobulins, serum albumins, acute-phase reactants, apolipoproteins, and others, which consume the majority of MS sequencing time (Anderson and Anderson, Mol Cell Proteomics. 2002;1(11):845-67; ​​Hortin et al., Clin Chem. 2008;54(10):1608-16; Chiu et al., Bioanalysis. 2009;1(4):847-55). For most laboratories, a typical sampling of undiluted human plasma using a single-shot DDA LC-MS yields 200–300 proteins, depending on the instrument configuration and detection sensitivity. Given the justification that signal saturation due to high-abundance blood proteins can be addressed by chemically washing proteins from the VAMS device prior to LC / MS, the efficacy of proteomic analysis and processing of DBS collections was determined using a VAMS device. This allows access to relatively low abundance proteins associated with circulating cells (i.e., leukocytes, platelets, circulating tumor cells, shed somatic cells) that are commonly lost from single-shot LC-MS proteomic analysis of plasma and may provide important precision medicine biomarkers.

[0191] Label-free quantification of three replicates was performed to characterize the detected proteins and their quantitative abundance for both whole blood (i.e., DBS) and dried plasma spots processed by the VAMS device using DDA on a nanoLC-MS H-FX orbitrap system. As shown in Figure 2, 1,438 proteins with a minimum of two peptides per protein were detected in two or more DBS replicates of WB (1,210 of which were unique to WB). Adding paired plasma to VAMS yielded 338 proteins in two or more replicates (110 of which were unique to plasma). 228 proteins from the VAMS device were common to both WB and plasma, including the majority of acute-phase reactants typically found in undepleted plasma LC-MS analysis (data not shown).

[0192] Notably, quantitative reproducibility (%CV) for analysis of DBS-derived WB proteins was highly acceptable according to established guidelines (Carr et al., Mol Cell Proteomics. 2014;13(3):907-17) and superior to analysis of plasma proteins added to VAMS (13.5% (interquartile range (IQR) 8.8-19.5) and 23.8% (IQR 14.2-35.8)). While analysis of plasma using VAMS is promising and enabled similar depth of the plasma proteome compared to conventional LC-MS analysis of undepleted plasma, analysis of WB as DBS provided access to the majority of these plasma proteins, in addition to hundreds of other cellular proteins not typically detected in single-shot LC-MS analysis of undepleted plasma. This included 54 proteins annotated in ProteinAtlas (Uhlen et al., Sci Signal. 2019;12(609)) as cluster of differentiation markers (CD antigens) and numerous others with important established roles in various pathologies (e.g., SOD1, HTT, MTOR, IDH2). Of the 1438 proteins detected in WB with two or more peptides in at least two of the three DDA experiments, 1180 were reported as previously detected in plasma / serum according to the Plasma Proteome Database (Nanjappa et al., Nucleic Acids Res. 2014;42 (Database issue):D959-65). 318 additional proteins were not described (data not shown).

[0193] As an example, demonstrating the potential of WB DBS for biomarker discovery, a product of PDZK1IP1, PDZK1-interacting protein 1 (MAP17), was identified, which has not been previously reported in plasma proteome databases and has not been detected in blood according to ProteinAtlas (Uhlen et al., Science. 2015;347(6220):1260419). ProteinAtlas interrogation revealed that MAP17 is enriched in renal tubules, and intracellular antibody staining shows cytosolic and nuclear speckle localization. MAP17 is involved in renal Na+ + MAP17 has been reported as an essential activator of the ATP / glucose cotransporter (Coady et al., J Am Soc Nephrol. 2017;28(1):85-93). MAP17 is overexpressed in various human cancers, and MAP17 tissue staining has been reported to predict response to neoadjuvant chemoradiotherapy in rectal cancer (Rivero et al., Oncotarget. 2018;9(68):32958-71) and response to cisplatin, carboplatin, and EGFR inhibitors in lung adenocarcinoma (Ferrer I et al., J Exp Clin Cancer Res. 2018;37(1):195). Detection of MAP17 by WB using DBS microsampling highlights the general utility of our novel approach and provides an opportunity to establish the clinical utility of this protein as a secreted cancer biomarker.

[0194] It was noted that washing WB on the VAMS device did not remove highly abundant red blood cell proteins, but was effective in removing sufficient amounts to facilitate deeper proteomic profiling compared to conventional plasma analysis using LC-MS. Thus, analysis of WB-derived proteins using the VAMS device holds promise for increased proteomic biomarker discovery using streamlined sample collection, preparation, and LC-MS acquisition workflows.

[0195] Taken together, Figure 2 demonstrates that the microsampling device provided herein can be used to isolate proteins from WB and plasma.

[0196] Differential protein extraction depending on wash conditions. The effect of varying DBS wash conditions was tested using NaCl as a reference reagent. As shown in Figure 3, different protein subsets could be detected depending on the wash conditions used. While 1,011 proteins were recovered across all wash conditions, ANOVA analysis followed by a post-hoc Tukey's test demonstrated that 2 M urea was effective in stripping additional proteins from DBS (351 fewer proteins compared to other conditions) (data not shown). Interestingly, 109 proteins were uniquely detected after the urea wash. Gene ontology enrichment indicated that RNA-binding proteins were selectively captured using the urea wash (37% compared to 16% in the total dataset) (data not shown). We washed the chip once or three times with NaCl and found that more thorough washing enabled the detection of an additional 91 proteins. Replacing NaCl with a LiCl wash in DBS recovered the most proteins (1,642). This result led to the use of LiCl for processing VAMS blood samples. These results highlight the potential of WB to partition a subset of proteins from a complex mixture. This simple and streamlined protocol enables deep proteomic analysis of WB from DBS. Nakajima et al. (J Proteome Res. 2020;19(7):2821-7.) achieved similar proteomic depth from DBS using DIA with an in-house chromatogram library, but a significant drawback of their approach is the use of multiple rounds of ultracentrifugation, which requires specialized equipment and substantial preparation time.

[0197] In summary, Figure 3A demonstrates that washing the microsampling device with LiCl prior to digestion recovers the most proteins, and Figure 3B demonstrates that the urea wash recovers a distinct subset of proteins compared to the PBS or NaCl washes.

[0198] VAMS DBS Compared to Paper DBS. An advantage of VAMS blood collection compared to conventional paper DBS is the highly controlled volumetric blood collection achieved, which facilitates accurate user sample collection while avoiding concerns about hematocrit, which can complicate analyte quantification; a well-known challenge in clinical chemistry assays (Lehmann et al., Crit Rev Clin Lab Sci. 2017;54(3):173-84; Zakaria et al., Ejifcc. 2016;27(4):288-317). Conventional DBS with paper and PBS or LiCl washes were compared to VAMS DBS and LiCl washes. Additionally, it was determined whether freshly added blood or frozen, stored blood had any significant effect on protein detection following the VAMS assay. As shown in Figure 4, LC-MS analysis of proteins recovered from VAMS identified approximately 200 more proteins than those detected from PBS-washed paper DBS, a significant finding (Tukey's post hoc q < 0.01) (data not shown). Interestingly, many of the lost proteins were recovered by replacing the PBS wash with a LiCl wash of the paper DBS. Only very small, non-significant differences were observed between fresh and frozen blood added to VAMS, but quantitative reproducibility improved with fresh addition of blood (14.9% CV and 17.9% CV, respectively). Despite the caveats of inconsistent blood volume control for field use of paper DBS microsampling, it was noted that excellent recovery and reproducibility could be achieved when blood was added in a laboratory setting using our optimized workflow with a pipette and LiCl wash (7.5% CV, IQR 4.6-12.4). This means that the washing and analysis pipeline disclosed herein is uniformly suitable for any of these DBS matrices, but it is anticipated that the elimination of hemocrit effects in microsampling will result in greater consistency with VAMS tips compared to filter paper in realistic settings.

[0199] Because DIA-MS is rapidly evolving as the method of choice for proteomic data acquisition (Nakajima et al., J Proteome Res. 2020;19(7):2821-7; O'Rourke et al., J Proteomics. 2021;231:103998), DIA-MS was used with the above-mentioned fresh and frozen blood samples on a VAMS chip washed with LiCl. Using Spectronaut search software with a library constructed from DDA runs of representative samples, detection of 1,892 proteins at 1% FDR was possible (data not shown). The DIA-MS approach enabled the detection of over 650 additional proteins compared to DDA analysis of these samples.

[0200] In summary, Figure 4A demonstrates that a microsampling device washed with LiCl prior to digestion recovers more proteins than washing conventional DBS filter paper with LiCl, and Figure 4B demonstrates that a microsampling device washed with LiCl recovers a different subset of proteins compared to conventional DBS filter paper washed with LiCl.

[0201] Application of VAMS DBS using long-term frozen whole blood cell pellets from cancer patients. VAMS may be useful for analyzing long-term frozen blood cell pellets. As a proof-of-concept, we obtained frozen blood cell pellets from five cancer patients and five matched controls (collected from age- and sex-matched healthy individuals) that had been stored at -20°C and -80°C for several years. The cell pellets contained minimal residual plasma and could be slowly loaded onto the VAMS device chip. The VAMS chip was washed with LiCl according to the method provided herein. The number of proteins detected by DDA of individual samples ranged from 381 to 1052, with an average of 703 proteins detected across 10 samples. After filtering and data imputation, 867 proteins were quantified in all 10 samples (data not shown). A reduction in the number of proteins detected in plasma compared to WB was observed, which was due to the absence of most plasma proteins (data not shown). Because specimens were prepared and acquired in a single batch, sample-to-sample variation was attributed to intrinsic biological and storage factors, rather than sample preparation methodology. Based on a 2x protein abundance threshold, 18 proteins were detected at higher abundance (upregulated) in cancer patient samples compared to controls (upper right quadrant of Figure 5), while one protein (MAP2) was lower (downregulated; upper left quadrant of Figure 5). We were unable to identify the cellular origin of these differentially abundant proteins, which may have arisen from leukocytes, somatic cells, or tumor cells. However, the majority of these proteins have previously been implicated in cancer prognosis or progression studies. Three of the proteins identified at higher abundance in cancer samples—ankyrin 3 (ANK3), vimentin (VIME), and monocarboxylate transporter 1 (MOT1)—have been identified as markers of poor cancer prognosis.

[0202] (Dauphin et al., Lung Cancer. 2013;81(1):117-22; Koukourakis et al., Cancer Biology & Therapy. 2007;6(9):1472-5; Liu Y et al. Onco Targets Ther. 2016;9:7397-407).

[0203] In summary, Figure 5 demonstrates that the microsampling device provided herein can recover proteins in frozen, long-term stored cell pellets compared to fresh WBC / plasma samples. This is ideal for longitudinal studies where samples are collected and stored over an extended period of time. It is also ideal for minimizing repeated sample collections.

[0204] Conclusions: The suitability of single-shot LC-MS for deep proteomic analysis of WB by affixing to VAMS DBS or paper DBS and processing with a lithium salt wash was demonstrated. Using the methods provided herein, up to 1,600 proteins were detected and quantified from a single run of dried whole blood compared to undepleted plasma. Several proteins not previously reported in blood, including MAP17, were detected using this approach.

[0205] The use of VAMS for microsampling offers the advantage over paper of a suitable collection device that circumvents hematocrit issues and serves as a useful matrix for sample preparation. This is an ideal situation for longitudinal blood sample collection, as required for precision medicine applications in oncology. Established protocols are easy to implement and compatible with single-shot DDA or DIA LC-MS runs, providing access to hundreds of potential biomarker candidates. Subsequent stages of biomarker research require quantitative validation in larger cohorts using stable isotope-labeled reference standards with targeted mass spectrometry acquisition methods (Carr et al., Mol Cell Proteomics. 2014;13(3):907-17). Future studies will integrate the use of VAMS DBS for sample collection and processing using SRM to enhance biomarker limits of quantification.

[0206] Example II: Method for fractionation and sequential sample preparation using an absorbent device The methods described herein allow cells and / or membranes suspended in a fluid to be absorbed into single or multiple small pieces of 3-D porous material, fractionated by centrifugation, and subsequently dried. The drying step traps the cell membranes in the 3-D porous material. The material can then be washed to remove undesired sample components such as non-membrane-associated proteins, salts, etc.

[0207] Example II, Part A Each piece of the 3-D porous material is dried and then extracted sequentially, allowing multiple assays from each piece of the 3-D porous material.

[0208] Mitra® 3-D porous material from Neoteryx (30 μL volume) was removed from the standard plastic handle and placed inside a plastic test tube as shown in Figure 13. The experimental flow chart is shown in Figure 17.

[0209] Extraction 1: Each piece of Mitra® 3-D porous material was extracted overnight in 90 μL of phosphate-buffered saline (PBS). The extraction solution was removed from the Mitra® 3-D porous material by a centrifugation step at 3000 g for 3 minutes in an empty spin column tube, as shown in Figure 16. The Mitra® 3-D porous material piece was retained in the upper part of the tube, and the extraction solution was collected in the lower tube.

[0210] Washing: After extraction 1, each piece of Mitra® 3-D porous material was suspended in 1000 μL of wash solution in a 1.5 mL Eppendorf tube and incubated at room temperature for 24 hours with gentle shaking (100 rpm). The wash solution was 0.1 M Tris-HCl containing 0.5 M LiCl. After incubation, each piece of Mitra® 3-D porous material was washed three times in fresh extraction solution by pulse centrifugation of the chip in the wash solution (3× pulse, 0–10,000 g), followed by centrifuging the wash solution out of the chip using a custom column insert device and resuspending them in fresh wash solution (3000 g, 3 min, full brake). After the final wash, the dried chips were placed in a new Eppendorf tube for storage until extraction 2 was performed.

[0211] Immunoassay of Extract 1: The immunoassays used were the MILLIPLEX MAP Human High Sensitivity T Cell Panel and the MILLIPLEX MAP Human Chemokine Panel. The assays were performed according to the manufacturer's instructions using an automated magnetic wash station (Bio-Plex Pro II, Bio-Rad) for the wash steps. The assays were performed using Luminex® 200 TM The system (Bio-Rad) was run and fluorescence values ​​were collected. Calibration curves for each cytokine were analyzed by five-parameter logistic regression using Bio-Plex manager software (ver. 5.0, Bio-Rad). Standard values ​​were considered acceptable if the points fell within 80–120% of the expected values.

[0212] Extraction 2: After extraction 1 and washing steps as described above, each piece of Mitra® 3-D porous material was incubated with 90 μL digestion solution (0.1 M triethylammonium bicarbonate, 1% (w / v) sodium deoxycholate (SDC), 10 mM TCEP, 40 mM chloroacetamide). The sample was then heated at 95°C for 10 minutes. 1 μg of trypsin was added and incubated at 37°C overnight. After incubation, the chip was removed from the solution, and the remaining liquid was centrifuged out of the chip and combined. Formic acid was added to quench the reaction and precipitate SDC. Peptides were desalted using a Stage-tip.

[0213] Mass spectrometry acquisition: Peptides were loaded onto a 75 μm × 15 cm 1.9 μm C18 (ReproSil-Pur C18-AQ) nanoLC column in 0.1% formic acid and eluted over a 90 min gradient up to 30% solvent B (80% ACN, 0.1% FA).

[0214] Peptides were detected using an HF-X orbitrap mass spectrometer (Thermo, CA, USA) operated in DDA mode with the following instrument settings: MS1 - AGC 3e6, resolution 60K, scan range 300–1650 m / z. MS2 - AGC 1e5, resolution 15K, loop count 15.

[0215] Protein Identification. Raw files were searched with MaxQuant V1.6.5 against the UniProt human proteome database (UP000005640, 20,350 screened protein entries) using the following settings: 1% FDR, reverse decoy mode, minimum peptides 2, FTMS mass tolerance 20 ppm, deletion truncations set to a maximum of 2, variable oxidation of methionine and fixed carbamidomethyl modification of Cys, enabled LFQ using default settings.

[0216] Quantification and statistical analysis. Perseus was used to remove reverse hits and peptide matches to single peptides (two peptides was the minimum required for protein quantification). Data were log2 transformed and median normalization was applied. Missing measurement values ​​were replaced by imputation with Perseus using default settings: values ​​sampled from a Gaussian distribution downshifted by 1.8 standard deviations with a width of 0.3.

[0217] Immunoassay analysis of Extract 1: The multiplex immunoassay results on the chemokine kit are shown in Table 3 below. The upper plasma / WBC fraction is labeled A plasma WBC. The lower RBC-rich fraction is labeled A RBC. As expected for fractionated blood, concentration differences are seen between the upper and lower fractions for most chemokines.

[0218] The multiplex immunoassay results on the T cell kit are shown in Table 4 below. The upper plasma / WBC fraction is labeled A plasma WBC. The lower RBC-rich fraction is labeled A RBC. As expected for fractionated blood, there are concentration differences between the upper and lower fractions for most cytokines. OOR< indicates that the concentration of that cytokine was below the reliable limit for quantification.

[0219] Mass Spectrometry of Extraction 2. MS analysis of Extraction 2 identified a total of 913 proteins across extractions of both pieces of Mitra® 3-D porous material (upper plasma / WBC and lower RBC). The upper plasma and WBC-rich fraction produced 452 protein identifications, while the lower RBC-rich fraction produced 461 protein identifications. The Venn diagram in Figure 18 shows a Venn diagram depicting the number of identified proteins unique to the plasma fraction (246 proteins) and the RBC-rich fraction (255 proteins), as well as the number of proteins identified in both fractions (206 proteins).

[0220] Protein ranking by intensity-abundance. Protein name-plasma and WBC-rich fraction. The 25 most abundant proteins identified by MS analysis are shown in Table 5, with RBC-specific proteins in bold.

[0221] Protein Names—RBC-Rich Fraction. The 25 most abundant proteins identified by MS analysis are shown in Table 6, with RBC-specific proteins in bold.

[0222] Carbonic anhydrases (CA1 and CA2) are among the top 25 most abundant proteins in the RBC fraction. These enzymes are known to be present at high levels in RBCs. These enzymes have not been listed as RBC proteins because they have high levels of expression in other tissues. In the plasma and WBC fractions, CA1 ranks 163rd in abundance, and CA2 ranks 360th. The large difference in RBC protein abundance between the two fractions reflects the separation and enrichment of RBCs in the lower fraction that occurs during centrifugation.

[0223] [Table 3]

[0224] [Table 4]

[0225] [Table 5]

[0226] [Table 6]

[0227] Example II, Part B The setup for the Mitra® 3-D porous material (30 μL volume) for this experiment was similar to that in Example II, Part A. The volume of blood used was similar; however, the device was configured with an increased dead volume below the lower Mitra®. This allowed a greater proportion of the WBC component to be removed from the upper Mitra® during centrifugation.

[0228] Two small pieces of Mitra® 3-D porous material were removed after centrifugation, and the drying steps and sequential extractions and analyses were the same as in Example II, part A.

[0229] Immunoassay analysis of Extract 1. The results of the multiplex immunoassay on the chemokine kit are shown in Table 7 below. The upper plasma / WBC fraction is labeled B Plasma WBC. The lower RBC-rich fraction is labeled B RBC. As expected for fractionated blood, concentration differences are observed between the upper and lower fractions for most chemokines.

[0230] The multiplex immunoassay results on the T cell kit are shown in Table 8 below. The upper plasma / WBC fraction is labeled B plasma WBC. The lower RBC-rich fraction is labeled B RBC. As expected for fractionated blood, there are concentration differences between the upper and lower fractions for most cytokines. OOR (out of range) indicates that the concentration of that cytokine was below the reliable limit for quantification.

[0231] MS analysis of Extract 2 identified a total of 414 proteins across extractions of both pieces of Mitra® 3-D porous material (top plasma and bottom RBC). The top plasma fraction produced 54 protein identifications, while the bottom RBC-rich fraction produced 351 protein identifications. The Venn diagram in Figure 19 shows the number of identified proteins unique to the plasma fraction (3 proteins) and the RBC-rich fraction (300 proteins), as well as the number of proteins identified in both fractions (51 proteins).

[0232] [Table 7]

[0233] [Table 8]

[0234] Example II, part B demonstrates that when cells are substantially removed from Mitra® during fractionation, very little protein remains after the extraction 1 and washing steps.

[0235] Example II, Part C The setup for the Mitra® 3-D porous material (30 μL volume) is shown in Figures 20 and 21. The device was placed in a 2 mL Eppendorf tube, which was filled with EDTA blood (approximately 1.7 mL).

[0236] Two small pieces of Mitra® 3-D porous material were removed after centrifugation, and the drying steps and sequential extractions and analyses were the same as in Example II, part A.

[0237] Mitra® 3-D porous material from Neoteryx (30 μL volume) was removed from the standard plastic handle and placed inside a plastic test tube as shown in FIG.

[0238] Immunoassay analysis of Extract 1. The multiplex immunoassay results on the chemokine kit are shown below in Table 9. The multiplex immunoassay results on the T cell kit are shown below in Table 10. As expected for fractionated blood, there are concentration differences between the upper and lower fractions for most chemokines.

[0239] The MS analysis of Example II, Part C (Extraction 2) identified a total of 1621 proteins across extractions of both pieces of Mitra® 3-D porous material (upper plasma and lower RBC). The upper plasma fraction produced 1279 protein identifications, and the lower RBC-rich fraction produced 342 protein identifications. The Venn diagram in Figure 22 shows the number of identified proteins unique to the plasma fraction (1017 proteins) and the RBC-rich fraction (80 proteins), as well as the number of proteins identified in both fractions (262 proteins).

[0240] Protein Names. The 25 most abundant proteins identified by MS analysis in plasma and WBC-rich fractions are shown in Table 13, with RBC-specific proteins shown in red.

[0241] Carbonic anhydrases (CA1 and CA2) are among the top 25 most abundant proteins in the RBC fraction. These enzymes are known to be present at high levels in RBCs. These enzymes have not been listed as RBC proteins because they have high levels of expression in other tissues. In the plasma and WBC fractions, CA1 ranked 781 in abundance, and CA2 was not identified. The large difference in RBC protein abundance between the two fractions represents the separation and enrichment of RBCs in the lower fraction that occurs during centrifugation.

[0242] Table 14 shows the names of the proteins in the RBC-rich fraction, with RBC proteins in bold.

[0243] Example II, Part D The setup for the Mitra® 3-D porous material (30 μL volume) is shown in Figures 20 and 21. The device was placed in a 2 mL Eppendorf tube, which was filled with EDTA blood (approximately 1.7 mL).

[0244] Two small pieces of Mitra® 3-D porous material were removed after centrifugation, and the drying steps and sequential extractions and analyses were the same as in Example II, part A.

[0245] Mitra® 3-D porous material from Neoteryx (30 μL volume) was removed from the standard plastic handle and placed inside a plastic test tube as shown in FIG.

[0246] Immunoassay analysis of Extract 1. The multiplex immunoassay results on the chemokine kit are shown below in Table 11. The multiplex immunoassay results on the T cell kit are shown below in Table 12. As expected for fractionated blood, there are concentration differences between the upper and lower fractions for most cytokines. OOR< indicates that the concentration of that cytokine was below the reliable limit for quantification.

[0247] The MS analysis of Example II, Part D (Extraction 2) identified a total of 900 proteins across extractions of both pieces of Mitra® 3-D porous material (upper plasma and lower RBC). The upper plasma fraction produced 831 protein identifications, and the lower RBC-rich fraction produced 69 protein identifications. The Venn diagram in Figure 23 shows the number of identified proteins unique to the plasma / WBC fraction (791 proteins) and the RBC-rich fraction (29 proteins), as well as the number of proteins identified in both fractions (40 proteins).

[0248] [Table 9]

[0249] [Table 10]

[0250] [Table 11]

[0251] [Table 12]

[0252] [Table 13]

[0253] [Table 14]

[0254] Example III: Label-Free Quantification (LFQ) Data Basic Label-Free Quantification (LFQ) Workflow. The LFQ workflow was performed as established by LFQ Analyst and DataSciencePlus.

[0255] Sample Handling. Frozen whole blood cell pellets (plasma removed) from five age- and sex-matched healthy donors and five stage III cancer donors were purchased from PrecisionMed (Solana Beach, CA, USA). Samples were collected between 2014 and 2018. The supplier indicated that samples from healthy participants were stored at -20°C for 35.6 to 263.0 weeks, and samples from cancer patients were stored for 43.0 to 165.4 weeks. Upon receipt at our laboratory, samples were stored at -80°C for 101 weeks prior to the analysis reported herein. Samples were thawed on ice and applied to the VAMS tip until completely filled. Samples were then allowed to dry at room temperature for 5 minutes before being transferred to a foil ziplock bag containing desiccant. All samples were stored at 4°C for a minimum of 24 hours before extraction. Odd numbered samples (1, 3, 5, 7 and 9) are controls and even numbered samples (2, 4, 6, 8 and 10) are cancer samples.

[0256] Filtering. Data were filtered to remove reverse hits and contaminants (REV / CON proteins), which removed 36 proteins. Data were also filtered to remove single unique / razor peptide proteins, which removed 141 proteins.

[0257] Quantification filtering: Data were filtered to remove rare proteins, which allowed for quantification of proteins with more than three quantified values ​​per group, and also proteins present with two samples in both groups.

[0258] This resulted in the removal of 1026 proteins. The filtering can be seen in Figure 24.

[0259] Data imputation for missing values ​​after filtering and median normalization. After removing proteins identified with few peptides and proteins with sparse quantification, the remaining protein quantifications still have missing values, which can be imputed using default Perseus-style imputation (random numbers drawn from a normal distribution with a 1.8 standard deviation downward shift and with a depth of 0.3 for each sample).

[0260] Median normalization was performed before data imputation; otherwise, relatively low abundance samples would be shifted up.

[0261] Differential expression using simple t-test or adjusted t-test using the limma package. After data imputation, the usual workflow for differential expression was performed. Data between groups were compared using two-sample t-test and / or adjusted t-test from the limma package. To find the number of up-regulated proteins in cancer samples, significance was determined by either simple t-test or adjusted t-test with a p-value of less than 0.05.

[0262] Types of protein comparisons. Functional comparisons of all identified proteins (LFQAll) as well as a relatively small set of proteins with full complement data (LFQImputed) can be identified in fresh or frozen samples. However, no major differences are observed, with slightly more cytosolic proteins (in terms of percentage) and slightly more nuclear proteins. Similarly, no changes were observed when looking at KEGG pathways.

[0263] Reliability of results: iterative imputation, bootstrap, and label randomization. For small datasets (n=5 cancer / health), results are easily obtained erroneously or due to chance alone. Because it is relatively difficult to obtain positive results by chance from larger experiments, the likelihood of such false positives decreases with larger experiment size.

[0264] While there is no substitute for obtaining larger samples, and randomization or bootstrapping on small sets does not guarantee reproducibility of results across biologically distinct sets, the quality of the results can be confirmed in several ways.

[0265] Example IV: Urea Cleaning Broad Objectives: The objective of the experiments described herein is to understand the differences between methods, optimal preparations, and to establish best procedures for such data / analysis in the future.

[0266] Differences in specificity. It is useful to note differences in specificity, perhaps especially to evaluate different methodologies. These can be done manually using the online tool Venny for Venn diagrams. When multiple sets are used simultaneously, scripting online tools can be used to create UpSet plots.

[0267] FIG. 9 shows an UpSet plot depicting that washing the VAM device three times with 1 mL of 0.1 M Tris-HCl containing 0.5 M NaCl resulted in the identification of 1465 proteins common to frozen whole blood (WB), WB and white blood cells (WBC), and subsequently 1233 proteins unique to WBC.

[0268] FIG. 10 shows an UpSet plot demonstrating that washing the Neoteryx chip twice with 1 mL of 2 M urea resulted in the identification of a distinct protein profile with approximately 550 fewer proteins identified compared to FIG. 9.

[0269] FIG. 11 shows an UpSet plot depicting that washing Neoteryx chips or filter paper with LiCl or NaCl identifies 1321 proteins present in filter paper washed with LiCl, filter paper washed with NaCl, Neoteryx chips washed with LiCl, and Neoteryx chips washed with NaCl.

[0270] Gene Ontology (GO) annotation: GO annotations can be retrieved live from Uniprot and analyzed via scripts (thereby eliminating the need to download annotation sheets and analyze them manually).

[0271] A number of categories of interest (as described below) were selected and summaries were obtained for each of the sets below, including all proteins present in the experiment. This could be re-run with different subsets and different GO categories depending on the question that needed to be asked.

[0272] The urea wash resulted in the identification of more plasma membrane and ribosomal proteins based on GO annotation. Table 17 shows the GO annotation.

[0273] [Table 15-17] Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation: The same process can be repeated using KEGG pathway annotation. Urea detergent results in the isolation and identification of functional proteins, including ribosomes. Figure 12 shows the KEGG annotation.

[0274] Abbreviations used throughout the detailed description and examples are as follows: epithelial-neutrophil-activating peptide / CXCL5 (ENA-78 (LIX)), growth-regulated oncogene alpha (Gro-α / KC), interleukin-8 (IL-8), interferon-γ-inducible protein 10 (IP-10), interferon-inducible T cell α chemoattractant (ITAC), monocyte chemoattractant 1 (MCP-1), C-X-C motif ligand 9 (MIG), macrophage inflammatory protein 1 alpha (MIP-1α), macrophage inflammatory protein 1 beta (MIP-1β), macrophage inflammatory protein 3 alpha (MIP-3α), Regulated upon Activation, Normal T Cell Expressed and Presumably Secreted (REG-C), and IL-10. Secreted) (RANTES), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-10 (IL-10), interleukin-13 (IL-13), interleukin-17α (IL-17A), interleukin-4 (IL-4), interleukin-23 (IL-23), interleukin-5 (IL-5), and interleukin-6 (IL-6).

[0275] Example V: Evaluation of Buffers and Peptide Purification Objective: To evaluate the effect of altering the digestion buffer and / or peptide cleanup procedure compared to the method described in Example II on peptide yield and / or protein identification after mass spectrometry analysis. The method of Example II provides a peptide yield recovery of approximately 0.8 μg / μL (approximately 16 μg total, which is near the maximum capacity of the Stage-tip; if the existing method is limited by the maximum capacity of the Stage-tip, the result will be missing some IDs in mass spectrometry analysis). The changes in the digestion buffer and / or peptide cleanup procedure include the following: an additional centrifugation step after trypsin digestion, the use of solid-phase extraction (SPE) instead of a Stage-tip for peptide desalting, and the use of a freshly prepared buffer ("FPB" buffer; 1% SDC, 10 mM TCEP, 40 mM iodoacetamide (IAA), 100 mM triethylammonium bicarbonate buffer (TEAB) pH 8) instead of the digestion solution shown above (master mix buffer, Example II, part A) for the trypsin digestion.

[0276] A series of whole blood (WB) samples were prepared according to Example II (30 μL VAMS / Mitra® tips) and as described in Molloy et al., “Proteomic Analysis of Whole Blood Using Volumetric Absorptive Microsampling for Precision Medicine Biomarker Studies,” J Proteomics Res. 2022;21(4):1196-1203; DOI: 10.1021 / acs.jproteome.1c00971. The following protocol combinations were used to evaluate changes in digestion buffer and / or peptide cleanup procedures compared to Example II.

[0277] Protocol as described in Example II: 1. Wash overnight with 1 mL of LiCl 2. Wash twice with 1 mL of LiCl by centrifugation pulses 3. Add 100 µL of master mix buffer (1% sodium deoxycholate (SDC), 10 mM tris(2-carboxyethyl)phosphine (TCEP), 40 mM 2-chloroacetamide (CLA), 100 mM trisaminomethane (Tris), pH 8) to each chip. 4. Heat at 95°C for 10 minutes with gentle stirring. 5. Add 1 μg (2 μL) of trypsin (0.5 μg / μL in master mix buffer) 6. Incubate overnight at 37°C for 19-21 hours. 7. Remove the tip from the test tube (slide the tip gently along the inside of the Eppendorf tube, allowing excess supernatant to flow back into the test tube) and collect all of the supernatant, including any solution that has accumulated in the probe attachment opening. 8. Precipitate sodium deoxycholate (SDC) with 2 µL of 100% formic acid 9. Centrifuge at 12,000g for 5 minutes and transfer the supernatant to a clean tube. 10. Purification through Stage-Tips using styrene divinylbenzene reversed-phase sulfonate (SDB-RPS) plugs (3-layer: essentially 3 Stage-Tips stacked in a single device to increase maximum capacity) 11. Centrifuge at 12,000 g for 5 minutes to prevent any solid SDC from accumulating on the Stage-Tip.

[0278] Protocol using FPB buffer: 1. Wash overnight with 1 mL of LiCl 2. Wash twice with 1 mL of LiCl by centrifugation pulses 3. Add 100 µL of FPB buffer (1% SDC, 10 mM TCEP, 40 mM iodoacetamide (IAA), 100 mM triethylammonium bicarbonate buffer (TEAB) pH 8) to each chip. 4. Heat at 95°C for 10 minutes with gentle stirring. 5. Add 1 μg (2 μL) of trypsin (0.5 μg / μL in FPB buffer) 6. Incubate overnight at 37°C for 19-21 hours. 7. Remove the tip from the test tube (slide the tip gently along the inside of the Eppendorf tube, allowing excess supernatant to flow back into the test tube) and collect all of the supernatant, including any solution that has accumulated in the probe attachment opening. 8. Precipitate sodium deoxycholate (SDC) with 2 µL of 100% formic acid 9. Centrifuge at 12,000g for 5 minutes and transfer the supernatant to a clean tube. 10. Clean up using the Stage-Tip protocol or Solid Phase Extraction (SPE) protocol below.

[0279] Protocol using Stage-Tip: 1. Prepare a Stage-Tip using a needle and syringe by puncturing three layers of SDB-RPS disk with the needle and packing it into a 200 μL pipette tip. Using gentle force from a 10 mL syringe with the plunger pulled, the disk can be ejected from the needle (located near the lower inner portion of the 200 μL pipette tip) into the tip. 2. A Stage-Tip holder (a lid required during centrifugation) can be made by using a screwdriver to drill a hole through the lid of a 1.5 mL test tube. 3. Place the Stage-Tip into its holder mounted in the Eppendorf tube and add 100 μL of 100% methanol. Centrifuge at 1000×g for 1 minute. 4. Add 100 μL of 0.2% trifluoroacetic acid (TFA) into each Stage-Tip for equilibration. Centrifuge at 1000×g for 3 minutes. 5. Add sample to each Stage-Tip. Centrifuge at 1000 x g for 3 minutes. 6. Wash the Stage-Tip with 100 μL of 0.2% TFA and centrifuge at 1000 × g for 3 minutes. 7. Repeat step 6 8. Place the Stage-Tip into a new 1.5 mL tube and elute the sample with 100 μL of 80% acetonitrile (ACN), 5% ammonium hydroxide, and centrifuge at 1000 × g for 3 minutes. 9. Dry the sample in a SpeedVac 10. Add 25 µL of 0.5% formic acid (FA) to the dried sample to reconstitute it prior to liquid chromatography-mass spectrometry (LC-MS) analysis 11. Estimate sample concentration using Nanodrop 12. Once the protein concentration is known, normalize each sample by taking an aliquot and further diluting it in loading buffer to ensure equal loading of the sample to the LC-MS. For a 75 μm column, an injection of 0.6-1 μg works well.

[0280] Protocol using SPE: 1. Dilute the sample with 700 µL of 0.5% formic acid (FA) 2. Centrifuge at 12,000 g for 5 minutes 3. Activate the column (Waters HLB 10 mg cc) with 500 μL of 90% acetonitrile (ACN), 0.5% FA (HLB is a type of SPE, see for example: https: / / www.sigmaaldrich.com / AU / en / technical-documents / technical-article / analytical-chemistry / solid-phase-extraction / supel-select-hlb-spe) 4. Wash with 800 μL of 0.5% FA 5. Add the sample 6. Wash with 1000 μL of 0.5% FA 7. Wash with 1000 μL of 0.5% FA 8. Elute with 300 μL of 70% ACN 0.5% FA 9. Elute with 150 μL of 90% ACN 0.5% FA 10. Drying 11. Resuspend in 25 μL of 0.5% FA 12. Sample concentrations can be estimated using Nanodrop.

[0281] Samples prepared and evaluated: Example V-1: Whole Blood Cells, Overnight Digestion, FPB Buffer, and Stage-Tip Example V-2: Whole Blood Cells, Overnight Digestion, FPB Buffer, and Solid Phase Extraction (SPE) Example V-3: Whole Blood Cells, Overnight Digestion, Master Mix Buffer, and SPE Example V-4: Whole blood cells, overnight digestion, master mix buffer, and Stage-Tip.

[0282] Results and Discussion: The resulting peptide yields recovered according to Examples V-1 to V-4 are shown in Figure 42. For comparison, the resulting peptide yield recovered according to Example II is approximately 0.8 μg / μL (approximately 16 μg total, which is near the maximum capacity of the Stage-tip). The number of protein and peptide IDs identified according to Examples V-1 to V-4 is shown in Figure 43. As illustrated in Figure 43, the use of the master mix buffer yielded superior peptide IDs than the FPB buffer when utilizing either the Stage-Tip or SPE protocols (samples run on a Mass Spec QEHFX4). In addition, the use of the SPE protocol yielded approximately 10% more protein IDs and approximately 15% more peptide IDs than when using the Stage-Tip protocol. The percentage of deletion truncations after trypsin digestion according to Examples V-1 to V-4 is shown in Figure 44. As illustrated in Figure 44, samples using FPB buffer had more deletion truncations than those using master mix buffer, indicating problems with digestion or cysteine ​​reduction / alkylation in FPB buffer.

[0283] In the method of Example II, after trypsin digestion, the porous plastic material (Mitra®) was removed from the solution and the solution was allowed to flow into a test tube, essentially by gravity, resulting in a recovery of 50% of the solution—50 μL (from 100 μL) in the test tube. Use of the Stage-Tip protocol in this method includes a centrifugation and washing step of the tip to remove residual trypsin digestion buffer. Centrifugal removal of the digest from the tip resulted in a near doubling of the liquid in the test tube and a large increase in peptide yield (compared to approximately 16 μg according to Example II). Use of the SPE protocol increased the total yield by approximately 30% (see Examples V-2 and V-3, compared to Example V-1).

[0284] Conclusion: Centrifugal removal of peptide digests results in higher yields of peptides compared to the protocol as described in Example II. Solid-phase extraction (SPE) results in higher yields of peptides compared to Stage-Tip cleanup of peptide digests.

[0285] Example VI: Evaluation of buffer changes and shorter trypsin digestion on PBMCs and whole blood Objective: To evaluate the effect of changing the digestion buffer and / or a shorter digestion time (2 hours versus overnight (19-21 hours)) for peripheral blood mononuclear cells (PBMCs) and whole blood (WB) compared to the method described in Example II on peptide yield and / or protein identification after mass spectrometry analysis.

[0286] Samples prepared and evaluated: Example VI-1: PBMCs in VAMS chip (30 μL), overnight digestion, FPB buffer Example VI-2: PBMCs in VAMS chip (30 μL), overnight digestion, FPB buffer Example VI-3: PBMCs in VAMS chip (30 μL), 2-hour digestion, FPB buffer Example VI-4: PBMCs in VAMS chip (30 μL), 2-hour digestion, FPB buffer Example VI-5: WB in VAMS chip (30 μL), overnight digestion, FPB buffer Example VI-6: WB in VAMS chip (30 μL), overnight digestion, FPB buffer Example VI-7: WB in VAMS chip (30 μL), 2-hour digestion, FPB buffer Example VI-8: WB in VAMS chip (30 μL), 2-hour digestion, FPB buffer Example VI-9: WB in VAMS tip (30 μL), control-overnight digest, master mix buffer Example VI-10: WB in VAMS tip (30 μL), control—overnight digestion, master mix buffer, no centrifugation.

[0287] PBMC preparation. PBMC chips were prepared in a Vacutainer CPT as per the manufacturer's protocol. TM Cells were prepared using cell preparation tubes containing heparin sodium (BD Vacutainer CPT TM , cell preparation tubes containing heparin sodium N, reference number 362753; https: / / www.rch.org.au / uploadedFiles / Main / Content / Specimen_Collection / Cell%20Preparation%20Tube%20CPT%20BD%20Vacutainer.pdf). After isolation, the white blood cell (WBC) count for the collected PBMC fraction was 21 × 10 3 The extracted PBMCs were 7 x 10 cells / µL. 3 Diluted 1:3 to reach a cell count of cells / μL.

[0288] VAMS 30 μL tips were dipped into the PBMC preparation or whole blood and allowed to dry for 24 hours.

[0289] Overnight Digestion Protocol 1. Wash overnight with 1 mL of LiCl 2. Wash twice with 1 mL of LiCl by centrifugation pulses 3. Add 100 µL of master mix buffer (1% SDC, 10 mM TCEP, 40 mM CLA, 100 mM Tris) or FPB buffer (1% SDC, 10 mM TCEP, 40 mM IAA, 100 mM TEAB) to each chip. 4. Heat at 95°C for 10 minutes with gentle agitation (the chip must be held in the proper position; any chips in the wrong orientation will be inverted). 5. Add 1 μg (2 μL) of trypsin (0.5 μg / μL in master mix buffer) 6. Incubate overnight at 37°C for 19-21 hours. 7. Centrifuge the tube to remove the liquid (this step was omitted in Example VI-10 to examine the yield / results without centrifugation) 8. Wash with 100 μL of 0.5% formic acid 9. Dilute with 700 μL of 0.5% formic acid 10. Centrifuge at 12,000g for 10 minutes and transfer the supernatant to a conditioned SPE cartridge. 11. Clean up using the SPE protocol as shown in Example V.

[0290] 2-hour digestion protocol 1. Wash overnight with 1 mL of LiCl 2. Wash twice with 1 mL of LiCl by pulse centrifugation 3. Add 50 µL of FPB buffer (1% SDC, 10 mM TCEP, 40 mM IAA, 100 mM TEAB) to each chip. 4. Heat at 95°C for 10 minutes with gentle agitation (the chip must be held in the proper position; any chips in the wrong orientation will be inverted). 5. Add 150 μg of Rapid Trypsin Digestion Buffer 6. Add 1 μg of trypsin / LysC mix 7. Incubate at 70°C for 2 hours on a thermomixer 8. Centrifuge the tube to remove the liquid 9. Wash with 100 μL of 0.5% formic acid 10. Dilute with 700 μL of 0.5% formic acid 11. Centrifuge at 12,000g for 10 minutes and transfer the supernatant to a conditioned SPE cartridge. 12. Clean up using SPE protocol as for Example V.

[0291] Results and Discussion: Total protein yields for all samples are shown in Figure 45. All PBMCs had less than 5 μg of total protein. Example VI-10, which did not include a centrifugation step, had a lower yield than the sample that did (Example VI-9). The 2-hour digestion samples (Examples VI-3, VI-4, VI-7, and VI-8) had lower yields compared to the overnight digestion samples, primarily for the WB samples (Examples VI-7 and VI-8). Figure 46 illustrates the number of protein and peptide IDs observed from the PBMC and WB samples. While the IDs for the FPB buffer (Examples VI-1 to VI-8) were still lower than those using the master mix buffer (Examples VI-9, VI-10, and Hela standard (using Hela cells in the master mix buffer)), the PBMC samples (Examples VI-1 to VI-4) showed a significant number of IDs. Overall, the rapid 2-hour digestion identified more peptides, although a greater number of truncated peptides were observed (as illustrated in Figure 47). The "no centrifugation" sample (Example VI-10) showed the greatest number of IDs. Samples that included a centrifugation step had a greater number of more highly charged / larger peptides.

[0292] The percentage of deletion truncations after trypsin digestion is shown graphically in FIG.

[0293] The number of alkylated peptides (containing carbamidomethyl modifications (CAM)) in the samples was also determined (see Figure 48). Samples using FPB buffer gave a higher percentage of CAM peptides.

[0294] A Venn diagram illustrating the overlap of protein IDs between PBMC and WB is provided in FIG.

[0295] The protein classes of proteins found exclusively in PBMCs are illustrated in Figure 50, and the protein classes of proteins found exclusively in WB are illustrated in Figure 51. Looking at proteins exclusive to either PBMCs or WB, the PBMC samples show significantly more RNA metabolism and translation proteins as well as some storage proteins that are not represented in the WB samples. The WB samples contain a greater proportion of protective / immune proteins.

[0296] Conclusion: The buffer change contributes to a relatively low amount of protein and peptide IDs. Centrifuging the digested solution off the chip results in a higher yield (see Example V), but results in lower protein and peptide IDs. The use of a 2-hour trypsin digestion produced a reasonable number of protein and peptide IDs, but also a higher percentage of truncated truncations.

[0297] Example VII: Evaluation of buffer changes and shorter trypsin digestion for PBMC, WB, and WB in Streck tubes Objective: To investigate the effects of various buffer modifications and to test a rapid trypsin / LysC 2-hour digestion using a relatively large amount of trypsin (to improve defective cleavage). Evaluations were performed using whole blood, whole blood in Streck tubes, and PBMC chips. Streck cell-free DNA BCT tubes contain a stabilizer for cell-free DNA, which provided an opportunity to evaluate protein recovery from Streck tubes (which cannot be used for immunoassays). It is noted that extensive disease sample collections at many biobanks utilize stabilization techniques similar to those used by Streck tubes. Therefore, stabilized samples, including nucleic acid-containing stabilized samples such as Streck tube samples or stabilized blood samples, may be suitable for proteomic analysis using the methods disclosed herein.

[0298] Test tubes designed to stabilize blood, such as Streck tubes, contain imidazolidinyl urea, which releases formaldehyde into solution after the blood sample enters the test tube. Formaldehyde has been used as a fixative and preservative for decades. Formaldehyde is reactive toward both proteins and DNA, forming intermolecular crosslinks between macromolecules as well as intramolecular chemical modifications. Formaldehyde's high reactivity, combined with its high permeability to cells and tissues, has led to its use in numerous applications in biology, biotechnology, and medicine. Because the sample does not contain individual macromolecules after stabilization, stabilized blood samples often produce erroneous results when assayed using immunoassays. Crosslinks form between proteins and nucleic acids, as well as between dissimilar molecules such as proteins and DNA (https: / / www.nature.com / articles / s41467-020-16935-w).

[0299] Samples prepared and evaluated (due to results from Example VI which included a post-digestion chip centrifugation step, each sample was run "no centrifugation"): Example VII-1: PBMCs in VAMS chip (30 μL), overnight digestion, FPB buffer Example VII-2: PBMCs in VAMS chip (30 μL), overnight digestion, master mix buffer Example VII-3: PBMCs in VAMS chip (30 μL), 2-hour digestion, master mix buffer Example VII-4: WB in VAMS chip (30 μL), overnight digestion, FPB buffer Example VII-5: WB in VAMS tip (30 μL), overnight digestion, master mix buffer Example VII-6: WB in VAMS chip (30 μL), 2-hour digestion, master mix buffer Example VII-7: Overnight digestion in WB-VAMS tip (30 μL) in Streck ("WB-Streck") tube, FPB buffer Example VII-8: Overnight digestion in WB-Streck tube-VAMS tip (30 μL), master mix buffer Example VII-9: 2 hour digestion in WB-Streck tube-VAMS tip (30 μL), master mix buffer.

[0300] Overnight Digestion Protocol 1. Wash overnight with 1 mL of LiCl 2. Wash twice with 1 mL of LiCl (centrifugation pulse) 3. Add 100 μL of master mix buffer (1% SDC, 10 mM TCEP, 40 mM CLA, 100 mM Tris) or FPB buffer (1% SDC, 10 mM TCEP, 40 mM CLA, 100 mM Tris) to each chip. 4. Heat at 95°C for 10 minutes with gentle agitation (the chip must be held in the proper position; any chips in the wrong orientation will be inverted). 5. Add 1 μg (2 μL) of trypsin (0.5 μg / μL in master mix buffer) 6. Incubate overnight at 37°C for 19-21 hours. 7. Pull the tip up and away from the side of the test tube 8. Dilute with 800 μL of 0.5% formic acid (SDC will precipitate) 9. Centrifuge at 12,000g for 10 minutes and transfer the supernatant to a conditioned SPE cartridge. 10. Clean up using the SPE protocol as described above for Example V.

[0301] 2-hour digestion protocol 1. Wash overnight with LiCl (1 mL) 2. Wash twice with 1 mL of LiCl (centrifugation pulse) 3. Add 100 µL of master mix buffer (1% SDC, 10 mM TCEP, 40 mM CLA, 100 mM Tris) to each chip. 4. Heat at 95°C for 10 minutes with gentle agitation (the chip must be held in the proper position; any chips in the wrong orientation will be inverted). 5. Add 200 μL of rapid trypsin digestion buffer 6. Add 3 μg of trypsin / LysC mix 7. Incubate at 70°C for 2 hours on a thermomixer 8. Pull the tip up and away from the side of the test tube 9. Dilute with 800 μL of 0.5% formic acid (SDC will precipitate) 10. Centrifuge at 12,000g for 10 minutes and transfer the supernatant to a conditioned SPE cartridge. 11. Clean up using the SPE protocol as described above for Example V.

[0302] Results and Discussion: The total protein yields for the samples of Examples VII-1 to VII-9 are shown in Figure 52. All PBMC samples (Examples VII-1 to VII-3) had less than 5 μg of total protein. Samples using 2-hour digestion showed relatively low yields for the WB samples, yet higher than for the PBMCs. Both WB rapid trypsin digestion samples (Examples VII-6 and VII-9) produced lower yields compared to the overnight digestion samples (Examples VII-5 and VII-8), respectively. The PBMC rapid trypsin digestion samples provided higher yields than the overnight digestion (Example VII-3 compared to Example VII-2, respectively).

[0303] The number of protein IDs and peptide IDs were measured as shown in Figures 53 and 54, respectively. As illustrated in Figure 53, the protein IDs for PBMCs were over 3,000. No problems were observed with the use of FPB buffer. The number of peptide IDs and protein IDs observed and recovered from the WB-Streck tubes (Examples VII-7 to VII-9) were comparable to those recovered and observed from the WB samples (Examples VII-4 to VII-6), respectively. The relative number of peptide IDs across samples was similar and reflected in the protein ID results.

[0304] The percentage of deletion cleavages after trypsin digestion is shown graphically in Figure 55. In this figure, the rapid digestion samples (e.g., Examples VII-3 and VII-6) produced even higher numbers of deletion cleavages compared to those in Example VI. Of note, in Example VI, 50 μL was added and diluted with 150 μL before adding trypsin, whereas in Example VII, 100 μL of buffer was added and diluted with 200 μL prior to adding trypsin.

[0305] A Venn diagram illustrating the overlap of protein IDs between WB and WB-Streck tube samples is provided in Figure 56. The WB and WB-Streck tubes had excellent overlap, indicating good recovery of samples. A Venn diagram illustrating the overlap of protein IDs between PBMC and WB samples is provided in Figure 57. WB and PBMC showed good overlap, with many additional IDs arising from PBMC. A Venn diagram illustrating the overlap of protein IDs between PBMC, WB, and WB-Streck tube samples is provided in Figure 58.

[0306] Figure 59 illustrates the protein classes of proteins found exclusively in PBMCs, and Figure 60 illustrates the protein classes of proteins found exclusively in WB and WB-Streck tube samples. Looking at proteins exclusive to PBMC, WB, or WB-Streck tube samples, the PBMC samples also show significantly more RNA metabolism and translation proteins, as well as some storage proteins that are not represented in the WB samples. The WB samples contain a greater proportion of protective / immune proteins.

[0307] Conclusion: Use of the methods disclosed herein resulted in a large number of protein and peptide IDs from PBMC, WB, and WB-Streck tube samples. Use of the buffer conditions provided in Example II produced similar results. Use of a 2-hour trypsin digestion resulted in a lower number of peptide and protein IDs, indicating a greater proportion of truncated forms.

[0308] Example VIII: Optimization of various washing conditions using buffy coat and trypsin digestion using whole blood Objective: To evaluate the effect of different initial wash mechanisms on chips immersed in buffy coat. Buffy coat is a fraction of an anticoagulated blood sample that contains the majority of white blood cells and platelets. The experiment also evaluated the use of LiCl washes and urea / thiourea washes. Using whole blood chips, the experiment also investigated the use of iodoacetamide compared to chloroacetamide for alkylation of proteins after reduction. For a 2-hour trypsin digestion, the volume of lysis buffer added prior to trypsin digestion was also evaluated.

[0309] Example VIII-1: Buffy Coat, LiCL Example VIII-2: Buffy Coat, Urea / Thiourea Example VIII-3: WB, urea / thiourea Example VIII-4: WB, Control Example VIII-5: WB, IAA Example VIII-6: WB, 50 μL RedAlk, overnight digestion Example VIII-7: WB, 50 μL RedAlk, 2 hour digestion.

[0310] Buffy Coat Tip Preparation. Whole blood was centrifuged at 1500 g for 10 minutes. Plasma was removed and the buffy coat layer was drawn off into a new tube. The tube was centrifuged again and the second separation was transferred to a new tube. A 30 μL VAMS tip was dipped into the buffy coat extract and allowed to dry overnight.

[0311] Overnight Digestion Protocol 1. Wash overnight with LiCl (1 mL) or with 7 M urea, 2 M thiourea (1 mL) 2. Wash twice with 1 mL of LiCl (centrifugation pulse) or twice with 1 mL of 7 M urea, 2 M thiourea (centrifugation pulse) 3. Add 100 μL of lysis buffer (1% SDC, 10 mM TCEP, 40 mM CLA, 100 mM Tris, pH 8) (for the sample in Example VIII-5, 40 mM IAA was used and 50 μL of lysis buffer was added to Example VIII-6) 4. Heat at 95°C for 10 minutes with gentle agitation (the chip must be held in the proper position; any chips in the wrong orientation will be inverted). 5. Add 1 μg (2 μL) of trypsin (0.5 μg / μL in master mix buffer) 6. Incubate overnight at 37°C for 19-21 hours. 7. Pull the tip up and away from the side of the test tube 8. Dilute with 800 μL of 0.5% formic acid (SDC will precipitate) 9. Centrifuge at 12,000g for 10 minutes and transfer the supernatant to a conditioned SPE cartridge. 10. Clean up using the SPE protocol as described above for Example V.

[0312] 2-hour digestion protocol used 1. Wash overnight with 1 mL of LiCl 2. Wash twice with 1 mL of LiCl by centrifugation pulses 3. Add 50 µL of master mix buffer (1% SDC, 10 mM TCEP, 40 mM CLA, 100 mM Tris) to each chip. 4. Heat at 95°C for 10 minutes with gentle agitation (the chip must be held in the proper position; any chips in the wrong orientation will be inverted). 5. Add 150 μL of Rapid Trypsin Digestion Buffer 6. Add 2 μg of trypsin / LysC mix 7. Incubate at 70°C for 2 hours on a thermomixer 8. Pull the tip up and away from the side of the test tube 9. Dilute with 800 μL of 0.5% formic acid (SDC will precipitate) 10. Centrifuge at 12,000g for 10 minutes and transfer the supernatant to a conditioned SPE cartridge. 11. Clean up using the SPE protocol as described above for Example V.

[0313] Results and Discussion: The total protein yields for the samples of Examples VIII-1 to VIII-7 are shown in Figure 61. The urea / thiourea washed samples had a large increase in total yield (μg / μL).

[0314] The number of protein IDs and peptide IDs was measured, as shown in Figures 62 and 63, respectively. The number of protein IDs and peptide IDs for the urea / thiourea washed samples (Examples VIII-2 and VIII-3) was greatly reduced. Such results could be due to incomplete digestion and / or the presence of more abundant proteins such as serum albumin and complement C3 (see section below). Alkylation with iodoacetamide (IAA) resulted in insufficient numbers of protein and peptide IDs. Lower volumes of reducing / alkylating buffer produced results similar and close to the control. The 2-hour digestion identified approximately the same number of proteins and peptides as the overnight digestion using the same volume of buffer, even though the digestion efficiency was still only about 50%.

[0315] The percentage of deletion cleavage products after trypsin digestion is shown graphically in Figure 64. In this figure, trypsin digestion was inefficient for samples washed with urea / thiourea. This could be due to the high concentration of residual urea in the chip, as urea at concentrations greater than 1 M can have a negative effect on trypsin digestion. Although the IDs were comparable, the 2-hour digestion resulted in a relatively low digestion efficiency.

[0316] A Venn diagram illustrating the overlap of protein IDs between buffy coat samples washed with LiCl and urea / thiourea is provided in Figure 65. A Venn diagram illustrating the overlap of protein IDs between WB samples washed with LiCl and urea / thiourea is provided in Figure 66. The results show that the majority of proteins identified in the urea / thiourea washed samples overlap with those identified in the LiCl washed samples for both the buffy coat and whole blood chip samples.

[0317] Figure 67 details the top protein IDs observed from the buffy coat sample with a LiCl wash, and Figure 68 details the top protein IDs observed from the buffy coat sample with a urea / thiourea wash. The buffy coat sample with a LiCl wash reduced the number of peptides for the highly abundant proteins serum albumin and complement C3 by approximately two-fold.

[0318] A Venn diagram illustrating the overlap of protein IDs between the buffy coat sample and the WB sample, each washed with LiCl, is provided in Figure 69. Significant overlap was observed between the WB and buffy coat chip samples, with a greater number of IDs provided by the buffy coat chip sample. A Venn diagram illustrating the overlap of protein IDs between the PBMC sample (Example VII) and the buffy coat sample is provided in Figure 70.

[0319] Conclusion: Using the method disclosed herein, the urea / thiourea washed sample had a significant increase in total yield; however, the number of protein and peptide IDs was greatly reduced. In addition, the method disclosed herein resulted in greater representation of high-abundance proteins, such as serum albumin and complement C3, with significant overlap between the urea / thiourea washed and LiCl washed samples. Digestion was inefficient for the urea / thiourea sample. Alkylation with iodoacetamide (IAA) produced insufficient numbers of protein and peptide IDs. A lower volume of reducing / alkylating buffer produced results similar to and close to the control. A 2-hour digestion identified nearly the same number of proteins and peptides as an overnight digestion using the same volume of buffer, even though the digestion efficiency was still only approximately 50%. Despite the much simpler preparation, the buffy coat chip sample produced a high number of protein and peptide IDs approaching those identified from the PBMC sample.

[0320] Example IX: Further optimization of various washing conditions using buffy coat and evaluation of freezing blood samples prior to fractionation using VAMS chips Objective: To examine different initial wash mechanisms for tips immersed in buffy coat. Buffy coat is the fraction of an anticoagulated blood sample that contains the majority of white blood cells and platelets. In this experiment, we performed variations on an initial LiCl wash, followed by a urea / thiourea wash: one containing a surfactant to help keep proteins soluble; one containing a reducing agent to help denature proteins; and urea / thiourea alone. We also examined the effect of freezing the blood prior to sample collection using the VAMS tip.

[0321] method Buffy Coat Tip Preparation. Whole blood was centrifuged at 1500 g for 10 minutes. Plasma was removed and the buffy coat layer was drawn off into a new tube. The tube was centrifuged again and the second separation was transferred to a new tube. A 30 μL VAMS tip was dipped into the buffy coat extract and allowed to dry overnight.

[0322] Frozen Blood Chip Preparation. Blood was collected in both Streck tubes and EDTA tubes. Whole blood was centrifuged at 1500g for 10 minutes to remove plasma. The remaining RBC pellet was frozen for a minimum of 24 hours and then diluted 1:2 in PBS. A 30-μL tip was then immersed in the thawed, diluted RBC blood. The thawed RBCs (total volume 1400 μL) were centrifuged at 16000g for 20 minutes to pellet the red blood cell membranes, and 700 μL of the supernatant was removed and used to fill a 30-μL tip. 700 μL of PBS was then added to further dilute the remaining supernatant, and the sample was centrifuged again at 16000g for 10 minutes. The supernatant was completely removed, and the pellet was resuspended in 700 μL PBS, into which a 30-μL tip was immersed. The tip was air-dried and then stored with desiccant for 24 hours.

[0323] Protocol used for overnight digestion Once dry, the chips were washed overnight with LiCl (1 mL), followed by two 30-minute washes with either: (a) 1 mL 7 M urea, 2 M thiourea; (b) 1 mL 2 M urea, 2 M thiourea, 1% sodium deoxycholate; or (c) 1 mL 7 M urea, 2 M thiourea, 1% sodium deoxycholate (SDC), tributylphosphine (TBP). Three centrifugation pulses (10,000 g) were performed at the beginning of each urea wash incubation. After the urea / thiourea wash, the chips were washed in 1 mL of water, followed by 100 μL lysis buffer (1% SDC, 10 mM TCEP, 40 mM CLA, 100 mM Tris, pH 8). The chips were heated at 95°C for 10 minutes with gentle agitation. Next, 1 μg (2 μL) of trypsin (0.5 μg / μL in 100 mM Tris) was added, and the solution was incubated overnight (19–21 h) at 37 °C. After incubation, the chip was removed, and the remaining digest was diluted with 800 μL of 0.5% formic acid. The solution was then centrifuged at 12,000 g for 10 min, and the supernatant was transferred to a conditioned SPE cartridge for clarification. Peptides were then analyzed using mass spectrometry.

[0324] Results and Discussion Total yield The total protein yield for the buffy coat extraction decreased with increasing washes, which is to be expected since each wash removes more protein. Streck tubes and WB averaged approximately 30-35 μg total protein. The total protein yields for the samples are shown in Figure 71.

[0325] Number of protein and peptide IDs Only the number of protein IDs decreased slightly with each wash and would not be considered significant. Frozen samples also yielded similar numbers of IDs as we had previously observed for fresh samples, demonstrating their successful practicality for processing and analysis using these methods. The number of protein IDs and peptide IDs was measured, as shown in Figure 72.

[0326] The results show that the majority of proteins found in the urea / thiourea wash overlap with those in the LiCl wash for both buffy coat and whole blood chips. A greater number of unique proteins appears to arise from the TBP-containing wash. Figure 73 illustrates the overlap of protein IDs for buffy coat (BC) chips washed with LiCl; urea / thiourea (UT); urea / thiourea / SDC; and urea / thiourea / SDC / TBP. Figures 74 and 75 show proteins expressed exclusively in TBP and LiCl, respectively.

[0327] Comparison of protein IDs for the three RBC fractions (RBC, RBC pellet, and RBC supernatant) revealed that the RBC pellet yielded the most distinct IDs. Figure 76 shows the overlap of protein IDs for RBC, RBC pellet, and supernatant.

[0328] Three DIA files for the frozen RBC fractions were searched using DIA-NN. Overall, 3588 proteins were found in the DIA-NN matrix. When comparing RBC frozen pellet proteins to RBC frozen sample proteins, 637 were downregulated (log2 fold change < -1.5 p-value 0.05) and 343 were upregulated (log2 fold change > 1.5 p-value 0.05). A list of protein classes (PantherDB) for the upregulated and downregulated groups is shown below.

[0329] [Table 16-18] Proteins found exclusively in the RBC pellet appeared to be cell-cell junction proteins, extracellular matrix proteins, and numerous metabolite interconverting enzymes. Figures 77 and 78 show proteins found exclusively in the RBC pellet and RBC supernatant, respectively.

[0330] Figure 79 is a Venn diagram showing the overlap of proteins for PBMCs and whole blood. Figures 80 and 81 show the top abundant proteins in whole blood and PBMCs, respectively. The top eight abundant proteins in whole blood are present at very low levels in PBMCs. Note the reverse: similar levels of the top proteins in PBMCs are found in whole blood. Hemoglobin (HBB) is the 20th most abundant peptide in PBMCs, but its abundance is a fraction of that of whole blood (third most abundant).

[0331] Conclusions: Washing with urea / thiourea alone and with SDC produced a profile similar to that of the LiCl wash, but with greatly reduced yields. Frozen blood yields were reduced (approximately half that of fresh). The most unique protein was produced from the PBS pellet.

[0332] Example X: Evaluation of buffer changes for WB and analysis of PBMCs digested in VAMS chips and in solution Objective: To highlight different initial wash mechanisms for chips submerged in whole blood. In previous experiments, we tested various wash steps using urea / thiourea with and without detergent and reducing agent on chips submerged in buffy coat. In this experiment, we further expanded using wash steps with whole blood and urea / thiourea / detergent (SDC) / reducing agent (TBP), and to include an alkylating agent (CLA). We also noted differences in the analysis of PBMCs in the chip or simply in solution.

[0333] method: PBMC preparation. PBMC chips were prepared using cell preparation tubes containing Vacutainer CPT sodium heparin as per the manufacturer's instructions. Extracted PBMCs were diluted 1:3 to reach a cell count of 7 x 10^3 cells / μL and then frozen at -80°C. Frozen PBMCs were then thawed. A 30 μL aliquot was taken for in-solution digestion, and another aliquot was added to a VAMS 30 μL tip and allowed to dry overnight.

[0334] Protocol used for overnight digestion. Once dry, chips were washed overnight with LiCl (1 mL), followed by either (a) 2× LiCl washes (with centrifugation pulses at 10,000 g) or (b) 2× 30 min washes with either (b1) 1 mL LiCl; (b2) 1 mL 7 M urea, 2 M thiourea, 1% sodium deoxycholate (SDC), tributylphosphine (TBP), 100 mM Tris; or (b3) 1 mL 7 M urea, 2 M thiourea, 1% sodium deoxycholate (SDC), tributylphosphine (TBP), choloractamide (CLA) in 100 mM Tris. At the beginning of each urea wash incubation, 3× centrifugation pulses at 10,000 g were performed. After the urea / thiourea wash, chips were washed in 1 mL water. The PBMCs in solution were not washed and digestion continued as per the remainder of the method. 100 μL lysis buffer (1% SDC, 10 mM TCEP, 40 mM CLA, 100 mM Tris, pH 8) was added to all samples. The chips were heated at 95°C for 10 minutes with gentle agitation. 1 μg (2 μL) of trypsin (0.5 μg / μL in 100 mM Tris) was then added, and the solution was incubated overnight at 37°C (19-21 hours). After incubation, the chips were removed, and the remaining digest was diluted with 800 μL of 0.5% formic acid. The solution was then centrifuged at 12,000 g for 10 minutes, and the supernatant was transferred to a conditioned SPE cartridge for clarification. Peptides were then analyzed using mass spectrometry.

[0335] Results and Discussion Total yield Total protein yield for whole blood extraction decreased with increasing washes, although the alkylating agent CLA increased it slightly. When preparing samples during the wash steps, visually, the CLA-containing detergent continued to extract color out of the chip during each successive wash; this did not occur with the LiCl washes, but only for the first wash with TBP alone. Yields for washed PBMCs in the Mitra® chip were only slightly lower than PBMCs that were not in the chip. TEAB buffer produced a slightly lower yield than Tris, but not significantly. Total protein yields for the samples are shown in Figure 82.

[0336] TEAB and Tris buffers performed very similarly in terms of protein and peptide ID and trypsin digestion efficiency. Figure 83 shows the protein and peptide ID for substitutions of TEAB and Tris.

[0337] PBMCs in solution and PBMCs in VAMS A comparison was made between PBMCs in solution and those collected into VAMS. Overall, 7006 proteins were found in the DIA-NN matrix, with more IDs from cells in solution compared to Mitra® samples. 622 proteins were downregulated (log2 fold change < -1.5 p-value 0.05) and 136 were upregulated (log2 fold change > 1.5 p-value 0.05). A list of protein classes (PantherDB) for the upregulated and downregulated groups is shown below. Thus, the addition of cells to VAMS and the additional steps of processing through multiple washes and extractions allowed for the collection of new subsets of proteins that would otherwise be lost with standard methods. Figure 84 shows protein and peptide IDs from DIA-NN for PBMCs digested directly in solution or in-chip after sequential extraction.

[0338] [Table 17-19] [Table 18-20] Figure 85 shows a summary of down-regulated proteins from PBMCs in VAMS. Figure 86 shows a summary of up-regulated proteins from PBMCs in VAMS.

[0339] Chip cleaning step using a urea / thiourea combination A slight modification to the washing step with LiCl (centrifugation pulse and 2 x 30 min incubations) appeared to give a higher number of proteins and peptide IDs. Washing WB samples with the addition of the alkylating agent CLA (SB044) also gave an increase in peptides.

[0340] Six DIA files for different washing conditions were searched using DIA-NN. Overall, 5091 proteins were found in the DIA-NN matrix. The table below shows the number of up- and down-regulated proteins compared to the control (LiCl).

[0341] [Table 19-21] Figure 87 shows protein and peptide IDs from DIA-NN for samples subjected to different washing conditions. Figures 88 and 89 are Venn diagrams displaying the overlap of up- and down-regulated proteins in TBP and CLA, respectively.

[0342] Example XI: Evaluation of samples obtained from healthy and diseased subjects Objective: To analyze a series of frozen samples from cancer patients and matched healthy controls.

[0343] method Sample preparation. Frozen whole blood cell pellets were thawed and diluted 1:2 in PBS. 30 μL of the diluted sample was applied to a VAMS tip and allowed to dry for 24 hours.

[0344] Protocol used for overnight digestion. Once dry, the chips were washed overnight with LiCl (1 mL), followed by 2x LiCl washes with a centrifugation pulse (10,000 g). 100 μL lysis buffer (1% SDC, 10 mM TCEP, 40 mM CLA, 100 mM TEAB, pH 8) was added to all samples. The chips were heated at 95 °C for 10 min with gentle agitation. 1 μg (2 μL) of trypsin (0.5 μg / μL in 100 mM TEAB) was then added, and the solution was incubated overnight at 37 °C (19–21 h). After incubation, the chips were removed, and the remaining digest was diluted with 800 μL of 0.5% formic acid. The solution was then centrifuged at 12,000 g for 10 min, and the supernatant was transferred to a conditioned SPE cartridge for clarification. Peptides were then analyzed using mass spectrometry.

[0345] result Overall, slightly more quantitative values ​​were seen in cancer samples, especially in female samples. There was no strong clear correlation of protein quantification numbers with age or sample age. Figure 90 shows the protein quantification plots for the various samples.

[0346] Using standard criteria (FDR corrected p-value <0.05), a large number of differentially expressed proteins are seen. Figure 91 shows a volcano plot depicting differential expression.

[0347] It was also found that there was a subset of female cancer samples that were very well distinguished from the rest of the samples. 508 differentially expressed proteins were identified, and applying filters to the data revealed a set of 13 differentially expressed proteins that were highly predictive of disease status. Figure 92 shows a heatmap of the selected 13 differentially expressed proteins that clustered well by each participant group.

[0348] Example XII: Evaluation of intra- and inter-assay reproducibility of the method Objective: To assess the reproducibility of sample preparation, extraction, and digestion methods across replicate DBS samples using mass spectrometry (intra-chip reproducibility). Intra-assay reproducibility was assessed by analyzing a single sample in triplicate.

[0349] method Sample preparation A total of ten 30 μL VAMS chips were immersed in whole blood and allowed to dry for a minimum of 24 hours. The chips were prepared in two batches, one week apart.

[0350] Protocol used for overnight digestion Once dry, the chips were washed overnight with LiCl (1 mL), followed by 2x LiCl washes with a centrifugation pulse (10,000 g). 100 μL lysis buffer (1% SDC, 10 mM TCEP, 40 mM CLA, 100 mM TEAB, pH 8) was added to all samples. The chips were heated at 95 °C for 10 min with gentle agitation. 1 μg (2 μL) of trypsin (0.5 μg / μL in 100 mM TEAB) was then added, and the solution was incubated overnight at 37 °C (19–21 h). After incubation, the chips were removed, and the remaining digest was diluted with 800 μL of 0.5% formic acid. The solution was then centrifuged at 12,000 g for 10 min, and the supernatant was transferred to a conditioned SPE cartridge for clarification. Peptides were then analyzed using mass spectrometry.

[0351] result Total yield Overall, the reproducibility of yields between chips is very good. Figure 93 shows the protein yields for individual chip replicates, and Figure 94 shows the total yield between batches.

[0352] The yield results from the two batches are statistically significant (t-test analysis), indicating that the length of time for drying affects the recovered protein yield.

[0353] DIA-NN analysis All 15 DIA files (from both batches) were searched using DIA-NN. Overall, the DIA-NN matrix contained a total of 4481 proteins and 49634 peptides. These all showed very similar numbers of protein and peptide IDs, averaging 3206 protein IDs and 34784 peptide IDs. Duplicate sample injections of WB_Tip1 show a drop in peptide IDs in the third injection. This could be due to injection issues, as all injections were made from the same well. For the third injection to MS, the drop could be considered significant and potentially removed as an outlier. Figure 95 shows the total protein and peptide IDs from DIA-NN.

[0354] Digestive Effectiveness Digestion efficiency was consistent between batches, with an average deletion cleavage value of 29%. Figure 96 shows the average deletion cleavage for individual chip replicates.

[0355] Intra- and inter-chip variation Coefficients of variation (CV) were calculated from raw peptide and protein intensities (DIA-NN output). Within a single instrument, intra-chip CVs (n=5) were similar for both batch 1 and batch 2, quantifying 2962 and 3088 proteins with 20% CV, respectively. For a total set of 10 chips, a total of 2200 proteins and 8884 peptides were quantified with CVs less than 20% across the two mass spectrometers. The greatest variation arose from the sample preparation and mass spectrometer rather than the VAMS device. Figure 97 shows the number of proteins with %CVs less than 20% (bottom) and greater than 20% (top) from analytical replicates using the same chip (intra-chip MS1), chips from each batch (inter-chip batch 1 or 2), chips from batch 1 reanalyzed when batch 2 was analyzed (intra-chip MS2), all chips on each instrument (inter-instrument), and finally all chips together (inter-chip total).

[0356] The median intra- and inter-chip protein CVs are less than 20%, an industry standard. Comparing data between two mass spectrometers without normalization yielded a median %CV of less than 30%.

[0357] Figure 98 shows box plots of %CV from analytical replicates using the same chip (intra-chip MS1), chips from each batch (inter-chip batch 1 or 2), chips from batch 1 reanalyzed when batch 2 was analyzed (intra-chip MS2), all chips on each instrument (inter-instrument), and finally all chips together (inter-chip).

[0358] The violin plots show the distribution of %CV within each batch (intra-batch) and between the two batches (inter-batch). Figure 99 shows, from left to right, violin plots of %CV for each batch (B1 and B2) and for both batches together (intra-batch). The dotted line indicates 20%CV. The dots indicate the median %CV.

[0359] Using PCA analysis, the data clustered by mass spectrometer rather than by preparation batch or chip variation, further indicating that the instrument contributes most to data variability. Figure 100 shows a PCA plot of chip and batch reproducibility.

[0360] Comparison to the literature Comparing these %CVs with those in the literature revealed that our data are consistent with other methods (Strategies to enable large-scale proteomics for reproducible research, R. Poulos et. al. Nature Communications volume 11, Article number: 3793 (2020)).

[0361] conclusion Most of the variation observed during the runs occurred within the mass spectrometer itself (within chip) and between instruments, with only minor variations added when compared to sample preparation / VAMS (between chips). The overall median %CV for individual batches is due to one instrument. The %CV for the entire set across the two instruments is still less than 30% without normalization, which is acceptable.

[0362] Example XIII. Evaluation of Alternative Drying / Storage Methods Objective: To evaluate the effect of a drying step at room temperature on protein yield and extraction.

[0363] Methods: Sample preparation Six VAMS 30 μL tips were immersed in whole blood. Half of the tips were dried over desiccant at room temperature for one week, while the other half were left for five minutes, then transferred to Eppendorf tubes and frozen at -80°C for one week. After that time, the samples were removed from the freezer, allowed to reach room temperature, and then refrozen, a total of three times.

[0364] Protocol used for overnight digestion. After 1 week of drying and / or storage, dried and frozen chips were washed overnight with LiCl (1 mL), followed by 2x LiCl washes with a centrifugation pulse (10,000 g). 100 μL lysis buffer (1% SDC, 10 mM TCEP, 40 mM CLA, 100 mM TEAB, pH 8) was added to all samples. The chips were heated at 95°C for 10 minutes with gentle agitation. 1 μg (2 μL) of trypsin (0.5 μg / μL in 100 mM TEAB) was then added, and the solution was incubated overnight at 37°C (19–21 hours). After incubation, the chips were removed, and the remaining digest was diluted with 800 μL of 0.5% formic acid. The solution was then centrifuged at 12,000 g for 10 minutes, and the supernatant was transferred to a conditioned SPE cartridge for clarification. The peptides were then analyzed using mass spectrometry.

[0365] result Total yield Without wishing to be bound by theory, the total yield results support the hypothesis that non-dried samples do not form protein gels within the chip, and therefore more protein should be removed via the washing steps, resulting in a lower overall total yield. It was also visually apparent that more hemoglobin was washed away (via the loss of color in the chip after washing).

[0366] Figure 101 shows the total yield from DBS dried at room temperature for 1 week (dry) and from DBS that were prevented from drying by freezing and cycling through 3x freeze-thaw.

[0367] DIA-NN analysis Dried samples yielded an average of 3200 proteins compared to 4300 in frozen samples. Figure 102 shows the protein IDs from DIA-NN for dried and frozen DBS. Figure 103 is a Venn diagram showing the overlap of protein IDs from dried and frozen VAMS.

[0368] Analysis of the %CV reveals that the dried tips were more reproducible than the frozen tips. Figure 104 shows the %CV of replicates for dried and frozen DBS.

[0369] The top most abundant proteins were highly reduced in frozen samples compared to dried samples, especially in the case of hemoglobin α and β subunits.

[0370] In the analysis of the most commonly found blood proteins, frozen samples contained higher amounts in virtually all instances, except for albumin and transthyretin.

[0371] Figure 105 shows the most abundant proteins in the dried and frozen samples. Figure 106 shows the most abundant blood proteins in the dried and frozen samples.

[0372] Additionally, frozen samples had a reduced dynamic range compared to dried samples (64% reduction), which contributes to why we observed more protein overall in frozen samples.

[0373] Figure 107 shows the dynamic range, defined as the area difference between the highest and lowest peaks from both room temperature dried and frozen DBS.

[0374] Conclusion: The results of this analysis demonstrate that different preservation methods can result in very different protein profiles. Based on visual observation, freezing the chips without drying and performing freeze-thaw cycles washed out more hemoglobin. This is confirmed by down-regulated proteins, including hemoglobin, which resulted in a fold change of 2.9. Frozen samples also yielded higher numbers of proteins and peptide IDs, most likely due to more efficient removal of high-abundance proteins. However, removal of high-abundance proteins does not appear to be consistent with albumin and transthyretin, which are the most common high-abundance proteins in blood. For example, vitronectin was substantially higher in frozen samples.

[0375] Example XIV. Evaluation of Alternative Preservation Methods To evaluate the effect of different extraction methods on DBS stored at various temperatures. Samples stored at 37°C were included to evaluate whether elevated temperatures accelerated the effects observed with room temperature stored samples. The requirement for cellular integrity was also evaluated by lysing the cells with a sonic probe prior to addition, and the requirement for cellular presence was also evaluated, as observed by evaluating plasma in VAMS at various temperatures.

[0376] method Sample preparation Six VAMS 30 μL tips were first dipped in whole blood. Two of the tips were dried over desiccant at room temperature for five days, two more at 37°C for five days, and the final two tips were dried for five minutes, then transferred to Eppendorf tubes and frozen at -80°C for five days. After that time, the samples were removed from the freezer, allowed to reach room temperature, and then refrozen, a total of three times.

[0377] A portion of the liquid whole blood was also lysed using a sonic probe (3x on ice). This was then added to two 30 μL VAMS tips. The tips were transferred to Eppendorf tubes and frozen at -80°C for 5 days. After that time, the samples were removed from the freezer, allowed to reach room temperature, and then refrozen, a total of three times.

[0378] Finally, plasma was added to four VAMS 30 μL tips. Two of the tips were dried over desiccant at room temperature for five days, and the final two tips were dried for five minutes, then transferred to Eppendorf tubes and frozen at -80°C for five days. After that time, the samples were removed from the freezer, allowed to reach room temperature, and then refrozen, a total of three times.

[0379] Protocol used for overnight digestion After the drying and / or storage period, dried and frozen chips were washed overnight with either (a) 2× LiCl washes with LiCl (1 mL) followed by a centrifugation pulse (10,000 g) or (b) 1 mL DUTRA (1% SDC, 7 M urea, 2 M thiourea, TBP, CLA, 100 mM Tris). At the beginning of each urea wash incubation, 3× centrifugation pulses (10,000 g) were performed. DUTRA samples were washed with 1 mL water at the end of the wash. 100 μL lysis buffer (1% SDC, 10 mM TCEP, 40 mM CLA, 100 mM TEAB, pH 8) was added to all samples. The chips were heated at 95 °C for 10 min with gentle agitation. Next, 1 μg (2 μL) of trypsin (0.5 μg / μL in 100 mM TEAB) was added, and the solution was incubated overnight (19–21 h) at 37 °C. After incubation, the chip was removed, and the remaining digest was diluted with 800 μL of 0.5% formic acid. The solution was then centrifuged at 12,000 g for 10 min, and the supernatant was transferred to a conditioned SPE cartridge for clarification. Peptides were then analyzed using mass spectrometry.

[0380] result Total yield The total yield follows the same trend observed previously, with relatively low total yield for frozen samples. Following the same trend, increasing the temperature to 37°C also increased the total yield. Drying the whole blood sample in the chip increased the yield by 40% over freezing, and drying at 37°C increased the yield by 70% over room temperature.

[0381] Figure 108 shows the total protein yield for whole blood DBS after storage at room temperature (dry), 37°C (dry 37°C), freezing at -80°C (freezing) or thawing and then freezing at -80°C (thaw-freezing).

[0382] Drying plasma samples in chips at room temperature also increased yield compared to freezing. The average yield was 34% higher for room temperature samples. Figure 109 shows the total protein yield for plasma DBS after storage at room temperature (drying) or frozen at -80°C (frozen).

[0383] Comparison 1: Dried and frozen whole blood Both dried samples (room temperature and 37°C) showed good overlap, as did the two frozen sample types (frozen and thawed-frozen). Only 331 proteins were found to be unique to the dried samples and 267 unique to the frozen samples. Figure 110 is a Venn diagram showing the overlap of dried and frozen whole blood DBS extracted using DUTRA detergent.

[0384] Figure 111 shows proteins found exclusively in the dried sample. Figure 112 shows proteins found exclusively in the frozen sample.

[0385] [Table 20-22] [Table 21-23] Comparison 2: DUTRA and LiCl Cleaning There is some overlap between the two different detergents in both up- and down-regulated proteins. There is an overlap of 214 proteins that are down-regulated by LiCl and up-regulated by DUTRA. Figure 113 is a Venn diagram showing the overlap of up- and down-regulated proteins in LiCl and DUTRA extractions.

[0386] Compared to LiCl extraction, no such dramatic difference in dynamic range was observed between drying and freezing for DUTRA-washed samples, although drying does have a slightly lower dynamic range. This is the opposite of what we observed with LiCl-washed samples. Figure 114 shows the dynamic range, defined as the area difference between the highest and lowest peaks from both room-temperature dried and frozen DBS followed by DUTRA extraction.

[0387] Comparison 3: Whole blood dried at 37°C and room temperature and frozen whole blood Overall, 610 up-regulated proteins and 603 down-regulated proteins were found in the 37°C whole blood samples when compared directly with the room temperature samples. There was good overlap between the 37°C and room temperature samples. The frozen samples were the most different in both up- and down-regulated proteins.

[0388] Figure 115 shows the overlap of down- and up-regulated protein IDs from VAMS stored at room temperature (RT), 37°C (37) or frozen at -80°C (Fz).

[0389] Comparison 4: Lysed and Unlysed Whole Blood Whole blood that was lysed prior to application to the DBS chip had 212 fewer IDs. Overall, the lysed samples produced the lowest yields and relatively low protein and peptide counts.

[0390] Figure 116 shows the overlap of proteins identified in whole blood (unlysed), chips (unlysed frozen), and whole blood that was lysed prior to addition to the chips (lysed frozen). Both sets of chips were stored frozen at -80°C.

[0391] Comparison 5: Dried and frozen plasma Similar abundance of protein IDs was observed between dried and frozen plasma, with the majority of plasma proteins found in the whole blood sample, which had the most unique set, containing 656 unique protein IDs.

[0392] Figure 117 shows the overlap of protein IDs from whole blood and plasma VAMS that were dried at room temperature or otherwise immediately frozen.

[0393] In plasma VAMS dried at room temperature, 378 proteins were upregulated compared to the frozen equivalent. 368 proteins were also downregulated. The protein classes within each group were significantly different from each other. Figures 118 and 119 show the protein classes that were upregulated and downregulated, respectively, in plasma VAMS dried at room temperature or frozen at -80°C.

[0394] Conclusion: Comparison 1: Dried and frozen whole blood The frozen samples (thawed and unthawed) were most similar to each other, as were the dried samples. Storing samples at -80°C significantly reduced total protein yield and resulted in substantially different protein profiles compared to storage at room temperature or 37°C.

[0395] Comparison 2: DUTRA and LiCl Cleaning The DUTRA wash resulted in fewer inherent differences in proteins between fresh and frozen compared to the LiCl wash. Regarding fold-change comparisons using the DUTRA wash, as was found with the LiCl wash, an overall similar number of up- and down-regulated proteins was seen when comparing dried and frozen. The dynamic range did not change dramatically with the DUTRA wash compared to the LiCl wash, showing an opposite trend of a smaller dynamic range in dried chips compared to frozen chips.

[0396] Comparison 3: Whole blood dried at 37°C and room temperature The 37°C drying step significantly increased yield compared to room temperature chips. The 37°C drying step also uniquely resulted in a significantly higher number of up-regulated cytoskeletal proteins compared to the other samples. There was a significant overlap in up- and down-regulated proteins between the 37°C and room temperature drying steps compared to freezing.

[0397] Comparison 4: Lysed and Unlysed Whole Blood Similar to the 37°C samples, numerous cytoskeletal proteins were upregulated in the lysed samples compared with the frozen samples. Downregulated proteins were primarily RNA metabolism and scaffold proteins.

[0398] Comparison 5: Dried and frozen plasma The majority of plasma proteins were also identified in whole blood. No significant number of unique proteins were identified in the comparison between dried and frozen plasma (compared to whole blood).

[0399] Example XV. Evaluation of Various Extraction Solutions, Including DNA Extraction Objectives: To evaluate the effect of various extraction solutions on overall protein yield and ID, and to demonstrate an example of sequential extraction in which the first extract is processed for DNA analysis and the subsequent extract is processed for protein MS analysis. Finally, to present an example of a preloaded VAMS tip to achieve protein extraction.

[0400] method Sample preparation Eleven VAMS 30 μL tips were immersed in whole blood. One portion was dried over desiccant at room temperature overnight. One tip was dried over desiccant at 37°C overnight. One tip was incubated at 99°C (lid open) for 10 minutes, then placed back into the spindle and dried over desiccant at room temperature overnight.

[0401] To preload the chip, one chip was immersed in a solution of benzonase (1 U / μL). The chip was dried for 10 minutes and stored in the refrigerator overnight. Next, whole blood was added and dried over a desiccant at room temperature overnight. Whole blood that had been treated with benzonase (1 U / μL) for 10 minutes prior to application to the chip was added to another chip. Fingertip whole blood was added to one chip and dried over a desiccant at room temperature overnight. Finally, plasma that had been double-centrifuged to ensure relatively high purity was added to one chip, followed by drying over a desiccant at room temperature overnight.

[0402] DNA extraction DNA was extracted from one of the whole blood chips according to the following method: A 30 μL VAMS chip containing whole blood was dispensed into a 1.5 mL microcentrifuge tube. 200 μL PBS was added to the chip and vortexed for 10 seconds. 200 μL Proteinase K and 300 μL Lysis Buffer B were then added and vortexed for 10 seconds. The solution was then incubated at 56°C for 20 minutes. 250 μL ethanol was added and vortexed to mix, after which the DBS chip (tup) was collected and processed for mass spectrometry analysis as described below. The remaining solution was processed according to the manufacturer's instructions for DNA preparation (QIAamp Blood Kit).

[0403] Protocol used for overnight digestion Once dry, the chips were washed using one of the following methods: (a) 500 mM LiCl (1 mL), 100 mM Tris overnight wash, followed by 2× LiCl washes using a centrifugation pulse (10,000 g) (b) An overnight wash in 2% CHAPS, 500 mM LiCl, 100 mM Tris overnight, followed by 2× washes with the same buffer with a centrifugation pulse (10,000 g). At the end of the wash, the chip was then washed three times in 1 mL of water. (c) Overnight wash in 10 mM citric acid, 500 mM LiCl overnight, followed by 2× washes with the same buffer with a centrifugation pulse (10,000 g). At the end of the wash, the chip was then washed three times in 1 mL of water. (d) 10 min with 25% trifluoroacetic acid (TFA), followed by overnight in 100 mM Tris, followed by 2× washes in 100 mM Tris with a centrifugation pulse (10,000 g ). (e) Washing overnight in methanol (MeOH) 40% v / v, followed by 2× washing with the same buffer with a centrifugation pulse (10,000 g ). (f) Add 1U benzonase in 100µL to the chip for 10 minutes, then bring to 1mL with 500mM LiCl, 100mM Tris overnight, followed by a 2x LiCl wash (using a centrifugation pulse at 10,000g). (g) Wash with 500 mM LiCl (1 mL), 100 mM Tris overnight, followed by 2× LiCl washes with sonication (3× sonic probe, each wash).

[0404] After this time, all chips were processed identically. 100 μL lysis buffer (1% SDC, 10 mM TCEP, 40 mM CLA, 100 mM TEAB, pH 8) was added to all samples. The chips were heated at 95°C for 10 minutes with gentle agitation. Next, 1 μg (2 μL) of trypsin (0.5 μg / μL in 100 mM TEAB) was added, and the solution was incubated overnight at 37°C (19–21 hours). After incubation, the chips were removed, and the remaining digest was diluted with 800 μL of 0.5% formic acid. The solution was then centrifuged at 12,000 g for 10 minutes, and the supernatant was transferred to a conditioned SPE cartridge for clarification. Peptides were then analyzed using mass spectrometry.

[0405] result Total yield Total yields for the heating experiments followed the same trend as seen previously, with drying at 37°C increasing yields by 170% and 99°C increasing yields by a further 580%. For the various washing methods, the CHAPS and sonicated LiCl tips provided the lowest yields. The two acid-treated tips provided similar results in yield, closely followed by the MeOH and DNA extraction tips. Fingerstick blood samples provided higher yields than the venous control, most likely due to clotting factors. The three benzonase-treated tips did not alter yields compared to the control. Double-centrifuged plasma provided yields similar to those from the previous experiment. Figure 120 shows the total protein yields from all samples.

[0406] A portion of the chips were visually different after extraction. Specifically, the chips heated to 99°C turned dark black and remained this color even after washing. The two acid-treated chips turned a similar color and remained dark even after washing. The MeOH chips were somewhere in between, while the CHAPS and sonicated LiCl were nearly white again after washing, even more so than the control.

[0407] These color differences are reflected in the total protein yield, with colored tips resulting in higher protein yields. Furthermore, tips that remained dark after washing generally exhibited relatively low ID, and mass spectrometry traces showed the majority of high-abundance peaks. The mass spectra of CHAPS-containing samples looked good, and ID was good. CHAPS is typically avoided in mass spectrometry experiments due to mass spectrometry artifacts; however, its use with these methods produced high-quality results.

[0408] Figure 121 shows the number of protein IDs from various processing methods.

[0409] Comparison 1: Temperature comparison Protein overlap from drying steps of whole blood chips at room temperature, 37°C, or 99°C. The room temperature drying step yielded the most unique proteins. The 99°C drying step yielded half the IDs of the room temperature and 37°C chips, primarily due to the higher abundance of high-abundance proteins in samples dried at elevated temperatures. This further demonstrates that drying and storage temperatures can be used to generate distinct protein profiles. Figure 122 is a Venn diagram showing the overlap of protein IDs from VAMS dried and stored at various temperatures: room temperature (control), 37°C (37), or 99°C (100).

[0410] [Table 22-24] [Table 23-25] [Table 24-26] [Table 25-27] Comparison 2: Fingerstick and venous blood There was a large amount of overlap between finger-prick and venous blood samples. Venous blood gave slightly more IDs overall and therefore had slightly more uniquely identified proteins. Therefore, both samples can be successfully collected and processed according to these methods. Figure 123 is a Venn diagram showing the overlap in protein IDs from venous and finger-prick blood. Figure 124 shows the protein classes unique to venous blood samples. Figure 125 shows the protein classes unique to finger-prick blood samples.

[0411] Comparison 3: DNA extracts and whole blood Initial extraction from DBS extract samples yielded 0.5 ng / μL DNA, an amount that will likely increase with further method optimization. When subsequently processed for protein extraction, the DNA extract samples had fewer protein IDs compared to whole blood processed using standard methods, but still had IDs greater than 1500. This method demonstrates that both DNA and protein can be extracted sequentially from a single VAMS chip.

[0412] Figure 126 is a Venn diagram showing the overlap of protein IDs from samples processed using standard methods and samples extracted first for DNA and then for protein.

[0413] Comparison 4: Testing various wash buffers Both LiCl and CHAPS produced the most unique protein profiles, followed by MeOH. Figure 127 is a Venn diagram showing protein ID overlap after using various wash buffers for extraction. Figure 128 is an upset plot of protein ID overlap from various wash and extraction methods, including sonication and benzonase during extraction.

[0414] Each washing / extraction method produced a different protein profile, but the profile of the 99°C heating step had similarities to the acid and solvent treatments, while the 37°C heating step had similarities to the salt treatments. CHAPS was the only washing solvent that produced a completely different profile of its own when compared to the salt, acid, and organic solvents.

[0415] Figure 129 shows a PCA plot comparing each of the washing and drying methods for whole blood VAMS. Figure 130 shows a summary of the protein peak areas for the most abundant proteins (most abundant proteins at positions 1-20) using the various washing methods, and Figure 131 shows a summary of the protein peak areas for the most abundant proteins (most abundant proteins at positions 21-30) using the various washing methods.

[0416] Comparison 5: Use of Benzonase Addition of benzonase at either step did not significantly change the extraction profile. Overall, it seemed to work better when added at the wash step, but this difference was minor.

[0417] Preloading the chip with benzonase did not have a significant effect on the protein profile, but does provide evidence that these chips can be preloaded with molecules and not interfere with the subsequent withdrawal of blood into the chip.

[0418] Figure 132 is a Venn diagram showing the overlap in protein IDs for samples treated with benzonase at different stages of extraction compared to untreated controls.

[0419] conclusion Comparison 1: Temperature comparison The heating step to 99°C had a detrimental effect on protein ID because it made it more difficult to wash away high-abundance proteins such as hemoglobin. The drying step at 37°C did not make a significant difference in overall ID; however, high-abundance proteins such as hemoglobin were upregulated by the elevated temperature, indicating that this temperature suffers from some of the problems associated with the higher 99°C temperature.

[0420] Comparison 2: Fingerstick and venous blood Venous blood gave slightly more unique IDs than finger stick blood, but overall they were a fairly comparable set, demonstrating usable samples for the type of analysis.

[0421] Comparison 3: DNA extracts and whole blood DNA and proteins have been successfully extracted sequentially from a single VAMS chip, and further optimization is likely to achieve better yields of both.

[0422] Comparison 4: Testing various wash buffers Of the various wash solutions, LiCl and CHAPS yielded the most unique proteins, all of which differed somewhat from each other.

[0423] Comparison 5: Use of Benzonase The use of benzonase generally gave results similar to the LiCl control but appeared to slightly increase the number of protein IDs, however this was not significant. Preloading of benzonase in the chip demonstrated that it is possible to preload molecules onto the VAMS and that the VAMS can still successfully collect blood samples.

[0424] Example XVI. Evaluation of various sample types, including spiked blood samples Objective: To investigate the sensitivity of mass spectrometry in detecting mouse brain cancer cells in human whole blood. To investigate the dissolution performance of the chip. To investigate the performance of sonication for protein quantification.

[0425] method Sample preparation Murine brain cancer cells (MBCC) and yeast cells were sonicated (4x100 AMP, on ice). The sonicated MBCC was added to one 30 μL VAMS tip and stored at room temperature. Another 30 μL aliquot of the sonicated MBCC was frozen at -80°C. The sample was thawed prior to digestion. The sonicated yeast cells were added to one 30 μL VAMS tip and stored at room temperature. Another 30 μL aliquot of the sonicated yeast cells was frozen at -80°C. The sample was thawed prior to digestion. Finally, a series of seven dilutions of sonicated MBCC in whole blood were prepared according to Table 10. One 30 μL VAMS tip was prepared from each dilution and stored at room temperature.

[0426] [Table 26-28] Protocol used for overnight digestion Once dry, the chips were washed overnight with LiCl (1 mL), followed by 2x LiCl washes with a centrifugation pulse (10,000 g). All samples (chips and liquid samples) were then processed according to the following method: 100 μL lysis buffer (1% SDC, 10 mM TCEP, 40 mM CLA, 100 mM TEAB, pH 8) was added to all samples. The chips were heated at 95°C for 10 minutes with gentle agitation. 1 μg (2 μL) of trypsin (0.5 μg / μL in 100 mM TEAB) was then added, and the solution was incubated overnight at 37°C (19–21 hours). After incubation, the chips were removed, and the remaining digest was diluted with 800 μL of 0.5% formic acid. The solution was then centrifuged at 12,000 g for 10 minutes, and the supernatant was transferred to a conditioned SPE cartridge for clarification. The peptides were then analyzed using mass spectrometry.

[0427] result Total yield The total yield across the dilution series was consistent. MBCC and yeast cells had relatively high yields from the in-chip samples and were higher in the liquid samples. Figure 133 shows the total protein yield from all digested samples.

[0428] DIA-NN analysis on chip and liquid samples Nearly similar protein and peptide identifications (IDs) were found between the digests and the chip; however, slightly more protein and peptide IDs were found for both MBCC (419 more IDs) and yeast (94 more IDs).

[0429] Figure 134 shows protein IDs from liquid samples (digests) or in-chip processed samples (chips) for MBCC and yeast cells.

[0430] Analysis of overlap revealed that the protein IDs for each sample type (liquid and DBS) were approximately 10% unique to MBCC and approximately 5% unique to yeast cells. This provides evidence that the sample collection and digestion method can generate a set of unique IDs that would otherwise be lost with conventional methods. Figure 135 shows the overlap of protein IDs between liquid samples (digests) and in-chip samples for MBCC and yeast cells. Figures 136 and 137 show proteins found exclusively in MBCC liquid samples and MBCC in-chip samples, respectively. Figures 138 and 139 show proteins found exclusively in yeast liquid samples and yeast in-chip samples.

[0431] Serial dilutions of MBCC in whole blood Mouse proteins were identified in all MBCC dilutions. A dilution series trend is observed for both mouse and human proteins. This trend begins to plateau at relatively low dilutions (1-6 μL). Figure 140 shows highly abundant mouse proteins across a dilution series of MBCCs in whole blood. Figure 141 shows highly abundant human proteins across a dilution series of MBCCs in whole blood.

[0432] conclusion Total protein yield concentrations were higher in the liquid samples compared to the chip; however, DIA-NN analysis revealed slightly more protein IDs for both MBCC and yeast. In addition, more unique proteins were identified in the chip samples, with overall improved coverage across protein classes. MBCC spiked into whole blood and collected in the chip was detectable and capable of distinguishing between mouse and human proteins. The level of detection for MBCC in whole blood was determined to be between 6 and 12 μL spikes, corresponding to between 318 and 636 cells / μL or 15,891 and 31,781 cells / 30 μL VAMS.

[0433] Example XVII. Evaluation of Various Extraction Solutions, Wash Buffers, Digestion, Porous Materials, and Pretreatments Objectives: To evaluate the feasibility of extracting proteins from previously extracted chips for DNA or immunoassays. To focus on different types of washes for whole blood chips, including various combinations, and the use of alternative enzymes to trypsin (Asp-N and Glu-C). To test the feasibility of enriching for phosphorylated peptides for peptides extracted from whole blood. To test relatively low-volume extractions (10 μL), the use of alternative porous materials, and protein quantification in saliva. Finally, to test the effect of preloading the sample with different solutions.

[0434] method Sample preparation Whole blood was added to eight 30 μL VAMS tubes and one 10 μL VAMS tube and allowed to dry for at least one week. Saliva was added to one 30 μL VAMS tube and allowed to dry overnight using a desiccant. To test alternative porous materials, two small 2 mm x 2 mm square PVDF membranes were cut and wetted with methanol. 30 μL of blood was added to the top of each. One was placed in an Eppendorf tube and stored at -80°C overnight, and the other was allowed to dry overnight using a desiccant. Double-centrifuged plasma was added to two 30 μL VAMS tubes. One was placed in an Eppendorf tube and stored at -80°C overnight, and the other was allowed to dry overnight using a desiccant.

[0435] Two 30 μL VAMS tubes were preloaded with BSA by incubating the empty chips in 10% BSA for 2 hours, followed by washing in either 500 mM LiCl and 100 mM Tris (without a drying step). The chips were then dried overnight at room temperature using a desiccant. Plasma was then added to the chips, and one tube was dried overnight using a desiccant, while the other was placed in an Eppendorf tube and stored at -80°C overnight.

[0436] Two 30 μL VAMS tubes were preloaded with SDS by incubating the empty chips in 2% SDS for 2 hours and then dried overnight at room temperature using a desiccant. Plasma was then added to the chips, and one tube was dried overnight using a desiccant, while the other was placed in an Eppendorf tube and stored overnight at -80°C.

[0437] Protocol used for overnight digestion Once dry, the chips were washed using one of the following methods: (a) 500 mM LiCl (1 mL), 100 mM Tris overnight wash, followed by 2× LiCl washes using a centrifugation pulse (10,000 g) (b) Overnight PBS and protease inhibitor (PI) extraction for DNA extraction and immunoassay, followed by overnight with 500 mM LiCl (1 mL) and 100 mM Tris, followed by 2x LiCl washes (using a centrifugation pulse (10,000 g)). (c) Addition of 1 U benzonase in 100 μL and incubation for 10 minutes, followed by an overnight wash in 2% CHAPS, 500 mM LiCl, and 100 mM Tris, followed by 2× washes with the same buffer with sonication (3× sonic probe, each wash). At the end of the washes, the chip was washed 3 times in 1 mL of water. (d) Overnight wash with 10 mM citric acid, 500 mM LiCl, 10 mM TBP, 40 mM CLA, followed by 2× washes in the same buffer using a centrifugation pulse (10,000 g). At the end of the washes, the chip was washed three times in 1 mL of water. (e) Washing overnight with 500 mM LiCl (1 mL) and 100 mM Tris, followed by washing (2x) with 2 M urea and 2% CHAPS with a centrifugation pulse (10,000 g). At the end of the washes, the chip was washed three times in 1 mL of water. (f) Washing with 500 mM LiCl (1 mL) and 100 mM Tris overnight, followed by a 30 min wash with 500 mM LiCl, 100 mM dithiothreitol (DTT), and a 30 min wash with 500 mM LiCl, 100 mM Tris, and 40 mM chloroacetamide (CLA) with a subsequent centrifugation pulse (10,000 g ).

[0438] 100 μL lysis buffer (1% SDC, 10 mM TCEP, 40 mM CLA, 100 mM TEAB, pH 8) was added to all samples, except for samples to be digested with Asp-N or Glu-C, to which only 50 μL buffer was added. All chips were heated at 95 °C for 10 min with gentle agitation. Subsequently, 1 μg (2 μL) of trypsin (0.5 μg / μL in 100 mM TEAB) was added to all samples, except when an alternative protease was used. For these, chips were resuspended to a final volume of 500 μL in 100 mM TEAB and 1 μg of Asp-N or 1 μg of Glu-C. All chips were then incubated overnight (19–21 h) at 37 °C. After incubation, the chip was removed, and the remaining digest was diluted with 800 μL of 0.5% formic acid for the alternative protease, followed by the addition of 505 μL of 1.5% formic acid. The solution was then centrifuged at 12,000 g for 10 minutes, and the supernatant was transferred to a conditioned SPE cartridge for clarification. Peptides were then analyzed using mass spectrometry.

[0439] Phosphorylated peptide enrichment For enrichment of phosphorylated peptides, digested peptides from previous experiments were pooled (all processed using LiCl washes and trypsin digestion) for a total of 175 μg of digested peptides. Samples were enriched for phosphorylated peptides using Zr-IMAC-HP beads (35 μL) according to the manufacturer's instructions. Peptides were then analyzed using mass spectrometry.

[0440] Data analysis All whole blood samples were combined into one DIA-NN search, all plasma samples were combined into one search, and Glu-C, Asp-N, saliva, and phosphorylated samples were searched separately. The phosphorylated samples were also searched using MS Fragger and MaxQant.

[0441] result Total yield The yields of the PBS extract (for DNA and immunoassay analysis) are very similar to the LiCl control. The combined detergents containing citric acid and CHAPS were both lower than those observed in Example XV, indicating that more was washed away in the initial detergent / extract. The yield for the 10 μL tip was roughly one-third that of the 30 μL tip. The PVDF membrane had a higher yield, and it was not visually apparent that much more was washed away.

[0442] For BSA-preloaded chips, dried samples had twice the yield of frozen samples, but all were significantly lower than the control. SDS-preloaded chips had higher yields than the control plasma samples, indicating further potential studies on the chips.

[0443] Protein ID Samples with high protein yield typically identified relatively few proteins due to higher levels of highly abundant proteins. This specifically affected the citrate detergent (Figure 142) and PDVF samples (Figure 143). Unexpectedly high amounts of protein IDs were found in the phosphopeptide-enriched samples (Figure 142) and saliva samples (Figure 143). While the trypsin samples produced more protein IDs, both Asp-N and Glu-C produced significant numbers of IDs (3100 for trypsin and 1500 for Asp-N and Glu-C). Combinations of proteases may still produce higher total ID numbers.

[0444] Principal component analysis (PCA) revealed that the standard LiCl method, PBS extraction for sequential analysis DNA / immunoassay prior to mass spectrometry, and 10 μL VAMS all clustered with similar profiles. However, the results for the PVDF membrane and the urea-CHAPS and citric acid detergents were different. This further supports the applicability of these methods to other porous materials and detergent solutions and that they can be used to generate specific desired profiles.

[0445] Comparison 1: Sequential extraction for DNA / immunoassay analysis followed by mass spectrometry There was significant overlap in protein IDs between the control and PBS extracts (DNA and immunoassay extracts), demonstrating that sequential extractions can be performed to maximize the number and type of analysis from a single sample without compromising sample quality for mass spectrometry (Figure 145).

[0446] Comparison 2: Use of different wash buffers and wash buffer combinations Each of the different wash buffers yielded a slightly different profile. The citrate wash had a high amount of hemoglobin and albumin and yielded reduced protein IDs. The 2M urea and 2% CHAPS washes enriched for a unique set of proteins with a high proportion of defense / immunity proteins and metabolite interconverting enzymes (Figures 146 and 147). Steps using DDT as a reducing agent also yielded metabolite interconverting proteins, but also protein-modifying enzymes, cytoskeletal proteins, and membrane transport proteins (Figures 146 and 148). The combination washes alternatively yielded primarily translation proteins, chromatin / chromatin-binding or regulatory proteins, and protein-modifying enzymes (Figures 146 and 149).

[0447] Combination 3: Use of different proteases Although there was minimal peptide overlap with different proteases, which is to be expected, substantial protein overlap was observed. Despite the overlap, Glu-C and Asp-N still identified 350 proteins that were not seen with trypsin (Figure 150).

[0448] Comparison 4: 10 μL VAMS and 30 μL VAMS The 10 μL VAMS and 30 μL had substantial protein overlap, as expected. The 10 μL VAMS had lower levels of hemoglobin than the 30 μL VAMS, which could be due to the larger ratio of wash buffer volume to VAMS maximum volume (1:100 and 1:33 for the 10 μL and 30 μL VAMS, respectively), since both tips were washed in a 1 mL volume. Figure 151 shows the overlap of proteins extracted from the 30 μL VAMS and 10 μL VAMS using the same protocol.

[0449] Comparison 5: Use of alternative porous materials Whole blood in VAMS yielded 10 more protein IDs compared to the PVDF membrane. This is hypothesized to be the result of high-abundance proteins not being fully removed. Nevertheless, the use of alternative porous materials still successfully extracted proteins and produced altered profiles relative to conventional VAMS. There was only a slight difference between drying and freezing the PVDF samples prior to extraction. Figure 152 shows the relative abundance of the top high-abundance proteins for whole blood samples collected either in 30 μL VAMS (Mitra®) or on a PVDF membrane.

[0450] Comparison 6: Chip Preload Preloading with BSA and SDS slowed the absorption of plasma into VAMS and reduced the overall protein ID count by approximately 40%. Overall, freezing the samples had little to no effect compared to drying the samples.

[0451] SDS-preloaded chips reduced many high-abundance proteins (i.e., vitronectin and apolipoproteins), but increased the intensity for fibrinogen (Figure 153). Eighty-nine significantly up-regulated proteins were found on BSA-preloaded chips compared to controls, with higher abundance of metabolite interconverters and cytoskeletal proteins. The 136 significantly down-regulated proteins consisted of protein-modifying enzymes and scaffold / adapter proteins. Seventy-seven significantly up-regulated proteins (mainly protein binding activity regulators) and 322 significantly down-regulated proteins (protective / immune proteins and protein-modifying enzymes) were found for SDS-preloaded samples compared to controls.

[0452] Comparison 7: Phosphorylation enrichment of dried blood spot samples The enriched phosphopeptide fraction was subjected to both DDA and DIA modes on a 90-minute gradient. DDA was analyzed using Max Quant and Fragpipe. Fragpipe results were also used to generate a spectral reference library for DIA analysis using DIANN.

[0453] In total, 4887 phosphopeptides were identified across all three platforms, with 1883 common to all three. Across each of the platforms, phosphopeptides accounted for approximately 50% of the total spectra.

[0454] Figure 154 shows the overlap of phosphopeptides identified across three analytical platforms: Fraggler, DIANN, and Max Quant.

[0455] Overall, successful enrichment and analysis of phosphorylated peptides from these samples was observed. Although there are no published phosphorylation studies on dried blood spots, these results demonstrate higher levels of detection than those reported for liquid blood samples.

[0456] Comparison 8: Saliva in VAMS Proteins were successfully detected from saliva collected in VAMS. DIANN quantified 3415 proteins from saliva. A prior literature search revealed an average of 2000 proteins detected from liquid saliva, so our method yields a much larger number. There is substantial overlap in protein IDs between whole blood, plasma, and saliva (Figure 155).

[0457] conclusion Comparison 1: Sequential extraction for DNA / immunoassay analysis followed by mass spectrometry The initial PBS extraction allows for analysis of DNA and immunoassays prior to standard mass spectrometry extraction and digestion. Mass spectrometry results from each protocol were very similar, indicating no adverse effects. This provides evidence that multiple extractions can be performed from a single sample.

[0458] Comparison 2: Use of different wash buffers and wash buffer combinations All of the wash buffers produced distinct profiles, as previously observed. The use of 2M urea and CHAPS in combination and the combination of benzonase, LiCl, CHAPS and sonication produced unique protein profiles.

[0459] Combination 3: Use of different proteases Both tested alternative proteases (Asp-N and Glu-C) successfully digested unique peptides and increased the total number of unique proteins identified when combined with the trypsin results.

[0460] Comparison 4: 10 μL VAMS and 30 μL VAMS Extraction from 10 μL VAMS was as successful as 30 μL VAMS.

[0461] Comparison 5: Use of alternative porous materials The PVDF membrane produced a different profile compared to that typically seen with VAMS, however, hemoglobin was still present in the sample, resulting in an overall lower protein ID.

[0462] Comparison 6: Chip Preload Chips preloaded with BSA and SDS yielded fewer protein IDs compared to control plasma, with more down-regulated proteins overall. A different profile was observed for preloaded samples, especially those preloaded with SDS.

[0463] Comparison 7: Phosphorylation enrichment of dried blood spot samples The phosphorylation enrichment was successful and a good number of phosphorylated peptide IDs were found.

[0464] Comparison 8: Saliva in VAMS This achieved excellent results compared to the literature on liquid samples. DIA quantified 3415 proteins from saliva samples.

[0465] Example XVIII. Evaluation of Various Extraction Solutions, Wash Buffers, Digestion, and Porous Materials Objectives: To evaluate the feasibility of extracting multiple samples from the chip for sequential mass spectrometry analysis, to look at different types of washes for the whole blood chip, including different salts, using a range of concentrations and the use of an alternative enzyme to trypsin (Glu-C). Finally, to test the use of alternative porous materials.

[0466] method Sample preparation Ten VAMS samples (30 μL) were loaded with whole blood; nine were dried overnight with desiccant, and the others were placed in Eppendorf tubes and stored overnight at −80° C. To test alternative porous materials, 30 μL of blood was loaded onto two pieces of filter paper; one was placed in an Eppendorf tube and stored overnight at −80° C., and the other was dried overnight with desiccant.

[0467] Protocol used for overnight digestion (a) Once dry, the chips were washed using one of the following methods: (b) Wash overnight with 250 mM LiCl + 100 mM Tris (1 mL), followed by 2× LiCl washes using a centrifugation pulse (10,000 g). (c) Wash overnight with 500 mM LiCl + 100 mM Tris (1 mL), followed by 2 × LiCl washes using a centrifugation pulse (10,000 g). (d) Wash overnight with 1 M LiCl + 100 mM Tris (1 mL), followed by 2 × LiCl washes using a centrifugation pulse (10,000 g). (e) Wash overnight with 500 mM CaCl2 + 100 mM Tris (1 mL), followed by 2x LiCl washes using a centrifugation pulse (10,000 g). (f) An overnight wash with 500 mM LiCl + 100 mM Tris (1 mL) was followed by a wash with 500 mM LiCl + 100 mM Tris (1 mL) with a centrifugation pulse (10,000 g). This extract was collected for immunoassay analysis (Detergent A). This was followed by a final wash with 7 M urea, 2 M thiourea, 1% sodium deoxycholate (SDC) + tributylphosphine (TBP) + choloractamide (CLA) + 100 mM Tris (1 mL) with a centrifugation pulse (10,000 g). This fraction was collected for digestion and mass spectrometry analysis (Detergent B). Detergents A and B were acetone precipitated with 10 mL of acetone for 2 hours. The pellets were resuspended in lysis buffer (1 mL for Detergent A and 100 μL for Detergent B), and 100 μL was taken from each for trypsin digestion as described below. The remaining chip was washed three times in 1 mL of water at the end of the third wash.

[0468] All samples received 100 μL of lysis buffer (1% SDC, 10 mM TCEP, 40 mM CLA, 100 mM TEAB, pH 8) except for the filter paper sample, which received 300 μL of lysis buffer. All samples were heated at 95°C for 10 minutes with gentle agitation. Subsequently, 1 μg (2 μL) of trypsin (0.5 μg / μL in lysis buffer) was added to all samples. After incubation, the samples were removed, and the remaining digest was diluted with 800 μL of 0.5% formic acid. The chips to be further digested were removed from the solution and diluted with 700 μL of 0.5% formic acid. An additional 100 μL of 100 mM TEAB was then added to the chip, followed by 1 μg of trypsin or 1 μg of Glu-C. These were incubated for an additional 2 hours, after which the digested material was pooled with the original 800 μL of collected digest. All samples were centrifuged at 12,000 g for 10 min and the supernatant was transferred to a conditioned SPE cartridge for clarification. Peptides were subsequently analyzed using mass spectrometry.

[0469] Data analysis All whole blood samples were combined into one DIA-NN search, except for the Glu-C sample, which was searched individually.

[0470] result Total yield and protein ID The CaCl2 washed samples had much higher yields than either of the other conditions, indicating that a number of relatively high-abundance proteins (i.e., hemoglobin) were not effectively removed. A minimum of 3000 IDs was found in each of the samples, except for the CaCl2 and sequentially extracted samples, which had only about 2250 IDs (Figure 156). The frozen filter papers had slightly higher IDs compared to the dried samples, the same trend observed from the frozen and dried VAMS. The filter papers had about 500 fewer IDs overall than the VAMS, but still produced good counts.

[0471] Comparison 1: Sequential extraction for multiple mass spectrometry analyses Each subsequent extraction yielded more IDs than the preceding extraction, resulting in a total of 3665 unique proteins identified (Figure 156). Of the unique proteins identified, 30% were found in all samples, and 96.5% were found in the final extraction (Figure 157). Although the first two contributed very few unique proteins, 36% and 50% of the total were also identified in the first and second extractions, respectively. This demonstrates that sequential extractions can be performed and do not have a significant effect on the final on-chip digestion and analysis. These results also highlight the value of a chip wash step before performing digestion, as simple liquid extractions are largely ineffective in maximizing protein IDs.

[0472] Comparison 2: LiCl concentration Washing steps using 250 mM, 500 mM, or 1 M LiCl yielded distinct protein profiles (Figure 158). The wash step using 250 mM LiCl yielded the highest number of unique proteins as well as the highest total number of IDs, while the 1 M comparison yielded the lowest number of both. Proteins unique to 250 mM LiCl were primarily metabolite interconverting enzymes and RNA metabolism proteins.

[0473] Comparison 3: Using CaCl2 instead of LiCl Compared to CaCl, more proteins were identified in the sample washed with LiCl, with 20% more IDs (Figure 159). This may be due to insufficient washing and high abundance proteins remaining; analysis revealed increased amounts of hemoglobin (subunits δ, β, α) compared to the LiCl sample.

[0474] A wash step with CaCl2 resulted in the identification of 44 unique proteins, which consisted of protein-modifying enzymes, chromatin / chromatin-binding or regulatory proteins, and defense / immunity proteins.

[0475] Comparison 4: Use of alternative porous materials The same trend of higher protein IDs from samples stored in the freezer compared to dried samples was observed for both the filter paper samples and the VAMS. Overall, many similarities were observed between the use of filter paper and VAMS, with approximately 84% overlap between sample types, indicating that these methods are compatible with both. Overall, more unique IDs were identified from the VAMS (Figure 160).

[0476] Compared with filter paper, proteins unique to VAMS consisted mostly of membrane interconverting enzymes, structural proteins, and translation proteins, whereas proteins unique to filter paper consisted of metabolite interconverting enzymes and intracellular signaling molecules.

[0477] Based on PCA, there is less variation between filter paper and VAMS than between dried and frozen samples immediately after collection (Figure 161). Similarly, heatmap hierarchical clustering clusters frozen and dried samples together, in contrast to VAMS and filter paper.

[0478] Comparison 5: Use of different proteases The data were searched using DIA-NN in two ways: first using Glu-C and trypsin to define cleavages at K, R, D, E, and second using trypsin alone. The trypsin-only search yielded many more peptides and proteins overall, while the Glu-C search yielded unique IDs for the Glu-C samples. Results from both searches are shown in Figure 162. Inclusion of Glu-C resulted in many more peptide IDs overall, but no significant protein IDs.

[0479] Proteins unique to Glu-C digested samples include protein-modifying ...

Claims

1. 1. A method for fractionating a sample, comprising: (a) introducing a sample into a porous material; (b) optionally centrifuging the porous material containing the sample; (c) drying the sample in the porous material; (d) extracting the first set of molecules from the dried sample-containing porous material, comprising: (1) incubating the dried sample-containing porous material in a first extraction solution; (2) separating the first extraction solution from the porous material; and (3) optionally washing the porous material; (e) digesting the proteins remaining in the porous material, comprising: (1) incubating the porous material in a digestion solution; and (2) separating the digestion solution from the porous material; and (f) detecting one or more molecules in the separated first extract solution; and (g) detecting one or more peptides or proteins in the digest solution A method comprising:

2. Following extraction of the first set of molecules and prior to the digestion step, (A) extracting a second set of molecules from the first extracted porous material, the second set of molecules comprising: (1) incubating the first extracted porous material in a second extraction solution; (2) separating the second extraction solution from the second incubated porous material; and (h) optionally washing the separated second extracted porous material; and (B) optionally detecting a second set of molecules in the separated second extraction solution; The method of claim 1 further comprising:

3. 3. The method of claim 2, further comprising sequentially extracting one or more additional sets of molecules from the second extracted porous material, comprising repeating steps (A)-(B) with one or more additional extraction solutions prior to the digestion step.

4. The method of claim 3, wherein the first set of molecules, the second set of molecules, or one or more further sets of molecules are selected from the group consisting of proteins, nucleic acids, and glycans.

5. 5. The method of claim 4, wherein each of the proteins in the first, second, or one or more further sets of molecules and / or one or more peptides or proteins in the digestion solution is a phosphoprotein or phosphopeptide.

6. 6. The method of claim 5, comprising a step of enriching for phosphorylated proteins and / or peptides in the separated first, second, or one or more extraction and / or digestion solutions.

7. The extraction solution comprises an extractant comprising a salt, a weak detergent, a strong detergent, a chaotropic agent, a reducing agent, a thiol-containing reducing agent, or an alkylating agent, an acid, an organic solvent, or an enzyme, or a combination thereof; and / or the digestion solution comprises a reducing agent, an alkylating agent, a buffering agent, a detergent, or a combination thereof, and optionally the digestion solution further comprises a salt, a weak detergent, a strong detergent, a chaotropic agent, or a thiol-containing reducing agent, an acid, an organic solvent, or an enzyme, or a combination thereof; The method of claim 1.

8. Salts include NaCl, LiCl, and CaCl 2 or Tris-HCl; The weak detergent is a non-ionic detergent, an ionic detergent, or a zwitterionic detergent, and optionally is or comprises: (i) a non-ionic detergent including PBS or Tween; (ii) an ionic detergent including sodium deoxycholate; or (iii) a zwitterionic detergent including sulfobetaine or amidosulfobetaine; the strong detergent is or comprises CTAB, CHAPS, or SDS; the chaotropic agent is or comprises urea, thiourea, or guanidine; the reducing agent is or comprises a phosphine, optionally the phosphine is or comprises TBP or TCEP; the thiol-containing reducing agent is or comprises β-mercaptoethanol or dithiothreitol; the alkylating agent is or comprises chloroacetamide, iodoacetamide, or acrylamide; the acid is or comprises citric acid or trifluoroacetic acid; the organic solvent is or comprises methanol; and / or the organic enzyme is or comprises benzonase; The method of claim 7.

9. the first, second, one or more further extraction solutions, and digestion solution are different; The selection of the first, second, one or more additional extraction solutions is for fractionating proteins contained in the porous material via differential solubility; Each successive extraction solution selected is characterized as having a relatively large solubility of the protein remaining in the porous material; or the first, second, third, and further extraction solutions contain increasing concentrations of the same extractant as one proceeds from one extraction to the next; The method of claim 3.

10. The concentration of salt in the extraction solution is 0.01 to 1 M, optionally the salt comprises LiCl and Tris-HCl, further optionally the concentration of LiCl is 0.5 M and the concentration of Tris-HCl is 0.1 M; The concentration of the chaotropic agent in the extraction solution is 0.01 to 8 M; and / or The extraction solution contains urea and thiourea at concentrations of 2, 5, 6, or 7 M; The method of claim 7.

11. 2. The method of claim 1, wherein the digestion solution comprises triethylammonium bicarbonate, sodium deoxycholate (SDC), TCEP, or chloroacetamide, or a combination thereof; optionally, triethylammonium bicarbonate is present in the digestion solution at a concentration of 0.1 M; sodium deoxycholate (SDC) is present in the digestion solution at a concentration of 0.05% to 10% (w / v); TCEP is present in the digestion solution at a concentration of 1 mM to 100 mM, and / or chloroacetamide is present in the digestion solution at a concentration of 5 mM to 100 mM; and / or the digestion solution comprises a protease or a combination of proteases, optionally wherein the protease is trypsin or the combination of proteases includes trypsin.

12. The method of claim 1, wherein the extraction solution contains an endoglycosidase, optionally wherein the endoglycosidase is peptide-N-glycosidase F (PNGase F) and / or O-glycosidase.

13. 2. The method of claim 1, wherein the sample is or comprises a body fluid sample, cells or tissue, cells suspended in a liquid, cultured cells suspended in a culture medium, blood, optionally a whole blood (WB) sample, a red blood cell (RBC) sample, a white blood cell (WBC) sample, a frozen blood sample, or a fresh blood sample, a blood fraction, plasma, urine, tears, wound fluid, CSF, bronchoalveolar lavage fluid, or ascites.

14. 10. The method of claim 1, wherein the sample has a volume within the range of 100 μL to 2 μL, 100 μL to 5 μL, 100 μL to 10 μL, 99 μL to 2 μL, 90 μL to 2 μL, 80 μL to 2 μL, 70 μL to 2 μL, 60 μL to 2 μL, 50 μL to 2 μL, 40 μL to 2 μL, 30 μL to 2 μL, 20 μL to 2 μL, 10 μL to 2 μL, or 5 μL to 2 μL.

15. The implementation steps are as follows: Absorption of the sample into a porous material; or Absorbing the sample into the porous material via a finger prick, dipping the porous material into the sample, or pipetting a known volume of sample into the porous material.

2. The method of claim 1, comprising:

16. drying the sample in the porous material, the drying step comprising drying for a period of time to adhere the sample to the porous material, and optionally the drying step comprising: comprising air-drying the sample within the porous material, wherein the drying step is for less than 30 minutes, preferably at least 5 minutes, or wherein the air-drying is for at least 1, 2, 4, 6, 8, 10, 12, 16, 20, or 24 hours, optionally wherein the drying step is in the presence of a desiccant; Centrifuging the sample within the porous material, preferably at a speed of 500g to 10,000g, more preferably for at least 1 to 15 minutes or up to 15 minutes; and / or drawing a vacuum on the sample within the porous material; The method of claim 1.

17. further comprising storing the sample-containing porous material for a period of time prior to the first extraction step, and optionally Storage of the sample-containing porous material is immediately after the drying step; The sample-containing porous material is frozen and stored; or The sample-containing porous material is stored at room temperature; The method of claim 1.

18. 10. The method of claim 1, wherein the drying, extraction, and digestion steps are performed using a sample-containing porous material in a test tube.

19. The method described in claim 1, wherein the extraction solution has a volume of less than 100 μL, a volume of less than 95 μL, a volume of less than 90 μL, a volume of 1 to 5 times the volume of the porous material, a volume of 1, 2, 3, 4 or 5 times the volume of the porous material, preferably a volume of 3 times the volume of the porous material.

20. The method of claim 1, wherein the incubation in the extraction solution is carried out for a time sufficient to extract one or more proteins contained within or adhered to the porous material, optionally for 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes, or for 1 to 48 hours, 1 to 42 hours, 1 to 36 hours, 1 to 30 hours, 1 to 24 hours, 1 to 18 hours, 1 to 12 hours, or 1 to 6 hours; wherein the incubation is carried out at ambient temperature, or wherein the incubation is carried out at a temperature elevated above ambient temperature.

21. 2. The method of claim 1, wherein the incubation in the digestion solution is carried out for a time long enough to digest one or more proteins contained within or adhered to the porous material, optionally the incubation is carried out for a period of between 1 minute and 48 hours, the incubation is carried out for 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes, the incubation is carried out for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours, and / or the incubation is carried out at ambient temperature, at an elevated temperature above ambient temperature, preferably at 37°C, and / or the incubation is carried out with stirring.

22. 2. The method of claim 1, further comprising washing the separated extracted porous material, wherein the separated extracted porous material is optionally washed with a washing volume of an extraction solution, a volume of an extraction solution different from the extraction solution, or a volume of water, respectively, wherein the selection of the different extraction solution is limited to a solution based on proteins remaining in the separated extracted porous material that have the same or lower solubility in the different extraction solution than the preceding extraction solution, and wherein the washing volume of the extraction solution is between 5 times the volume of the porous material and 3 mL, wherein the washing volume of the extraction solution is 0.05 to 3 mL, and / or the washing step is repeated once, twice, or three times.

23. The method of claim 22, wherein detecting one or more molecules in the separated first extraction solution comprises: (A) detecting proteins in the separated first extract solution, optionally including: Protein detection is via immunoassay, Western blot, or ELISA; the detection comprises Luminex and proximity extension assays, optionally the Luminex and proximity extension assays comprise slow off-rate modified aptamer (SOMAmer) reagents; and / or wherein detecting the proteins is via mass spectrometry (MS), optionally wherein the MS-mediated detection comprises subjecting the extract solution to digestion prior to said detection via mass spectrometry (MS); or (B) detecting glycans in the separated first extraction solution, optionally wherein the detection of glycans is via high performance liquid chromatography (HPLC); and / or detection of one or more peptides or proteins in the digest solution via mass spectrometry (MS); The method of claim 1.

24. The MS is LC-MS; The MS is selected reaction monitoring mass spectrometry (SRM-MS); The MS is a data-dependent acquisition MS (DDA-MS), the MS is a data-independent acquisition MS (DIA-MS), or The MS may be selected from the group consisting of matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) MS; MALDI-TOF post-source decay (PSD); MALDI-TOF / TOF; surface-enhanced laser desorption / ionization time-of-flight mass spectrometry (SELDI-TOF) MS; electrospray ionization mass spectrometry (ESI-MS); ESI-MS / MS; ESI-MS / (MS)n (n is an integer greater than zero); ESI selected from the group consisting of 3D or linear (2D) ion trap MS; ESI triple quadrupole MS; ESI quadrupole orthogonal time-of-flight (Q-TOF); ESI Fourier transform MS systems; desorption / ionization on silicon (DIOS); secondary ion mass spectrometry (SIMS); atmospheric pressure chemical ionization mass spectrometry (APCI-MS); APCI-MS; APCI-(MS)n; ion mobility spectrometry (IMS); inductively coupled plasma mass spectrometry (ICP-MS), atmospheric pressure photoionization mass spectrometry (APPI-MS), APPI-MS / MS; and APPI-(MS)n, 24. The method of claim 23.

25. 10. The method of claim 1, wherein the porous material is a hydrophilic polymeric material, optionally wherein the porous material comprises a plastic and / or wherein the porous material comprises a sponge or is an absorbent material tip of a volumetric absorption microsampling (VAMS) device, and optionally wherein the method uses multiple porous materials.

26. The porous material is (i) a protease inhibitor; (ii) an enzyme, preferably a nuclease, more preferably a benzonase; (iii) a detergent, preferably SDS; or (iv) an anticoagulant, preferably EDTA or heparin 26. The method of claim 25, wherein the

27. 2. The method of claim 1, wherein one or more proteins detected in the set of molecules or one or more proteins detected in the separated digest solution are or comprise non-membrane / soluble protein complexes.

28. 2. The method of claim 1, wherein one or more proteins detected in the set of molecules or one or more proteins detected in the separated digestion solution are or comprise plasma membrane-associated proteins, and optionally the plasma membrane-associated proteins are plasma membrane-bound proteins, plasma membrane-integral proteins or membrane protein complexes.

29. 2. The method of claim 1, wherein a fraction of non-membrane / soluble protein complexes not obtained by conventional microsampling or a fraction of membrane protein complexes not obtained by conventional microsampling analysis is recovered from a red blood cell (RBC) sample, a plasma sample, or a whole blood sample.

30. (i) the porous material is a hydrophilic polymer tip of a volumetric absorption microsampling (VAMS) device, the first extraction solution comprises 100 mM to 500 mM LiCl and 100 mM Tris, and the method comprises washing the porous material in step (d)(3) with a wash solution comprising 100 mM to 500 mM LiCl and 100 mM Tris, and the digestion solution comprises 1 μg trypsin in a buffer comprising 1% sodium deoxycholate (SDC), 10 mM tris(2-carboxyethyl)phosphine (TCEP), 40 mM 2-chloroacetamide (CLA), 100 mM, and 100 mM triethylammonium bicarbonate buffer (TEAB); (ii) the porous material is a hydrophilic polymer tip of a volumetric absorption microsampling (VAMS) device, the first extraction solution comprises a PBS solution containing a protease inhibitor, and the volume of the first extraction solution is 100 μL or less; the method comprises washing the porous material in step (d)(3) with a wash solution comprising 100 mM to 500 mM LiCl and 100 mM Tris; the digestion solution comprises 1 μg trypsin in a buffer comprising 1% sodium deoxycholate (SDC), 10 mM tris(2-carboxyethyl)phosphine (TCEP), 40 mM 2-chloroacetamide (CLA), 100 mM, and 100 mM triethylammonium bicarbonate buffer (TEAB); and the method comprises detecting one or more molecules in the separated first extraction solution, wherein the one or more molecules are one or more proteins, and the detection is via an immunoassay; or (iii) the porous material is a hydrophilic polymer tip of a volumetric absorption microsampling (VAMS) device, the first extraction solution comprises a PBS solution containing a protease inhibitor, and the volume of the first extraction solution is 100 μL or less; the method comprises washing the porous material in step (d)(3) with a wash solution comprising 100 mM to 500 mM LiCl and 100 mM Tris; the digestion solution comprises 1 μg trypsin in a buffer comprising 1% sodium deoxycholate (SDC), 10 mM tris(2-carboxyethyl)phosphine (TCEP), 40 mM 2-chloroacetamide (CLA), 100 mM, and 100 mM triethylammonium bicarbonate buffer (TEAB); and the method comprises detecting one or more molecules in the separated first extraction solution, the one or more molecules being one or more proteins, and the detection is via an immunoassay, following extraction of the first set of molecules and prior to the digestion step. (A) extracting a second set of molecules from the first extracted porous material, the second set of molecules comprising: (1) incubating the first extracted porous material in a second extraction solution; (2) separating the second extraction solution from the second incubated porous material; and (h) optionally washing the separated second extracted porous material; and (B) optionally detecting a second set of molecules in the separated second extraction solution; the second extraction solution comprises PNGase F in a buffer comprising 1% sodium deoxycholate (SDC), 10 mM tris(2-carboxyethyl)phosphine (TCEP), 40 mM 2-chloroacetamide (CLA), 100 mM and 100 mM triethylammonium bicarbonate buffer (TEAB); and the method comprises detecting the second set of molecules in the separated second extraction solution, wherein one or more molecules are one or more glycans. The method of claim 1.

31. 4. The method of claim 3, wherein the one or more additional extraction solutions comprise PNGase F in a buffer comprising 1% sodium deoxycholate (SDC), 10 mM tris(2-carboxyethyl)phosphine (TCEP), 40 mM 2-chloroacetamide (CLA), 100 mM, and 100 mM triethylammonium bicarbonate buffer (TEAB); and the method comprises detecting a further set of molecules in the separated additional extraction solution, wherein the one or more molecules are one or more glycans.

32. 32. The method of claim 31, further comprising dividing the porous material prior to digestion step (e) and analyzing a portion of the porous material for residual glycans.

33. 10. A method for generating a protein profile, comprising the method of claim 1, optionally wherein the protein profile is generated by an immunoassay workflow, a proteomics workflow, or a combination of an immunoassay workflow and a proteomics workflow.

34. (a) obtaining at least one protein profile generated in accordance with claim 1 from a sample obtained from a subject having a disease or disorder; (b) obtaining at least one protein profile generated in accordance with claim 1 from a sample obtained from at least one subject who does not have the disease or disorder; (c) comparing the protein profile of the subject with the disease or disorder to the protein profile of at least one subject without the disease or disorder; and (d) generating a disease protein profile from the comparison, the generated disease protein profile comprising one or more proteins that have a different presence or level in the protein profile from the subject with the disease or disorder compared to the protein profile of at least one subject without the disease or disorder. A method for generating a protein profile, comprising:

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