Mass spectrometry sample processing methods, chromatography devices, and data analysis techniques for biomarker analysis

Abstract

In certain aspects, the present disclosure is directed to methods, devices, and platforms including components thereof for processing samples for mass spectrometry.In other aspects, provided herein is an analysis platform for analyzing mass spectrometry data, including data obtained from mass spectrometry of samples obtained from the methods and devices described herein.In other aspects, provided is a determined proteomic signature of a condition in an individual, such as a coronary artery disease (CAD) proteomic signature.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 125,955, filed December 15, 2020, which is incorporated by reference in its entirety.

[0002] Technical Field In certain aspects, the present disclosure is directed to methods, devices, and platforms including components thereof for processing samples for mass spectrometry. In other aspects, provided herein are analytical platforms for analyzing mass spectrometry data, including data obtained from mass spectrometry of samples obtained from the methods and devices described herein. [Background technology]

[0003] background Mass spectrometry is a useful tool for analyzing samples that contain a range of different types of components, ranging from small molecules to nucleic acids to polypeptides. Samples, such as those of biological or environmental origin, can be highly complex and contain components at vastly different concentrations with different physical and chemical properties. For example, a typical sample is known to contain components with a dynamic range of over 10 orders of magnitude, composed of hydrophilic and hydrophobic peptides and proteins, primary and secondary metabolites, natural peptides, small molecule metabolites, and nucleic acids such as RNA and DNA, including microRNA, circular and long non-coding RNA, and mitochondrial RNA. Existing methods are not entirely satisfactory in unbiased capture of a wide range of proteins and other biomolecules in fluid samples. Improved methods are needed for the discovery of biomolecules from biological samples as biomarkers associated with biological phenomena such as disease. The embodiments provided address these needs. Summary of the Invention

[0004] overview In some aspects, provided herein are methods for processing a test sample, the methods comprising: (a) subjecting the test sample to a size exclusion chromatography (SEC) technique using a SEC microfluidic device, where the test sample comprises one or more biomolecules and a chaotropic agent, and where the SEC microfluidic device comprises a plurality of interconnected channels; (b) collecting one or more fractions eluted from the SEC microfluidic device; (c) subjecting one or more of the fractions from the SEC microfluidic device to a proteolytic technique; and (d) subjecting one or more of the fractions to a reversed-phase liquid chromatography (RPLC) technique to prepare fractions for introduction into a mass spectrometer, where the one or more RPLC fractions comprise: (i) zero or more fractions obtained from the SEC microfluidic device; and (ii) zero or more fractions subjected to the proteolytic technique.

[0005] In some aspects, provided herein are methods of processing a test sample for mass spectrometry analysis, the methods including: (a) subjecting the test sample to a size exclusion chromatography (SEC) technique using a SEC microfluidic device, the test sample comprising one or more biomolecules and a chaotropic agent, the SEC microfluidic device comprising a plurality of interconnected channels; (b) collecting a plurality of fractions eluted from the SEC microfluidic device; (c) subjecting one or more of the plurality of fractions from the SEC microfluidic device to a proteolytic technique; and (d) individually subjecting one or more fractions from one or both of steps (b) and (c) to a reversed-phase liquid chromatography (RPLC) technique using an RPLC microfluidic device under conditions to prepare respective components of the one or more fractions for introduction into a mass spectrometer, the RPLC microfluidic device comprising a plurality of interconnected channels comprising a reversed-phase medium, the RPLC microfluidic device being coupled to an electrospray ionization source.

[0006] In some embodiments, the test sample is a biological sample. In some embodiments, the test sample is from an individual. In some embodiments, the test sample has a chaotropic agent concentration of about 5M to about 8M. In some embodiments, the chaotropic agent comprises guanidine or a salt thereof, guanidinium or a salt thereof, potassium or a salt thereof, lithium or a salt thereof, magnesium or a salt thereof, or sodium or a salt thereof. In some embodiments, the chaotropic agent is guanidine hydrochloride or guanidinium chloride. In some embodiments, the chaotropic agent in the test sample is from a liquid fixative.

[0007] In some embodiments, the test sample has a concentration of viscosity modifier from about 5% to about 40%. In some embodiments, the viscosity modifier is glycerol. In some embodiments, the test sample comprises at least about 6M guanidine and about 10% to about 30% glycerol.

[0008] In some embodiments, the test sample subjected to the SEC technique using the SEC microfluidic device has a volume of about 1 μL to about 200 μL. In some embodiments, the range of concentrations of the mobile phase chaotropic agent of the SEC technique is within about ±40% of the predetermined concentration of the chaotropic agent of the test sample. In some embodiments, the SEC technique includes the use of a SEC mobile phase having a concentration of the mobile phase chaotropic agent within the range of the chaotropic agent in the test sample.

[0009] In some embodiments, the mobile phase chaotropic agent of the SEC technique is the same as the chaotropic agent of the test sample. In some embodiments, the mobile phase chaotropic agent of the SEC technique is different from the chaotropic agent of the test sample.

[0010] In some embodiments, the SEC mobile phase comprises a mobile phase chaotropic agent at a concentration of about 4 M to about 8 M. In some embodiments, the mobile phase chaotropic agent of the SEC technique comprises guanidine or a salt thereof, guanidinium or a salt thereof, lithium or a salt thereof, magnesium or a salt thereof, or sodium or a salt thereof. In some embodiments, the mobile phase chaotropic agent of the SEC technique is selected from the group consisting of guanidine hydrochloride, guanidinium chloride, guanidinium thiocyanate, lithium perchlorate, lithium acetate, magnesium chloride, potassium acetate, and sodium iodide.

[0011] In some embodiments, the SEC mobile phase comprises a mobile phase viscosity modifier. In some embodiments, the mobile phase viscosity modifier of the SEC technique has a concentration of about 5% to about 40%. In some embodiments, the viscosity modifier is glycerol. In some embodiments, the mobile phase viscosity modifier of the SEC technique is the same as the viscosity modifier of the liquid fixing agent. In some embodiments, the mobile phase viscosity modifier of the SEC technique is different from the viscosity modifier of the liquid fixing agent. In some embodiments, the test sample comprises at least about 6M guanidine and about 10% to about 30% glycerol.

[0012] In some embodiments, the SEC technique is an isocratic SEC technique. In some embodiments, the SEC technique includes the use of a mobile phase flow rate of about 1 μL / min to about 5 μL / min.

[0013] In some embodiments, the SEC technique is performed at an elevated temperature. In some embodiments, the SEC technique is performed at a temperature of about 45° C. to about 60° C. In some embodiments, the SEC technique is performed at a substantially constant temperature.

[0014] In some embodiments, the SEC microfluidic device includes a SEC medium. In some embodiments, the SEC medium is a material having an average pore size of about 10 nm to about 500 nm. In some embodiments, the SEC medium is an inner surface of each of a plurality of interconnected channels. In some embodiments, the inner surface material of a plurality of interconnected channels of the SEC microfluidic device has a thickness of about 0.5 μm to about 2 μm.

[0015] In some embodiments, the plurality of interconnected channels of the SEC microfluidic device is configured in an open tubular format. In some embodiments, the plurality of interconnected channels of the SEC microfluidic device comprises 8 or more channels. In some embodiments, the plurality of interconnected channels of the SEC microfluidic device comprises 32 channels. In some embodiments, the plurality of interconnected channels of the SEC microfluidic device comprises 64 channels.

[0016] In some embodiments, each of the multiple interconnected channels of the SEC microfluidic device is in fluid communication with an input port of the SEC microfluidic device via an upstream network of connecting channels. In some embodiments, the upstream network of connecting channels or a portion thereof is connected to a proximal region of each of the multiple interconnected channels. In some embodiments, the upstream network of connecting channels comprises a series of branching channels configured to split the flow of fluid from the input port of the SEC microfluidic device to each of the multiple interconnected channels.

[0017] In some embodiments, each of the multiple interconnected channels of the SEC microfluidic device is in fluid communication with the output port of the SEC microfluidic device through a downstream network of connecting channels. In some embodiments, the downstream network of connecting channels or a portion thereof is connected to a distal region of each of the multiple interconnected channels. In some embodiments, the downstream network of connecting channels comprises a series of converging channels configured to combine the fluid flow from the multiple interconnected channels of the SEC microfluidic device to the output port. In some embodiments, the multiple interconnected channels of the SEC microfluidic device are only connected through an upstream network of connecting channels or a downstream network of connecting channels.

[0018] In some embodiments, each of the multiple interconnected channels of the SEC microfluidic device has a length of about 2 cm to about 50 cm. In some embodiments, each of the multiple interconnected channels of the SEC microfluidic device has a width of about 1 μm to about 15 μm.

[0019] In some embodiments, each of the multiple interconnected channels of the SEC microfluidic device has a depth of about 1 μm to about 15 μm.

[0020] In some embodiments, the plurality of interconnected channels of the SEC microfluidic device are formed by a pillar array. In some embodiments, the pillar array is an amorphous pillar array. In some embodiments, the pillar array is a non-amorphous pillar array. In some embodiments, the pillar array forms an inner surface of each of the plurality of interconnected channels of the SEC microfluidic device.

[0021] In some embodiments, the SEC microfluidic device comprises a quartz substrate. In some embodiments, the SEC microfluidic device comprises a monolithic substrate forming a plurality of interconnected channels. In some embodiments, the SEC microfluidic device comprises a quartz monolithic substrate.

[0022] In some embodiments, the SEC microfluidic device comprises a three-dimensional (3D) printed substrate.

[0023] In some embodiments, collecting the plurality of fractions eluted from the SEC microfluidic device is performed using a fraction collector. In some embodiments, each of the plurality of fractions is collected from the SEC microfluidic device based on time. In some embodiments, each of the plurality of fractions is collected from the SEC microfluidic device over a period of about 30 seconds to about 5 minutes. In some embodiments, each of the plurality of fractions is collected from the SEC microfluidic device over a uniform period of time. In some embodiments, a portion of the plurality of fractions is collected from the SEC microfluidic device over a different period of time than another portion of the plurality of fractions.

[0024] In some embodiments, each of the plurality of fractions is collected from the SEC microfluidic device based on the volume of the eluate from the SEC microfluidic device. In some embodiments, each of the plurality of fractions collected from the SEC microfluidic device has a volume of about 1 μL to about 20 μL. In some embodiments, the plurality of fractions collected from the SEC microfluidic device have a uniform volume. In some embodiments, one fraction of the plurality of fractions collected from the SEC microfluidic device has a different volume from another fraction of the plurality of fractions.

[0025] In some embodiments, the plurality of fractions is from about 5 to about 50 fractions. In some embodiments, the plurality of fractions is from about 12 to about 24 fractions.

[0026] In some embodiments, the proteolytic techniques include enzyme-based digestion techniques, hi some aspects, the enzyme-based digestion techniques include the use of enzymes selected from the group consisting of trypsin, chymotrypsin, pepsin, LysC, LysN, AspN, GluC and ArgC, or combinations thereof.

[0027] In some embodiments, the enzyme-based digestion technique includes diluting the fraction eluted from the SEC microfluidic device. In some embodiments, the dilution includes mixing the fraction eluted from the SEC microfluidic device with water to reach a concentration of chaotropic agent. In some embodiments, the final concentration of chaotropic agent for the enzyme digestion is about 0.5M.

[0028] In some embodiments, the enzyme-based digestion technique does not include a buffer exchange step. In some embodiments, the enzyme-based digestion technique does not include an alkylation step. In some embodiments, the enzyme-based digestion technique does not include a reduction step.

[0029] In some embodiments, proteolytic techniques include non-enzyme-based approaches.

[0030] In some embodiments, the method further comprises subjecting one or more of the plurality of fractions from the SEC microfluidic device and / or one or more of the plurality of fractions subjected to the proteolytic technique to a quantitative labeling technique, wherein the quantitative labeling technique is performed prior to a reversed-phase liquid chromatography (RPLC) technique using the RPLC microfluidic device.

[0031] In some embodiments, the quantitative labeling technique comprises the use of isobaric mass tags. In some embodiments, the quantitative labeling technique comprises the use of tandem mass tags (TMT).

[0032] In some embodiments, the quantitative labeling technique includes a desalting step.

[0033] In some embodiments, the method further comprises mixing an internal standard with one or more of the plurality of fractions from the SEC microfluidic device and / or one or more of the plurality of fractions subjected to the proteolytic technique, and the mixing of the internal standard is performed prior to the reversed-phase liquid chromatography (RPLC) technique using the RPLC microfluidic device. In some embodiments, the internal standard is an isotope-labeled peptide.

[0034] In some embodiments, the one or more fractions subjected to the RPLC technique comprise one or more fractions, or a portion thereof, obtained from (i) zero or more fractions obtained from a SEC microfluidic device, (ii) one or more of said plurality of fractions subjected to said proteolytic technique, In some embodiments, each of the one or more fractions subjected to the RPLC technique comprises the respective source fraction mixed with an aqueous solution.

[0035] In some embodiments, the fractions subjected to the RPLC technique have a volume of about 1 μL to about 50 μL.

[0036] In some embodiments, the RPLC technique comprises the use of an RPLC mobile phase. In some embodiments, the RPLC technique comprises a mobile phase flow rate of the RPLC mobile phase of about 0.05 μL / min to about 2 μL / min. In some embodiments, the RPLC technique is a gradient RPLC technique.

[0037] In some embodiments, the RPLC technique is performed at an elevated temperature. In some embodiments, the RPLC technique is performed at a temperature between about 30° C. and about 100° C. In some embodiments, the RPLC technique is performed at a substantially constant temperature.

[0038] In some embodiments, the reverse phase medium comprises the following alkyl moieties: C2, C4, C8, and C 18In some embodiments, the RPLC partial mixture comprises two or more of the following alkyl moieties: C2, C4, C8, and C 18 In some embodiments, the RPLC moiety mixture comprises three or more of the following alkyl moieties: C2, C4, C8, and C 18 In some embodiments, the alkyl moieties of the RPLC moiety mixture are present in equimolar amounts.

[0039] In some embodiments, the alkyl moieties of the RPLC moiety mixture are covalently attached to a surface of each of a plurality of interconnected channels of the RPLC microfluidic device. In some embodiments, the surface of each of the plurality of interconnected channels comprises silica (SiO2).

[0040] In some embodiments, the plurality of interconnected channels of the RPLC microfluidic device comprises 8 or more channels. In some embodiments, the plurality of interconnected channels of the RPLC microfluidic device comprises 32 channels. In some embodiments, the plurality of interconnected channels of the RPLC microfluidic device comprises 64 channels.

[0041] In some embodiments, each of the plurality of interconnected channels of the RPLC microfluidic device is in fluid communication with an input port of the RPLC microfluidic device via an upstream network of connecting channels. In some embodiments, the upstream network of connecting channels, or a portion thereof, is connected to a proximal region of each of the plurality of interconnected channels. In some embodiments, the upstream network of connecting channels comprises a series of branching channels configured to split the flow of fluid from the input port of the RPLC microfluidic device to each of the plurality of interconnected channels.

[0042] In some embodiments, each of the plurality of interconnected channels of the RPLC microfluidic device is in fluid communication with an output port of the RPLC microfluidic device via a downstream network of connecting channels. In some embodiments, the downstream network of connecting channels or a portion thereof is connected to a distal region of each of the plurality of interconnected channels. In some embodiments, the downstream network of connecting channels comprises a series of converging channels configured to combine fluid flow from the plurality of interconnected channels of the RPLC microfluidic device to the output port. In some embodiments, the plurality of interconnected channels of the RPLC microfluidic device are only connected via an upstream network of connecting channels or a downstream network of connecting channels.

[0043] In some embodiments, each of the multiple interconnected channels of the RPLC microfluidic device has a length of about 2 cm to about 50 cm. In some embodiments, each of the multiple interconnected channels of the RPLC microfluidic device has a width of about 1 μm to about 15 μm. In some embodiments, each of the multiple interconnected channels of the RPLC microfluidic device has a depth of about 1 μm to about 15 μm.

[0044] In some embodiments, the plurality of interconnected channels of the RPLC microfluidic device are formed by a pillar array. In some embodiments, the pillar array is an amorphous pillar array. In some embodiments, the pillar array is a non-amorphous pillar array. In some embodiments, the pillar array forms an interior surface of each of the plurality of interconnected channels that the RPLC microfluidic device comprises.

[0045] In some embodiments, the RPLC microfluidic device includes an online shunting mechanism. In some embodiments, the online shunting mechanism is a valve and / or a channel. In some embodiments, the online shunting mechanism is disposed between a plurality of interconnected channels of the RPLC microfluidic device and an electrospray ionization device.

[0046] In some embodiments, the RPLC microfluidic device comprises a quartz substrate. In some embodiments, the RPLC microfluidic device comprises a monolithic substrate forming a plurality of interconnected channels. In some embodiments, the RPLC microfluidic device comprises a quartz monolithic substrate.

[0047] In some embodiments, the RPLC microfluidic device comprises a three-dimensional (3D) printed substrate.

[0048] In some embodiments, the RPLC microfluidic device is configured in an open tubular format.

[0049] In some embodiments, the RPLC microfluidic device is configured for online desalting.

[0050] In some embodiments, the ionization source is a nano-electrospray ionization source, hi some embodiments, the electrospray ionization source is a heated electrospray ionization source.

[0051] In some embodiments, the sample is selected from the group consisting of a blood sample, a cerebrospinal fluid (CSF) sample, a peritoneal fluid sample, a seminal plasma sample, and a nipple aspirate sample. In some embodiments, the sample has a volume of about 10 μL to about 200 μL. In some embodiments, the sample is a blood sample.

[0052] In some embodiments, where the sample from the individual is a blood sample, the method further comprises preparing a plasma sample. In some embodiments, preparing the plasma sample comprises subjecting the blood sample to a plasma generation technique. In some embodiments, the plasma generation technique comprises subjecting the sample to a polysulfone medium. In some embodiments, the polysulfone medium is an asymmetric polysulfone material.

[0053] In some embodiments, the plasma generation technique is a capillary action filtration technique. In some embodiments, the volume of the blood sample subjected to the plasma generation technique is about 10 μL to about 200 μL.

[0054] In some embodiments, the method further comprises mixing the generated plasma sample with a liquid fixative to generate a test sample. In some embodiments, the test sample is not further depleted prior to subjecting the test sample to the SEC technique.

[0055] In some embodiments, the plasma generation technique is performed at ambient temperature.

[0056] In some embodiments, the sample is not subjected to a depletion step prior to the plasma generation technique.

[0057] In some embodiments, the method further comprises subjecting the components or products thereof eluted from the RPLC microfluidic device to a mass spectrometer. In some embodiments, the method further comprises performing mass spectrometry of the components or products thereof of the sample using a mass spectrometer. In some embodiments, the mass spectrometry comprises analysis of each fraction subjected to the RPLC technique using the RPLC microfluidic device. In some embodiments, the mass spectrometry comprises obtaining one or more data sets comprising information obtained from the mass spectrometer for each fraction subjected to the RPLC technique using the RPLC microfluidic device.

[0058] In some embodiments, the single data set comprises information obtained from a mass spectrometer from a single fraction subjected to an RPLC technique using an RPLC microfluidic device, in some embodiments, each of the one or more data sets comprises mass to charge ratio (m / z) and abundance information of ions of the components or their products introduced into the mass spectrometer.

[0059] In some embodiments, each composition of the collection of compositions obtained from any of the methods described herein is an RPLC microfluidic device eluate.

[0060] In some aspects, provided herein is a method of analyzing a composition comprising: (a) subjecting the composition to a mass spectrometer; and (b) performing mass spectrometry analysis of said composition, wherein said composition is obtained from a processing technique comprising fractionation of a sample using a SEC technique, including the use of a SEC microfluidic device, followed by application of each fraction or a product thereof to an RPLC technique.

[0061] In some aspects, provided herein is a method of analyzing a collection of compositions using mass spectrometry, the method comprising: (a) subjecting each composition of the collection of compositions to a mass spectrometer; and (b) performing mass spectrometry on each composition of said collection of compositions, wherein said collection of compositions is obtained from a processing technique comprising fractionation of a test sample using a SEC technique, comprising use of a SEC microfluidic device, and subsequently applying each fraction or a product thereof to an RPLC technique, comprising use of an RPLC microfluidic device.

[0062] In some embodiments, the SEC fraction is further processed by proteolytic techniques.

[0063] In some embodiments, the method further comprises determining an identity of each of the plurality of one or more biomolecules in the test sample based on at least one of the one or more datasets. In some embodiments, the method further comprises measuring an amount of each of the plurality of one or more biomolecules in the test sample based on at least one of the one or more datasets.

[0064] In some embodiments, the method further comprises identifying a signature comprising one or more identified biomolecules from the determined identities. In some embodiments, the identifying further comprises selecting a subset of the one or more identified biomolecules based on a measured amount of the one or more identified biomolecules. In some embodiments, the subset of the one or more identified biomolecules is selected based on a differential measured amount of the one or more identified biomolecules compared to a reference sample.

[0065] In some embodiments, the method further includes identifying a signature comprising the one or more identified biomolecules, where the identifying includes measuring an amount of each of the plurality of one or more biomolecules in the test sample based on at least one of the one or more datasets, selecting a plurality of subsets of the one or more biomolecules in the sample based on the measured amounts, and determining an identity of each of the subset of the plurality of one or more biomolecules in the test sample.

[0066] In some embodiments, a subset of the plurality of one or more biomolecules in the test sample is selected based on a differential measurement amount of the plurality of one or more biomolecules in the test sample compared to a reference sample. In some embodiments, the test sample is a sample from a diseased subject, and the reference sample is a sample from a healthy subject or a control subject. In some embodiments, the test sample is a sample from a subject with a disease-related precondition, and the reference sample is a sample from a healthy subject or a control subject. In some embodiments, the test sample is a sample from a subject with a disease in an active state, and the reference sample is a sample from a subject with a disease in an inactive state, optionally, the inactive state is remission. In some embodiments, the test sample is a sample from a subject with a disease in an advanced stage, and the reference sample is a sample from a subject with an early stage disease.

[0067] In some embodiments, a signature comprising a plurality of identified biomolecules or a subset thereof is identified by a method described herein. In some embodiments, a signature comprising a subset of the identified biomolecules is identified by a method described herein.

[0068] In some embodiments, the method further includes providing all or a subset of the signature-identified biomolecules as input to one or more processes configured to perform gene enrichment analysis, one or more processes configured to perform pathway analysis, and / or one or more processes configured to perform network analysis.

[0069] In some embodiments, a method of analyzing biomolecules of a sample includes providing the identified biomolecules of the signature as input to one or more processes configured to perform gene enrichment analysis, one or more processes configured to perform pathway analysis, and / or one or more processes configured to perform network analysis. In some embodiments, the identified biomolecules of one or more molecular types of the signature are provided as input. In some embodiments, the one or more molecular types include proteins. In some embodiments, the one or more molecular types consist exclusively of proteins.

[0070] In some aspects, provided herein are methods of analyzing signatures of identified components, comprising performing gene enrichment analysis, pathway analysis, and network analysis in any order, where the signatures of identified components include a protein set, a transcriptome set, a peptide set, and / or a metabolite set, and performing includes a process configured to perform gene enrichment analysis, a process performing pathway analysis, a process configured to perform gene enrichment analysis, and a process configured to perform network analysis to identify drug targets.

[0071] In some embodiments, the one or more processes configured to perform gene enrichment analysis include a process configured to identify one or more gene ontologies associated with at least one of the identified biomolecules of the signature provided as input or at least one of their products. In some embodiments, the one or more processes configured to perform gene enrichment analysis include a process configured to identify one or more cellular component gene ontologies associated with at least one of the identified biomolecules of the signature provided as input or at least one of their products, respectively, or a process configured to identify one or more molecular pathway gene ontologies associated with at least one of the identified biomolecules of the signature provided as input or at least one of their products, respectively, and / or a process configured to identify one or more biological process gene ontologies associated with at least one of the identified biomolecules of the signature provided as input or at least one of their products. In some embodiments, the one or more processes configured to perform gene enrichment analysis include a process configured to identify one or more regulators of at least one of the identified biomolecules of the signature provided as input or at least one of their products.

[0072] In some embodiments, the one or more processes configured to perform gene enrichment analysis include a process configured to identify one or more transcription factors that regulate at least one of the identified biomolecules of the signature provided as input or at least one of their products, and / or a process configured to identify one or more kinases that regulate at least one of the identified biomolecules of the signature provided as input or at least one of their products.

[0073] In some embodiments, the one or more processes configured to perform pathway analysis include a process configured to identify one or more pathways associated with at least one of the identified biomolecules of the signature provided as input or at least one of their products. In some embodiments, the one or more processes configured to perform pathway analysis include a process configured to identify one or more molecular pathways associated with at least one of the identified biomolecules of the signature provided as input or at least one of their products, respectively, a process configured to identify one or more signal transduction pathways associated with at least one of the identified biomolecules of the signature provided as input or at least one of their products, respectively, and / or a process configured to identify one or more metabolic pathways associated with at least one of the identified biomolecules of the signature provided as input or at least one of their products, respectively.

[0074] In some embodiments, the one or more processes configured to perform network analysis include a process configured to identify one or more networks associated with at least one of the identified biomolecules of the signature provided as input or at least one of their products. In some embodiments, the one or more processes configured to perform network analysis include a process configured to identify one or more molecular pathways associated with at least one of the identified biomolecules of the signature provided as input or at least one of their products, respectively, and / or a process configured to identify one or more signaling pathways associated with at least one of the identified biomolecules of the signature provided as input or at least one of their products, respectively.

[0075] In some embodiments, the one or more processes configured to perform network analysis include a process configured to identify one or more hubs of one or more networks associated with at least one of the identified biomolecules of the signature provided as input or at least one of their products. In some embodiments, the one or more processes configured to perform network analysis include a process configured to identify one or more drugs that target at least one of the identified biomolecules of the signature provided as input or at least one of their products, respectively. Here, the process is configured to identify one or more drugs that target at least one hub of the network including the multiple identified biomolecules of the signature provided as input. In some embodiments, the one or more processes configured to perform network analysis include two processes configured to identify one or more drugs that target at least one of the identified biomolecules of the signature provided as input or at least one of their products, respectively, and optionally, the two processes are configured to identify one or more drugs that target at least one hub of the network including the multiple identified biomolecules of the signature provided as input, respectively.

[0076] In some aspects, provided herein is a method of analyzing signatures of identified biomolecules, comprising providing a plurality of identified biomolecules to each of a plurality of processes each configured to perform a gene enrichment analysis, a pathway analysis, or a network analysis, performed in any order, where the plurality of identified biomolecules comprises a protein set, a transcriptome set, a peptide set, and / or a metabolite set, the plurality of processes including a process configured to perform a gene enrichment analysis to identify one or more gene ontologies associated with at least one of the plurality of identified biomolecules provided as input or at least one of their products, a process configured to perform a pathway analysis to identify one or more signaling pathways associated with at least one of the plurality of identified biomolecules provided as input or at least one of their products, a process configured to perform a pathway analysis to identify one or more transcription factors regulating at least one of the plurality of identified biomolecules provided as input or at least one of their products. the process configured to perform a gene enrichment analysis to identify one or more kinases that regulate at least one gene product or at least one of the products of at least one of the plurality of identified biomolecules provided as input; a process configured to perform a gene enrichment analysis to identify one or more networks associated with at least one of the plurality of identified biomolecules provided as input or at least one of their products, respectively; and two processes configured to perform a network analysis to identify one or more drugs that target at least one of the plurality of identified biomolecules provided as input or at least one of their products, respectively.

[0077] In some embodiments, provided herein is a method of analyzing a protein signature, the method comprising providing a plurality of proteins to each of a plurality of processes, each configured to perform a gene enrichment analysis, a pathway analysis, or a network analysis, the providing being performed in any order, the plurality of processes being a process configured to perform a gene enrichment analysis to identify one or more gene ontologies associated with at least one of the plurality of proteins provided as input or at least one of their products, respectively, or a process configured to perform a pathway analysis to identify one or more signaling pathways associated with at least one of the plurality of proteins provided as input or at least one of their products, respectively, ... gene enrichment analysis to identify one or more gene ontologies associated with at least one of the plurality of proteins provided as input or at least one of their products, respectively, a process configured to perform a gene enrichment analysis to identify one or more signaling pathways associated with at least one of the plurality of proteins provided as input or at least one of their products, respectively, a process configured to perform gene enrichment analysis to identify one or more transcription factors that regulate at least one of the plurality of proteins provided as input, a process configured to perform gene enrichment analysis to identify one or more kinases that regulate a gene product of at least one of the plurality of proteins or at least one of their products provided as input, a process configured to perform network analysis to identify one or more networks associated with at least one of the plurality of proteins provided as input or at least one of their products, respectively, and each of two processes configured to perform network analysis to identify one or more drugs that target at least one of the plurality of proteins provided as input or at least one of their products, respectively.

[0078] In some aspects, provided herein is a size exclusion chromatography (SEC) microfluidic device, the device comprising an input port, an upstream network of connecting channels, and a plurality of interconnected channels, each channel of the plurality of interconnected channels being in an open tubular format, each channel of the plurality of interconnected channels comprising an inner surface comprising a SEC medium, and each channel of the plurality of interconnected channels being in fluid communication with the input port via the upstream network of connecting channels.

[0079] In some embodiments, the inner surface of the SEC microfluidic device that contains the SEC medium has a thickness of about 0.5 μm to about 2 μm. In some embodiments, the SEC medium of the SEC microfluidic device is a material having an average pore size of about 10 nm to about 500 nm.

[0080] In some embodiments, the plurality of interconnected channels of the SEC microfluidic device comprises between 8 and 100 interconnected channels. In some embodiments, the plurality of interconnected channels of the SEC microfluidic device comprises 8 or more interconnected channels. In some embodiments, the plurality of interconnected channels of the SEC microfluidic device comprises 32 interconnected channels. In some embodiments, the plurality of interconnected channels of the SEC microfluidic device comprises 64 interconnected channels.

[0081] In some embodiments, the upstream network of connecting channels of the SEC microfluidic device or a portion thereof is connected to the proximal region of each of the plurality of interconnected channels. In some embodiments, the upstream network of connecting channels of the SEC microfluidic device comprises a series of branching channels configured to split the fluid flow from the input port of the SEC microfluidic device to each of the plurality of interconnected channels. In some embodiments, each of the plurality of interconnected channels of the SEC microfluidic device is in fluid communication with the output port of the SEC microfluidic device via the downstream network of connecting channels. In some embodiments, the downstream network of connecting channels of the SEC microfluidic device comprises a series of converging channels configured to combine the fluid flow from the plurality of interconnected channels of the SEC microfluidic device to the output port.

[0082] In some embodiments, each of the multiple interconnected channels of the SEC microfluidic device has a length of about 2 cm to about 50 cm. In some embodiments, each of the multiple interconnected channels of the SEC microfluidic device has a width of about 1 μm to about 15 μm. In some embodiments, each of the multiple interconnected channels of the SEC microfluidic device has a depth of about 1 μm to about 15 μm.

[0083] In some embodiments, the plurality of interconnected channels of the SEC microfluidic device are formed by a pillar array. In some embodiments, the pillar array of the SEC microfluidic device is an amorphous pillar array. In some embodiments, the pillar array of the SEC microfluidic device is a non-amorphous pillar array. In some embodiments, the pillar array of the SEC microfluidic device forms an inner surface of each of the plurality of interconnected channels of the SEC microfluidic device.

[0084] In some embodiments, the SEC microfluidic device comprises a quartz substrate. In some embodiments, the SEC microfluidic device comprises a monolithic substrate forming a plurality of interconnected channels. In some embodiments, the SEC microfluidic device comprises a quartz monolithic substrate.

[0085] In some embodiments, the SEC microfluidic device comprises a three-dimensional (3D) printed substrate.

[0086] In some aspects, provided herein is a reversed-phase liquid chromatography (RPLC) microfluidic device comprising an input port, an upstream network of connecting channels, and a plurality of interconnected channels, each channel of the plurality of interconnected channels being in an open tubular format, each channel of the plurality of interconnected channels comprising an inner surface comprising an RPLC medium, and each channel of the plurality of interconnected channels being in fluid communication with the input port via the upstream network of connecting channels.

[0087] In some embodiments, the RPLC medium of the RPLC microfluidic device comprises an alkyl moiety having from about 2 to about 20 carbons. In some embodiments, the RPLC medium of the RPLC microfluidic device comprises an alkyl moiety having from about 2 to about 20 carbons. 18 In some embodiments, the RPLC medium of the RPLC microfluidic device comprises one or more of the following alkyl moieties: C2, C4, C8, and C 18 In some embodiments, the RPLC microfluidic device comprises an RPLC partial mixture that includes two or more of the following alkyl moieties: C2, C4, C8, and C 18 In some embodiments, the RPLC moiety mixture of the RPLC microfluidic device comprises three or more of the following alkyl moieties: C2, C4, C8, and C 18 In some embodiments, the alkyl moieties of the RPLC moiety mixture of the RPLC microfluidic device are present in equimolar amounts.

[0088] In some embodiments, the RPLC medium of the RPLC microfluidic device is coupled to an inner surface of each channel of the plurality of interconnected channels via silica (SiO2). In some embodiments, the plurality of interconnected channels of the RPLC microfluidic device includes between 8 and 100 interconnected channels. In some embodiments, the plurality of interconnected channels of the RPLC microfluidic device includes 8 or more interconnected channels. In some embodiments, the plurality of interconnected channels of the RPLC microfluidic device includes 32 interconnected channels. In some embodiments, the plurality of interconnected channels of the RPLC microfluidic device includes 64 interconnected channels.

[0089] In some embodiments, the upstream network of connecting channels of the RPLC microfluidic device, or a portion thereof, is connected to a proximal region of each of the plurality of interconnected channels, hi some embodiments, the upstream network of connecting channels of the RPLC microfluidic device comprises a series of branching channels configured to split the flow of fluid from an input port of the RPLC microfluidic device to each of the plurality of interconnected channels.

[0090] In some embodiments, each of the plurality of interconnected channels of the RPLC microfluidic device is in fluid communication with an output port of the RPLC microfluidic device via a downstream network of connecting channels, hi some embodiments, the downstream network of connecting channels of the RPLC microfluidic device comprises a series of converging channels configured to combine fluid flow from the plurality of interconnected channels to the output port of the RPLC microfluidic device.

[0091] In some embodiments, each of the multiple interconnected channels of the RPLC microfluidic device has a length of about 2 cm to about 50 cm. In some embodiments, each of the multiple interconnected channels of the RPLC microfluidic device has a width of about 1 μm to about 15 μm. In some embodiments, each of the multiple interconnected channels of the RPLC microfluidic device has a depth of about 1 μm to about 15 μm.

[0092] In some embodiments, the plurality of interconnected channels of the RPLC microfluidic device are formed by a pillar array. In some embodiments, the pillar array of the RPLC microfluidic device is an amorphous pillar array. In some embodiments, the pillar array of the RPLC microfluidic device is a non-amorphous pillar array. In some embodiments, the pillar array of the RPLC microfluidic device forms an interior surface of each of the plurality of interconnected channels of the RPLC microfluidic device.

[0093] In some embodiments, the RPLC microfluidic device comprises a quartz substrate. In some embodiments, the RPLC microfluidic device comprises a monolithic substrate forming a plurality of interconnected channels. In some embodiments, the RPLC microfluidic device comprises a quartz monolithic substrate.

[0094] In some embodiments, the RPLC microfluidic device comprises a three-dimensional (3D) printed substrate.

[0095] In another aspect, provided herein is a method of analyzing a signature of an identified component comprising performing gene enrichment analysis, pathway analysis, and network analysis in any order, where the signature of the identified component comprises a protein set, a transcriptome set, a peptide set, and / or a metabolite set, said performing including a process configured to perform gene enrichment analysis, a process performing pathway analysis, a process configured to perform gene enrichment analysis, and a process configured to perform network analysis to identify drug targets.

[0096] In another aspect, provided herein are methods of subjecting an individual to a determination of a coronary artery disease (CAD) diagnosis, the methods comprising: (a) obtaining mass spectrometry (MS) data from a sample or a derivative thereof obtained from the individual; (b) analyzing the MS data according to a CAD proteomic signature, the CAD proteomic signature comprising one or more biomarkers in Table 1; and (c) determining whether the individual has the CAD proteomic signature. In some embodiments, if the individual has the CAD proteomic signature, the individual is diagnosed with CAD.

[0097] In another aspect, provided herein is a method of diagnosing an individual as having coronary artery disease (CAD), the method comprising: (a) obtaining mass spectrometry (MS) data from a sample or a derivative thereof obtained from the individual; (b) analyzing the MS data according to a CAD proteomic signature, wherein the CAD proteomic signature comprises one or more biomarkers in Table 1; and (c) diagnosing the individual as having CAD based on the presence of the CAD proteomic signature.

[0098] In another aspect, provided herein is a method of treating an individual having coronary artery disease (CAD), the method comprising: (a) diagnosing the individual as having CAD according to the presence of a CAD proteomic signature in a sample obtained from the individual, or a derivative thereof, where the CAD proteomic signature comprises one or more biomarkers in Table 1; and (b) administering a CAD treatment to the individual.

[0099] In some embodiments, the presence of a CAD proteomic signature is determined by analyzing the MS data according to the CAD proteomic signature.

[0100] In some embodiments, the method further comprises obtaining MS data from a sample obtained from the individual, or a derivative thereof.

[0101] In some embodiments, the CAD treatment comprises lifestyle adjustments. In some embodiments, the CAD treatment comprises pharmaceutical intervention. In some embodiments, the pharmaceutical intervention comprises administration of a drug selected from the group consisting of calcium channel blockers, histone deacetylase (HDAC) inhibitors (such as HDAC6), Ca2+ / calmodulin (CaM)-dependent protein kinase II (CaMKII) inhibitors, guanylyl cyclase (sGC) activators, MMP inhibitors, statins, and antihypertensive drugs. In some embodiments, the pharmaceutical intervention comprises a drug selected from the group consisting of amlodipine, tubastatin-a, forskolin, trichostatin A, KN-93, CFM-1571, ilomastat, CAY-10603, and rosuvastatin, or a pharmaceutical salt thereof. In some embodiments, the drug is BRD-K52306726, BRD-K71361154, acetazolamide, rolipram, ruxolitinib, BRD-A59808129-001-01-7, BRD-K76876037, ZM336372, trehalose, SCHEMBL3092652, BMS-387032, BRD-K01425431, 4-hydroxyretinoic acid, CHEMBL585951, CHEMBL1673039, HY-11007, primidone, BRD-K81417919, SPECTRUM_000826, tamoxifen, BRD-K00544996, CID67066889, CX-5 461, BRD-K63944563, SCHEMBL6851809, BRD-A86146706, FR-180204, CHEMBL552425, hexachlorophene, Aggc, SUGA1_008424, BRD-K96640811, anastrozole, wortmannin, vandetanib, AC1NWALF, OTSSP167, WZ3105, dihydroergotamine, BRD-K99839793, SR33805 oxalate, AT-7519, sulfadoxine, SPECTRUM_001319, MLS003329219, trichostatin A, and rotenone, or a pharmaceutical salt thereof.

[0102] In another aspect, a method is provided for detecting a coronary artery disease (CAD) proteomic signature in an individual, comprising: (a) obtaining mass spectrometry (MS) data from a sample obtained from the individual and derivatives thereof; and (b) analyzing the MS data according to a CAD proteomic signature to detect a CAD proteomic signature, the CAD proteomic signature comprising one or more biomarkers in Table 1. In some embodiments, the individual is suspected of having CAD.

[0103] In some embodiments, the CAD proteomic signature comprises increased expression of one or more biomarkers according to Table 1 compared to a reference. In some embodiments, the CAD proteomic signature comprises decreased expression of one or more biomarkers according to Table 1 compared to a reference.

[0104] In some embodiments, the CAD proteomic signature comprises one or more biomarkers associated with calcium signaling pathway, histone regulation, HIF-1 signaling pathway, cAMP signaling pathway, beta adrenergic signaling pathway, PI3K-Akt signaling pathway, complement and / or coagulation cascade, sphingolipid signaling pathway, natural killer cell-mediated cytotoxicity, adipocytokine signaling pathway, DNA damage, calcium energy, metabolism, cell adhesion, inflammation, hypoxia, and histone methylation. In some embodiments, the one or more biomarkers comprise a subset thereof comprising one or more biomarkers associated with transcription factors. In some embodiments, the one or more biomarkers comprise a subset thereof comprising one or more biomarkers associated with kinases.

[0105] In some embodiments, the one or more biomarkers include at least 10 biomarkers in Table 1. In some embodiments, the one or more biomarkers include at least 25 biomarkers in Table 1. In some embodiments, the one or more biomarkers include at least 50 biomarkers. In some embodiments, the one or more biomarkers include all of the biomarkers in Table 1.

[0106] In some embodiments, the method further comprises obtaining a sample from the individual. In some embodiments, the sample or derivative thereof is a blood sample or a derivative thereof. In some embodiments, the sample or derivative thereof is a plasma sample. In some embodiments, the sample or derivative thereof comprises a liquid fixative.

[0107] In some embodiments, obtaining MS data from the sample or a derivative thereof comprises performing mass spectrometry of the sample or a derivative thereof using a mass spectrometer. In some embodiments, the mass spectrometry is performed according to any of the methods provided herein for performing mass spectrometry. In some embodiments, the mass spectrometry is performed according to the methods of embodiments 140-143. In some embodiments, analyzing the MS data according to the CAD proteomic signature comprises subjecting the MS data to the method of any one of embodiments 161-177. In some embodiments, analyzing the MS data according to the CAD proteomic signature comprises assessing the presence or absence or level of each of the one or more biomarkers of the CAD proteomic signature in the MS data.

[0108] In some embodiments, the method further comprises performing one or more of the following factorial assessments of the individual: gender, age, body mass index (BMI), systolic blood pressure, diastolic blood pressure, total cholesterol, HDL, LDL, triglycerides, hyperlipidemia, hypertension, diabetes mellitus, insulin resistance, kidney disease, smoking status, level of physical activity, level of sleep, or quality of nutrition.

[0109] In some embodiments, the method further comprises administering a medical procedure to the individual to assess the presence of CAD. [Brief description of the drawings]

[0110] [Figure 1] An exemplary workflow 100 for acquiring a sample and analyzing components therein using mass spectrometry is shown. As shown in FIG. 1, the exemplary workflow 100 includes sample acquisition 105, preliminary sample processing 110, liquid chromatography and optional proteolysis 115, ionization for mass spectrometry 120, mass spectrometry data acquisition 125, and mass spectrometry data analysis 130. [Diagram 2] As shown in FIG. 2 , which illustrates an exemplary workflow 200 for obtaining a sample and analyzing components therein using mass spectrometry, the exemplary workflow 200 includes blood sample acquisition 205, plasma generation 210, size exclusion chromatography 215, proteolysis using enzymatic digestion 220, reversed-phase liquid chromatography (RPLC) combined with online ionization for mass spectrometry 225, mass spectrometry data acquisition 230, and mass spectrometry data analysis 235. [Diagram 3] FIG. 3 shows a schematic diagram of an exemplary microfluidic device 300 configured for separation of components of a sample. [Figure 4] Representative size exclusion tracks of undepleted human plasma are shown, with fraction sizes illustrated using dashed lines. [Diagram 5] FIG. 1 shows a schematic diagram of an exemplary size-exclusion chromatography microfluidic device. [Figure 6] 1 shows an exemplary cellular component analysis of a 292 protein CAD signature using ToppGene software. [Figure 7] 1 shows an exemplary molecular pathway analysis of the 292 protein CAD signature using ToppGene software. [Figure 8] 1 shows an exemplary transcription factor enrichment analysis (TFEA) algorithm of the 292 protein CAD signature. [Figure 9] An exemplary kinase enrichment analysis (KEA) of the 292 protein CAD signature is shown. [Figure 10] 1 shows an exemplary 292 protein CAD signature interaction network generated from 292 protein CAD signatures using the GeneMANIA algorithm. [Figure 11] 1 shows an exemplary CAD complement pathway protein interaction sub-network generated using the GeneMANIA algorithm from a 292 protein CAD signature. [Figure 12] FIG. 1 shows an exemplary CAD histone-regulated protein interaction sub-network generated using the GeneMANIA algorithm from a 292-protein CAD signature. [Figure 13] FIG. 1 shows an exemplary CAD DNA damage protein interaction sub-network generated using the GeneMANIA algorithm from the 292 protein CAD signature. [Figure 14] FIG. 1 shows an exemplary CAD calcium-energy protein interaction sub-network generated using the GeneMANIA algorithm from a 292 protein CAD signature. [Figure 15] 1 shows an exemplary CAD metabolomic protein interaction sub-network generated using the GeneMANIA algorithm from 292 protein CAD signatures. [Figure 16] 1 shows an exemplary CAD cell adhesion protein interaction sub-network generated using the GeneMANIA algorithm from a 292 protein CAD signature. [Figure 17] FIG. 1 shows an exemplary CAD-inflammatory protein interaction sub-network generated using the GeneMANIA algorithm from a 292-protein CAD signature. [Figure 18] FIG. 1 shows an exemplary CAD hypoxia protein interaction sub-network generated using the GeneMANIA algorithm from a 292 protein CAD signature. [Figure 19] FIG. 1 shows an exemplary CAD histone methylation protein interaction sub-network generated using the GeneMANIA algorithm from a 292 protein CAD signature. [Figure 20] 1 shows an exemplary L1000FWD algorithm analysis identifying FDA-approved drugs that target hubs (B) of a protein network represented by a 292-protein CAD signature (A). [Figure 21] 1 shows an exemplary ILINC chemical perturbation algorithm analysis to identify novel drugs targeting hubs of a protein network represented by a 292 protein CAD signature. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0111] Detailed Description In some embodiments, provided herein are methods of processing test samples for mass spectrometry. In other embodiments, provided herein are microfluidic devices useful for separating components, such as size-exclusion chromatography microfluidic devices or reversed-phase liquid chromatography microfluidic devices. In other embodiments, provided herein are collections of compositions obtained using the methods and / or devices described herein. In other embodiments, provided herein are methods of analyzing collections of compositions using mass spectrometry techniques. In other aspects, provided herein are methods of identifying signatures comprising one or more identified biomolecules. In other embodiments, provided herein are signatures identified using the methods and / or devices disclosed herein. In other embodiments, provided herein are methods of analyzing components of a signature for functions, activities, and / or attributes.

[0112] The provided embodiments relate to non-a priori agnostic methods using mass spectrometry to achieve high proteomic coverage, including capture of a diverse set of proteins, such as secreted endogenous cleavage products, soluble proteins, and exosome or lipid microvesicle enriched proteins, as well as other non-proteinaceous components of a sample. These biomolecules can span a large linear dynamic range (e.g., typically 12 orders of magnitude or more). The analytical strategies, as achieved by the provided methods and / or devices, allow for unbiased capture and analysis of a wide range of proteins with diverse physicochemical and biological properties, as well as other non-proteinaceous components of a sample. The provided methods also minimize a priori analytical variables to reproducibly analyze the majority of observable components of a sample, such as proteomics, including proteins naturally occurring at low abundance levels.

[0113] In some embodiments, the provided methods and / or devices can be used for unbiased discovery and tracking targeted analysis of specific molecular signatures, including protein biosignals (e.g., disease-specific protein biosignals), from small biological samples, including blood specimens procured in simple prick tests. Plasma extraction from a single drop of blood can be achieved by capillary action filtration with commercially available materials and directly mixed with a chaotropic liquid fixative. In some embodiments, the liquid fixative solubilizes and preserves proteins and other biological analytes from the blood sample, including primary and secondary metabolites, natural peptides, and microRNAs. Due to its strong chaotropic activity, this liquid fixative eliminates protease activity, achieves maximum preservation of metabolite chemical integrity, eliminates protein-protein binding, and provides maximum hydrodynamic radius and liquid viscosity for their efficient size-exclusion chromatography (SEC) separation. Furthermore, the specimen procurement and preservation device completely neutralizes all human pathogens (e.g., viruses, bacteria, fungi, etc.) with minimal chemical or toxicological hazards. This configuration is suitable for a point-of-care device for procurement and chemical immobilization of plasma or serum and its protein, natural peptide, metabolite content, and nucleic acid, eg, RNA content.

[0114] In some embodiments, the methods and / or devices provide microfluidic size-exclusion chromatography that achieves efficient flow dynamics (minimal turbulence), low operating backpressure, optimal surface-to-volume ratios, and provides excellent sampling of the wide range of hydrodynamic radii or molecular weights observed for a diverse set of biomolecular species found in samples such as total non-depleted processed plasma / serum, including proteins, endogenous peptides, metabolites, and nucleic acids, e.g., RNA. Importantly, such microfluidic-based partitioning utilizes liquid fixatives from sample procurement to create a highly integrated, orthogonal pipeline.

[0115] In some embodiments, the biomarker discovery methods provided herein further include relative quantitative analysis of the fractionated samples by stoichiometrically normalized isobaric stable isotope tagging. In some embodiments, the methods are amendable to label-free approaches. In contrast to standard protein digestion with proteases, reduction and / or alkylation steps are not required due to the liquid immobilization properties present in the sample or fractions thereof subjected to proteolysis. The fractions generated from the original sample can be further separated using modified reversed-phase liquid chromatography devices with open tubular configurations provided herein. The devices described herein can be useful, for example, for the separation of proteolytic peptides derived from proteins, natural peptides (e.g., MHC class I and II, insulin, glucagon, troponin, etc.), as well as primary (e.g., enzyme cofactors, sugars, amino acids, nucleic acids, lipids, etc.) or secondary metabolites (e.g., derived from drugs or other xenobiotics, etc.) and nucleic acids, e.g., RNA species. The ability to co-analyze native peptides, metabolites and RNA species as they occur in exosomes or other lipid microvesicles naturally occurring in biological fluids such as plasma or serum may help decipher and validate the functional status of enzymes or kinase cofactors and thus act as surrogate markers for them. In some embodiments, open tubular reversed-phase liquid chromatography may be configured and performed on a lab-on-a-chip device.

[0116] As described in this disclosure, in some embodiments, an open tubular reversed-phase liquid chromatography microfluidic device includes a long bonded column length and is constructed from quartz material, as well as C2, C4, C8, and C 18 The open tubular reversed-phase liquid chromatography microfluidic device described herein provides increased theoretical plate numbers and therefore separation efficiency at higher binding capacities, as well as the ability to separate a wide range of hydrophobic, amphipathic and hydrophobic peptides, thus facilitating their downstream analysis (e.g., electrospray ionization and mass spectrometry).

[0117] In some embodiments, methods are provided herein for the robust and comprehensive discovery of biomarkers associated with specific biological phenomena, such as disease, stage or severity, responsiveness to a particular drug, and are important for enabling the evaluation, monitoring or prediction of biological phenomena. In particular, biomarkers can serve as diagnostic, prognostic or stratification markers. For example, biomarkers are important for the assessment of disease risk and progression, as well as for monitoring or predicting patient response to treatment. The ability to co-analyze natural peptides, metabolites and RNA species, as they are present in exosomes or other lipid microvesicles naturally present in biological fluids, such as plasma or serum, may constitute enzymes or kinase cofactors and thus help decipher and validate their functional status and serve as their surrogate markers. Proteins can regulate biochemical reactions in the human body and integrate the effects of genes with epigenetic factors related to environment, age, comorbidities, behavior and drugs. Thus, proteins show great endophenotypic biomarker potential.

[0118] Nevertheless, proteins used in the clinic as biomarkers are only a small part of circulating proteomics. Moreover, other biomolecules, such as certain metabolites in fluid samples such as blood, may also be relevant biomarkers of biological phenomena such as disease. Thus, existing methods generally fail to capture the extent of the range of relevant biomarkers. Moreover, despite advances in the development of multidimensional data integration algorithms and other computer-based machine learning tools, the flexibility, effectiveness and robustness of data integration to extract mechanistic insights into biomarkers remains limited. Thus, many existing methods fail to capture proteins present in biological samples that are pathophysiologically relevant to a particular biological phenomenon such as a particular disease. Thus, the available approaches for biomarker discovery and mechanistic analysis are not entirely satisfactory.

[0119] Furthermore, the usefulness of existing mass spectrometry methods is limited by a number of aspects, including the ability to introduce sample constituent species (such as a low-abundance population of a single type of peptide from a sample) into a mass spectrometer in a concentrated form such that the constituent species reach the mass spectrometer's detector and are analyzed. This challenge is confounded in the presence of highly abundant constituent species, such as human blood samples, and relatively high concentrations of, for example, albumin, IgG, antitrypsin, IgA, transferrin, haptoglobin, and fibrinogen. In addition to the challenge of efficiently separating and concentrating the components of a sample, many components can be lost during sample preparation prior to mass spectrometry.

[0120] The embodiments provided address one or more of these problems. For example, in some aspects, described herein is a comprehensive plasma discovery and validation pipeline that is completely independent of affinity depletion and enrichment steps and represents a quantitative application to a variety of biomedical applications in undepleted serum and / or plasma. Furthermore, the identified components can be analyzed according to the methods described herein to identify and / or use disease-specific biosignatures as novel and highly accurate tools, e.g., with diagnostic and / or prognostic value.

[0121] Furthermore, the methods and devices provided herein include technology platforms suitable for automation and scale-up. Such a premise is essential to achieve statistical power by comprehensive analysis of hundreds or thousands of samples. The large-scale and reproducible analysis of samples, such as plasma proteomics, achieved by the provided embodiments, allows for the full exploitation of the diversity of artificial intelligence, machine learning algorithms that can decipher, for example, functional and clinically relevant endophenotypic evidence at the protein and derivative metabolite level (e.g., the incorporated proteometabolomics profile described herein) despite the large heterogeneity of clinical symptoms of high-risk patients in the early, initiation phase and subsequent safe and effective treatment.

[0122] In some aspects, an additional advantage of the platform embodied by the provided methods is that its technology components constitute a single vertically integrated pipeline, given their highly complementary operating principles. Moreover, the pipeline is highly amenable to automation and can therefore be scaled up to increase analytical capacity with minimal human intervention. Such features collectively facilitate effective and comprehensive analysis of protein biosignals in plasma derived from any disease. Importantly, the platform can operate in both a discovery mode for unbiased or agnostic quantification of a wide range of components such as proteins, as they are disease-specific differentially expressed / present, or in a targeted absolute quantitative analysis mode for high-throughput parallel interrogation of components identified from the discovery analysis. Both the discovery and derived targeted analysis modes of the platform do not utilize expensive and unreliable antibody and / or aptamer-based protein depletion or enrichment prior to measurement.

[0123] The result of the disclosed methods and / or devices is a platform that provides sensitive, robust, and reproducible results that can identify and / or quantify components from samples such as proteins, including challenging ones from exosomes, etc. Furthermore, the methods and / or devices are amenable to miniaturization and integration, including into integrated lab-on-a-chip devices, if desired.

[0124] Thus, in some aspects, provided herein is a method of processing a test sample for mass spectrometry comprising: (a) subjecting the test sample to a size exclusion chromatography (SEC) technique using a SEC microfluidic device, where the test sample comprises one or more biomolecules and a chaotropic agent, and where the SEC microfluidic device comprises a plurality of interconnected channels; (b) collecting a plurality of fractions eluted from the SEC microfluidic device; (c) subjecting one or more of the plurality of fractions from the SEC microfluidic device to a proteolytic technique; and (d) individually subjecting one or more fractions from one or both of steps (b) and (c) to a reversed-phase liquid chromatography (RPLC) technique using an RPLC microfluidic device under conditions that prepare each component of the one or more fractions for introduction into a mass spectrometer, where the RPLC microfluidic device comprises a plurality of interconnected channels comprising a reversed-phase medium, and where the RPLC microfluidic device is coupled to an electrospray ionization source.

[0125] In some aspects, provided herein is a method for processing components of a sample, such as biological samples, or its products, for mass spectrometry.In some embodiments, the method includes: (a) subjecting a test sample to a size exclusion chromatography (SEC) technique using a SEC microfluidic device; (b) collecting a plurality of fractions eluted from the SEC microfluidic device; (c) subjecting one or more of the plurality of fractions from the SEC microfluidic device to a proteolytic technique; and (d) subjecting a series of fractions that can be subjected to reversed phase liquid chromatography (RPLC) to a reversed phase liquid chromatography (RPLC) technique using a reversed phase liquid chromatography (RPLC) microfluidic device to prepare the components of the sample, or its products, that are introduced into a mass spectrometer.

[0126] In another aspect, provided herein is a method of processing components of a biological sample or products thereof for mass spectrometry analysis, the method comprising: (a) subjecting a test sample to size exclusion chromatography (SEC) using a SEC microfluidic device, the test sample comprising a sample mixed with a liquid fixative, the test sample having a predetermined concentration of a chaotropic agent derived from the liquid fixative, the SEC technique comprising use of a SEC mobile phase having a concentration of a mobile phase chaotropic agent within a range of the predetermined concentration of the chaotropic agent in the test sample, the SEC microfluidic device comprising a plurality of interconnected channels, each channel configured in an open tubular format having an inner surface material having an average pore size of about 10 nm to about 500 nm; (b) collecting a plurality of fractions eluted from the SEC microfluidic device; and (c) collecting a plurality of fractions eluted from the SEC microfluidic device. and (d) subjecting each of the set of RPLC-compatible fractions to a reverse phase liquid chromatography (RPLC) technique using an RPLC microfluidic device to prepare components of the sample or products thereof for introduction into a mass spectrometer, the set of RPLC-compatible fractions including (i) zero or more of the plurality of fractions from the SEC microfluidic device and (ii) fractions obtained from one or more of the plurality of fractions subjected to the proteolytic technique, wherein the RPLC technique and the RPLC microfluidic device are configured for online desalting, the RPLC microfluidic device includes a plurality of interconnected channels, each channel configured in an open tubular format having an inner surface material that includes a reverse phase medium, and the RPLC microfluidic device is coupled to an electrospray ionization source.

[0127] In another aspect, provided herein is a collection of compositions obtained from any of the methods described herein. In some embodiments, each composition of the collection of compositions is an RPLC microfluidic device eluate.

[0128] In another aspect, provided herein is a method of analyzing a collection of compositions using mass spectrometry, comprising: (a) subjecting each composition of the collection of compositions to a mass spectrometer; and (b) performing mass spectrometry on each composition of the collection of compositions, wherein said collection of compositions is obtained from a processing technique comprising fractionation of a test sample using a SEC technique, comprising use of a SEC microfluidic device, and subsequently applying each fraction or a product thereof to an RPLC technique, comprising use of an RPLC microfluidic device.

[0129] In another aspect, provided herein is a size exclusion chromatography (SEC) microfluidic device comprising: an input port; an upstream network of connecting channels; and a plurality of interconnected channels, each channel of the plurality of interconnected channels being in an open tubular format, each channel of the plurality of interconnected channels comprising an inner surface comprising a SEC medium, and each channel of the plurality of interconnected channels being in fluid communication with said input port via the upstream network of connecting channels.

[0130] In another aspect, provided herein is a reversed-phase liquid chromatography (RPLC) microfluidic device comprising: an input port; an upstream network of connecting channels; and a plurality of interconnected channels, each channel of the plurality of interconnected channels being in an open tubular format, each channel of the plurality of interconnected channels comprising an inner surface comprising an RPLC medium, and each channel of the plurality of interconnected channels being in fluid communication with said input port via said upstream network of connecting channels.

[0131] All publications, including patent documents, scientific articles, and databases, referred to in this specification are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, including a patent, patent application, or scientific article, was specifically and individually indicated to be incorporated by reference. If a definition set forth herein is contrary to or inconsistent with a definition set forth in a patent, application, published application, or other publication incorporated herein by reference, the definition set forth herein shall take precedence over the definition incorporated herein by reference.

[0132] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0133] I. Definition Unless otherwise defined, all technical terms, notations, and other technical and scientific terms or terminology used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference to what is commonly understood in the art.

[0134] The terms "polypeptide" and "protein" as used herein can be used interchangeably to refer to polymers comprising amino acid residues and are not limited to a minimum length. Such polymers may contain natural or non-natural amino acid residues or combinations thereof, including, but not limited to, peptides, polypeptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Full-length polypeptides or proteins, and fragments thereof, are encompassed by this definition. The term also includes modifications thereof, such as post-translational modifications of one or more residues, such as methylation, phosphorylation, glycosylation, sialylation, or acetylation.

[0135] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is for convenience and brevity only and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Thus, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values ​​within that range. For example, when a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit between the upper and lower limits of that range, and any other stated or intervening value within that stated range, is encompassed within the disclosure, subject to any specifically excluded limit within the stated range. When a stated range includes one or both limits, ranges excluding either or both of those included limits are also included in the disclosure. In some embodiments, two opposing open-ended ranges are provided for a feature, and such descriptions contemplate that a combination of these two ranges is provided herein. For example, in some embodiments, a feature is described as being greater than about 10 units and then (in a separate sentence, etc.) the feature is described as being less than about 20 units, and thus a range of about 10 units to about 20 units is described herein.

[0136] The term "about" as used herein refers to the normal error range of each value, which is readily known in the art. Reference herein to "about" a value or parameter includes (and describes) the variation for that value or parameter itself. For example, a description that refers to "about X" includes the description of "X".

[0137] As used herein, including the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise. For example, "a" or "an" means "at least one" or "one or more." It is understood that aspects and variations described herein include embodiments that "consist of" and / or "consist essentially of" such aspects and variations.

[0138] As used herein, a "subject" or an "individual" are used interchangeably and are mammals. In some embodiments, a "mammal" includes humans, non-human primates, farm and zoo animals, sport animals, or pets such as dogs, rabbits, cows, pigs, hamsters, gerbils, ferrets, mice, cats, monkeys, etc. In some embodiments, the subject or individual is a human.

[0139] As used herein, the terms "treating" and "treatment" include administering to a subject an effective amount of an agent described herein, or prescribing lifestyle adjustments, such as cessation of smoking, such that the subject has a beneficial or desired clinical outcome, such as a reduction in at least one symptom of the disease or an improvement in the disease. For purposes of this technology, beneficial or desired clinical outcomes include, but are not limited to, alleviation of one or more symptoms, whether detectable or undetectable, attenuation of the extent of the disease, a stabilized (i.e., not worsening) state of the disease, a delay or slowing of disease progression, an improvement or alleviation of the disease state, and remission (whether partial or total). Treating may refer to prolonging survival compared to expected survival in the absence of treatment. Thus, one of skill in the art recognizes that treatment may improve the disease state, but may not be a complete cure of the disease. In some embodiments, one or more symptoms of the disease or disorder are alleviated by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% upon treatment of the disease.

[0140] Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of the present disclosure. The following description is illustrative of the disclosure and, of course, should not be construed as in any way limiting the scope of the invention described herein.

[0141] II. Methods for Processing Components of a Sample or Their Products In some aspects, provided herein are methods of processing components or products of a sample to at least partially separate them from one another for downstream applications. In some embodiments, the processing methods described herein are useful for efficiently and effectively separating and concentrating components or products thereof for mass spectrometry analysis.

[0142] In some embodiments, the method of processing a component of a sample or its products for mass analysis includes all steps from sample acquisition to the introduction of the component or its products into a mass spectrometer. In some embodiments, the method described herein includes certain aspects involved in the overall processing of the component or its products for mass analysis, such as one or more liquid chromatography steps and / or pre-processing steps. In some embodiments, the methods for processing described herein are configured to interface, such as immediately before a downstream application including mass analysis. Aspects of the methods disclosed herein are described in more detail below in a modular manner. Such presentation should not be construed as limiting the scope of combinations of various aspects encompassed by the disclosure of the present application to form a method of processing a component of a sample or its products.

[0143] A. The Sample, Its Components, Sample Acquisition, and Sample Pretreatment The methods disclosed herein are useful for processing a variety of sample components or products thereof from a diverse array of sources containing many different combinations of components.

[0144] In some embodiments, the sample is a biological sample, such as a sample that includes an organism or a part or product thereof. In some embodiments, the biological sample is from an individual, such as a human. In some embodiments, the individual is a mammal, such as a human, cow, horse, cat, dog, rodent, or primate. In some embodiments, the sample is a human sample. In some embodiments, the biological sample includes material from an organism classified in the kingdoms Eubacteria, Archaea, Protista, Plantae, Fungi, or Animalia. In some embodiments, the sample is an environmental sample.

[0145] In some embodiments, the sample comprises a fluid and / or solid (e.g., cells) of an individual. In some embodiments, the sample is a liquid biopsy. In some embodiments, the sample comprises a bodily fluid, such as a sample including a blood sample, a serum sample, a convalescent plasma sample, an oropharyngeal sample (including one obtained from an oropharyngeal swab), a nasopharyngeal sample (including one obtained from a nasopharyngeal swab), an oral sample, a bronchoalveolar lavage sample (including one obtained from an endotracheal aspirator), a sweat sample, a sputum sample, a saliva sample, a tear sample, a bodily excretion sample, or a cerebrospinal fluid sample. In some embodiments, the sample comprises a solid, such as a fecal sample. In some aspects, the sample is selected from the group consisting of a blood sample, a cerebrospinal fluid (CSF) sample, a peritoneal fluid sample (proximal fluid adjacent to an organ), a seminal plasma sample, and a nipple aspirate sample.

[0146] In some embodiments, the sample is a complex sample, such as a complex biological sample. In some embodiments, the sample includes components having concentrations that span at least about two orders of magnitude, such as at least about any of 3, 4, 5, 6, 7, 8, 9, or 10 orders of magnitude.

[0147] In some embodiments, a sample includes components such as biomolecules or derivatives thereof. In some embodiments, characteristics of a sample and / or any fraction (such as a portion of a fluid obtained from a method step and / or device described herein) may be described as components thereof (such as in their processed and / or labeled form), such as proteins, peptides, nucleic acids, metabolites, or derivatives.

[0148] In some embodiments, the components are polypeptides (such as proteins, natural peptides, or endogenous protein cleavage products), polynucleotides (such as DNA or RNA), or metabolites. In some embodiments, the samples include proteins, natural peptides, and metabolites. In some embodiments, the components include post-translational modifications.

[0149] In some embodiments, a product of a component of a sample is any derivative of the component generated during or after obtaining the sample. For example, in some embodiments, a product of a protein component of a sample includes any modifications to the protein component or resulting moieties that occur during and / or as a result of sample processing, including protein components with altered physical structure or composition (e.g., with post-translational modifications), polypeptides or peptides resulting from proteolysis of protein components, and polypeptides or peptides with altered physical structure of composition (e.g., with post-translational modifications and / or quantitative labels).

[0150] In some embodiments, the sample is a non-depleted sample, e.g., a sample that has not been treated to remove certain components thereof, such as abundant proteins.

[0151] In some embodiments, the sample is a blood sample or a sample derived therefrom, such as a plasma sample. In some embodiments, the sample comprises a blood sample. In some embodiments, the blood sample is a whole blood sample. In some embodiments, the blood sample is a non-depleted blood sample, e.g., a blood sample that has not been treated to remove certain components, such as highly abundant proteins. In some embodiments, the sample is a plasma sample. In some embodiments, the plasma sample is a non-depleted plasma sample, e.g., a plasma sample that has not been treated to remove certain components, such as highly abundant proteins, but has been treated to remove other components that are typically removed when generating a plasma sample from a whole blood sample. In some embodiments, the blood sample comprises a serum sample. In some embodiments, the serum sample is a non-depleted serum sample, e.g., a serum sample that has not been treated to remove certain components, such as highly abundant proteins. In some embodiments, the blood sample, including the plasma sample or a serum sample derived therefrom, has not been treated to remove any one or more of the seven common highly abundant blood proteins (albumin, IgG, antitrypsin, IgA, transferrin, haptoglobin, fibrinogen). In some embodiments, the blood sample, including the plasma sample or a serum sample derived therefrom, has not been processed to remove any one or more of the 14 common highly abundant blood proteins (albumin, IgG, antitrypsin, IgA, transferrin, haptoglobin, fibrinogen, α2-macroglobulin, α1-acid glycoprotein, IgM, apolipoprotein AI, apolipoprotein AII, complement C3, transthyretin).

[0152] In some embodiments, the sample has a volume (e.g., the volume of a sample obtained from an individual) of about 10 μL to about 200 μL, e.g., about 10 μL to about 100 μL, about 10 μL to about 75 μL, about 25 μL to about 75 μL, or about 30 μL to about 60 μL. In some embodiments, the sample has a volume of at least about 10 μL, e.g., at least about any of 15 μL, 20 μL, 25 μL, 30 μL, 35 μL, 40 μL, 45 μL, 50 μL, 55 μL, 60 μL, 65 μL, 70 μL, 75 μL, 80 μL, 85 μL, 90 μL, 95 μL, 100 μL, 105 μL, 110 μL, 115 μL, 120 μL, 125 μL, 130 μL, 135 μL, 140 μL, 145 μL, 150 μL, 155 μL, 160 μL, 165 μL, 170 μL, 175 μL, 180 μL, 185 μL, 190 μL, 195 μL, or 200 μL. In some embodiments, the sample has a volume of less than about 200 μL, e.g., less than about any of 195 μL, 190 μL, 185 μL, 180 μL, 175 μL, 170 μL, 165 μL, 160 μL, 155 μL, 150 μL, 145 μL, 140 μL, 135 μL, 130 μL, 125 μL, 120 μL, 115 μL, 110 μL, 105 μL, 100 μL, 95 μL, 90 μL, 85 μL, 80 μL, 75 μL, 70 μL, 65 μL, 60 μL, 55 μL, 50 μL, 45 μL, 40 μL, 35 μL, 30 μL, 25 μL, 20 μL, 15 μL, or 10 μL. In some embodiments, the sample has a volume of about any of 10 μL, 15 μL, 20 μL, 25 μL, 30 μL, 35 μL, 40 μL, 45 μL, 50 μL, 55 μL, 60 μL, 65 μL, 70 μL, 75 μL, 80 μL, 85 μL, 90 μL, 95 μL, 100 μL, 105 μL, 110 μL, 115 μL, 120 μL, 125 μL, 130 μL, 135 μL, 140 μL, 145 μL, 150 μL, 155 μL, 160 μL, 165 μL, 170 μL, 175 μL, 180 μL, 185 μL, 190 μL, 195 μL, or 200 μL.

[0153] In some embodiments, the method includes obtaining a sample from an individual. In some embodiments, the method includes one or more preliminary sample processing steps. In some embodiments, the preliminary sample processing step includes mixing the sample with an agent that preserves the sample in a condition for later analysis. In some embodiments, the preliminary sample processing step includes mixing the sample (such as a blood sample) with an anticoagulant. In some embodiments, the preliminary sample processing step includes mixing the sample (such as a blood sample) with an enzyme inhibitor, e.g., a protease inhibitor. In some embodiments, the preliminary sample processing step includes subjecting the sample to conditions to preserve the sample in a condition for later analysis. In some embodiments, the preliminary sample processing step includes subjecting the sample to a low temperature, such as a temperature of about 10° C. or less, 4° C. or less, 0° C. or less, −20° C. or less, or −80° C. or less.

[0154] In some embodiments, the sample is obtained at the point of care.

[0155] In some embodiments, the preliminary sample processing step includes mixing the sample with a liquid fixative to generate a test sample. In some embodiments, the liquid fixative components and / or their concentrations and / or the ratio of sample volume to liquid fixative volume can be adjusted to meet the needs of the methods described herein, for example to achieve a predetermined concentration of one or more components of the liquid fixative in the test sample.

[0156] In some embodiments, the liquid fixative comprises a chaotropic agent. In some embodiments, the chaotropic agent comprises guanidine or a salt thereof, guanidinium or a salt thereof, potassium or a salt thereof, lithium or a salt thereof, magnesium or a salt thereof, or sodium or a salt thereof. In some embodiments, the chaotropic agent is selected from the group consisting of guanidine hydrochloride, guanidinium chloride, guanidinium thiocyanate, lithium perchlorate, lithium acetate, magnesium chloride, potassium acetate, and sodium iodide. In some embodiments, the chaotropic agent is a guanidine salt. In some embodiments, the chaotropic agent is guanidine hydrochloride.

[0157] In some embodiments, the test sample comprises a concentration of chaotropic agent derived from any of the liquid fixatives of about 5M to about 8M, e.g., about 5.5M to about 8M, about 5.5M to about 7M, or about 5.5M to about 6.5M. In some embodiments, the test sample comprises a concentration of chaotropic agent derived from any of the liquid fixatives of at least about 5.5M, e.g., at least about 6M, 6.5M, 7M, 7.5M, or 8M. In some embodiments, the test sample comprises a concentration of chaotropic agent derived from any of the liquid fixatives of about 8M or less, e.g., about 7.5M or less, 6.5M or less, or 6M or less. In some embodiments, the test sample comprises a concentration of chaotropic agent derived from any of the liquid fixatives of about 5.5M, 6M, 6.5M, 7M, 7.5M, or 8M.

[0158] In some embodiments, the liquid fixative comprises a viscosity modifier. In some embodiments, the polyol is selected from the group consisting of glycerol, propylene glycol, sorbitol, and polyethylene glycol (PEG). In some embodiments, the viscosity modifier is glycerol.

[0159] In some embodiments, the test sample comprises a concentration of viscosity modifier derived from the liquid fixative of about 5% to about 40%, e.g., about 5% to about 20%, about 10% to about 30%, about 20% to about 30%, or about 20% to about 40%. In some embodiments, the test sample comprises a concentration of viscosity modifier derived from the liquid fixative of at least about 5%, e.g., at least about 10%, 15%, 20%, 25%, 30%, 35%, or 40%. In some embodiments, the test sample comprises a concentration of viscosity modifier derived from the liquid fixative of about 40% or less, e.g., about 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less. In some embodiments, the test sample comprises a concentration of viscosity modifier derived from the liquid fixative of about any of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%. In some embodiments, the amount of viscosity modifier in the test sample is based on the desired viscosity of the test sample (e.g., for processing by aspects of the methods described herein, including SEC microfluidic devices).

[0160] In some embodiments, the test sample comprises a concentration of a chaotropic agent (e.g., guanidine hydrochloride) derived from any of the liquid fixatives of about 5M to about 8M, e.g., about 5.5M to about 7.5M, about 5.5M to about 7M, or about 5.5M to about 6.5M, and a concentration of a viscosity modifier (e.g., glycerol) derived from any of the liquid fixatives of about 5% to about 40%, e.g., about 5% to about 20%, about 10% to about 30%, about 20% to about 30%, or about 20% to about 40%. In some embodiments, the test sample comprises a concentration of a chaotropic agent (such as guanidine hydrochloride) from the liquid fixative of at least about 5M, e.g., at least about 5.5M, 6M, 6.5M, 7M, 7.5M, or 8M, and a concentration of a viscosity modifier (such as glycerol) from the liquid fixative of at least about 5%, e.g., at least about 10%, 15%, 20%, 25%, 30%, 35%, or 40%. In some embodiments, the test sample comprises a concentration of a chaotropic agent (such as guanidine hydrochloride) from the liquid fixative of no more than about 8M, e.g., no more than about 7.5M, no more than 6.5M, or no more than 6M, and a concentration of a viscosity modifier (such as glycerol) from the liquid fixative of no more than about 40%, e.g., no more than about 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 10%, or no more than 5%. In some embodiments, the test sample comprises a concentration of a chaotropic agent (such as guanidine hydrochloride) from the liquid fixative of about 5M, 5.5M, 6M, 6.5M, 7M, 7.5M, or 8M, and a concentration of a viscosity adjusting agent (such as glycerol) from the liquid fixative of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%.

[0161] In some embodiments, the test sample comprises a chaotropic agent (e.g., guanidine hydrochloride) from the liquid fixative at a concentration of about 5.5M to about 8M, e.g., about 6M or more, and a viscosity modifier (e.g., glycerol) from the liquid fixative at a concentration of about 5% to about 40%, e.g., about 10% to about 30%.

[0162] In some embodiments, the test sample is a non-depleted sample, e.g., a test sample that has not been treated to remove certain components, such as high-abundance proteins. In some embodiments, the test sample, including the test sample obtained from a blood sample, plasma sample, or serum sample, has not been in a process to remove any one or more of the seven common highly abundant blood proteins (albumin, IgG, antitrypsin, IgA, transferrin, haptoglobin, fibrinogen). In some embodiments, the test sample, including the test sample obtained from a blood sample, plasma sample, or serum sample, has not been in a process to remove any one or more of the fourteen common highly abundant blood proteins (albumin, IgG, antitrypsin, IgA, transferrin, haptoglobin, fibrinogen, α2-macroglobulin, α1-acid glycoprotein, IgM, apolipoprotein AI, apolipoprotein AII, complement C3, transthyretin).

[0163] In some embodiments, the liquid fixative can be diluted with a solution such as water to reach a desired concentration, such as when prepared from a stock formulation (wet or dry). In some embodiments, a viscosity modifier of the liquid fixative is mixed with water to achieve a desired concentration of the liquid fixative. For example, in some embodiments, the liquid fixative includes 7M chaotropic agent mixed in a 10% viscosity modifier / 90% aqueous solution.

[0164] As discussed herein, the concentration of one or more components of the liquid fixative may be based on a desired component concentration from the liquid fixative in a test sample and / or the ratio of sample volume to liquid fixative volume. For example, in some embodiments, the liquid fixative comprises a chaotropic agent and / or a viscosity modifier at a concentration such that when mixed with the sample to generate a test sample, the chaotropic agent and / or viscosity modifier from the liquid fixative is at a concentration described herein.

[0165] In some aspects, provided herein is a method of preparing a test sample of plasma from a blood sample of an individual. In some embodiments, the method for preparing a test sample of plasma is integrated with other methods described herein. In some embodiments, when the sample from the individual is a blood sample, the method further comprises preparing a plasma sample. In some embodiments, preparing the plasma sample comprises subjecting the blood sample to a plasma generation technique. In some embodiments, the plasma generation technique comprises subjecting the sample to a polysulfone medium. In some embodiments, the polysulfone medium is an asymmetric polysulfone material. In some embodiments, the plasma generation technique is a capillary action filtration technique. In some embodiments, the plasma generation technique is a polysulfone (such as asymmetric polysulfone) capillary action filtration technique.

[0166] Additional techniques for generating plasma samples are known in the art, and such techniques may be used in the methods described herein. For example, in some embodiments, the plasma generation technique includes subjecting a blood sample from an individual to centrifugation, where centrifugation of the blood sample is performed in the presence of an anticoagulant (e.g., any one or more of ethylenediaminetetraacetic acid (EDTA), heparin, and citrate) to allow separation of plasma from whole blood. In some embodiments, the plasma generation technique includes subjecting a blood sample from an individual to aggregation. In some embodiments, the plasma generation technique includes subjecting a blood sample from an individual to passive or active microfluidic-based separation. In some embodiments, the plasma generation technique includes subjecting a blood sample from an individual to a medium comprising any one or more of polysulfone, polyethersulfone, and cellulose acetate.

[0167] In some embodiments, the volume of the blood sample subjected to the plasma generation technique is about 10 μL to about 200 μL, such as 10 μL to about 100 μL, for example, about 25 μL to about 75 μL. In some embodiments, the volume of the blood sample subjected to the plasma generation technique is at least about 10 μL, for example, at least about 20 μL, 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, 80 μL, 90 μL, 100 μL, 110 μL, 120 μL, 130 μL, 140 μL, 150 μL, 160 μL, 170 μL, 180 μL, 190 μL, or 200 μL. In some embodiments, the volume of the blood sample subjected to the plasma generation technique is at least about 10 μL, e.g., at least about any of 20 μL, 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, 80 μL, 90 μL, 100 μL, 110 μL, 120 μL, 130 μL, 140 μL, 150 μL, 160 μL, 170 μL, 180 μL, 190 μL, or 200 μL, and less than about 500 μL. In some embodiments, the volume of the blood sample subjected to the plasma generation technique is less than about 200 μL, e.g., less than any of 190 μL, 180 μL, 170 μL, 160 μL, 150 μL, 140 μL, 130 μL, 120 μL, 110 μL, 100 μL, 90 μL, 80 μL, 70 μL, 60 μL, 50 μL, 40 μL, 30 μL, 20 μL, or 10 μL. In some embodiments, the volume of the blood sample subjected to the plasma generation technique is about any of 10 μL, 20 μL, 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, 80 μL, 90 μL, 100 μL, 110 μL, 120 μL, 130 μL, 140 μL, 150 μL, 160 μL, 170 μL, 180 μL, 190 μL, or 200 μL.

[0168] In some embodiments, the volume of the plasma produced is between about 1 μL and about 100 μL. In some embodiments, the volume of the plasma produced is at least about 1 μL, such as at least about 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, 15 μL, 20 μL, 25 μL, 30 μL, 35 μL, 40 μL, 45 μL, 50 μL, 55 μL, 60 μL, 65 μL, 70 μL, 75 μL, 80 μL, 85 μL, 90 μL, 95 μL, or 100 μL. In some embodiments, the volume of plasma produced is about any of 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, 15 μL, 20 μL, 25 μL, 30 μL, 35 μL, 40 μL, 45 μL, 50 μL, 55 μL, 60 μL, 65 μL, 70 μL, 75 μL, 80 μL, 85 μL, 90 μL, 95 μL, or 100 μL.

[0169] In some embodiments, a plasma sample, such as a produced plasma sample, is mixed with a liquid fixative to generate a test sample (test plasma sample). In some embodiments, the plasma sample is mixed with a liquid fixative immediately (e.g., immediately) after preparation of the plasma sample. In some embodiments, the method includes mixing a plasma sample (such as a produced plasma sample) with a liquid fixative to generate a test sample (test plasma sample). As discussed herein, the concentration of a component of the liquid fixative can be adjusted based at least in part on a desired concentration of a component (such as a chaotropic agent and / or a viscosity modifier) ​​in the test sample derived from the liquid fixative. In some embodiments, the volume of the sample (such as a plasma sample) relative to the liquid fixative mixed in the methods described herein may be based at least in part on a desired concentration of a component (such as a chaotropic agent and / or a viscosity modifier) ​​in the test sample derived from the liquid fixative, a desired final volume of the test sample, and / or a limit on the concentration of a particular component in the liquid fixative.

[0170] In some embodiments, test samples, such as test plasma samples, generated using the methods described herein are not further depleted before subjecting the test samples to separation techniques described herein, such as SEC techniques using SEC microfluidic devices. In some embodiments, such depletion methods include the use of any one or more of antibodies, aptamers, other affinity reagents, and molecular membrane ultrafiltration. In some embodiments, test samples, such as test plasma samples, generated using the methods described herein are not further depleted to remove any one or more of the seven common highly abundant blood proteins (albumin, IgG, antitrypsin, IgA, transferrin, haptoglobin, fibrinogen) before subjecting the test samples to separation techniques described herein, such as SEC techniques using SEC microfluidic devices. In some embodiments, a test sample, such as a test plasma sample generated using the methods described herein, is not further depleted to remove any one or more of the 14 common high abundance blood proteins (albumin, IgG, antitrypsin, IgA, transferrin, haptoglobin, fibrinogen, α2-macroglobulin, α1-acid glycoprotein, IgM, apolipoprotein AI, apolipoprotein AII, complement C3, transthyretin) prior to subjecting the test sample to a separation technique described herein, such as a SEC technique using a SEC microfluidic device.

[0171] In some embodiments, the plasma generation technique is performed at ambient temperature, such as at or near room temperature. In some embodiments, the plasma generation technique is performed at a temperature of about 20° C. to about 40° C. In some embodiments, the plasma generation technique is performed at a temperature of about any of 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., or 40° C.

[0172] B. Liquid Chromatography In certain aspects, the methods described herein include liquid chromatography methods (such as liquid chromatography steps) designed to separate and / or concentrate components of a sample or products thereof. In some embodiments, the methods for processing components of a biological sample, such as a sample from an individual, or products thereof, for mass spectrometry analysis include one or more dimensions of chromatography, including two, three, and four dimensions of chromatography. In some embodiments, for methods that include two or more dimensions of chromatography, the chromatographic dimensions are performed offline and may optionally include one or more processing steps before, after, or between. In some embodiments, for methods that include multiple dimensions of chromatography, the chromatographic dimensions are performed online. In some embodiments, the chromatographic dimensions of the methods described herein are orthogonal. In some embodiments, the liquid chromatography methods described herein are completed using a microfluidic device having multiple interconnected channels as described herein.

[0173] In some embodiments, two or more chromatography steps (such as a size-exclusion chromatography step followed by a reversed-phase chromatography step) are completed sequentially, e.g., on the same chip, for applications that do not require an intermediate proteolytic step (e.g., for analysis of native peptides or metabolites that can serve as surrogate markers for protein pathways and networks). In some embodiments, such an integrated two-dimensional μSEC-RP lab chip can be directly interfaced to the atmospheric pressure ionization source of a mass spectrometer.

[0174] i. Size Exclusion Chromatography (SEC) In certain aspects, methods are provided herein that include size exclusion chromatography (SEC) techniques, such as SEC techniques, which are completed using the SEC microfluidic device described herein.In some embodiments, the SEC technique includes introducing a fluid input into the SEC microfluidic device.In some embodiments, the fluid input is a test sample or a derivative thereof, such as the product of any further processing step.

[0175] In some embodiments, the fluid input, such as a test sample, subjected to a SEC technique using a SEC microfluidic device has a volume of about 1 μL to about 200 μL. In some embodiments, the fluid input, such as a test sample, subjected to a SEC technique using a SEC microfluidic device has a volume of at least about 1 μL, for example at least about 5 μL, 10 μL, 15 μL, 20 μL, 25 μL, 30 μL, 35 μL, 40 μL, 45 μL, 50 μL, 55 μL, 60 μL, 65 μL, 70 μL, 75 μL, 80 μL, 85 μL, 90 μL, 95 μL, 100 μL, 110 μL, 120 μL, 130 μL, 140 μL, 150 μL, 160 μL, 170 μL, 180 μL, or 190 μL. In some embodiments, a fluid input, such as a test sample, subjected to a SEC technique using a SEC microfluidic device has a volume of less than about 200 μL, e.g., less than any of about 190 μL, 180 μL, 170 μL, 160 μL, 150 μL, 140 μL, 130 μL, 120 μL, 110 μL, 100 μL, 95 μL, 90 μL, 85 μL, 80 μL, 75 μL, 70 μL, 65 μL, 60 μL, 55 μL, 50 μL, 45 μL, 40 μL, 35 μL, 30 μL, 20 μL, 10 μL, or 5 μL. In some embodiments, a fluid input, such as a test sample, subjected to a SEC technique using a SEC microfluidic device has a volume of about any of 1 μL, 5 μL, 10 μL, 15 μL, 20 μL, 25 μL, 30 μL, 35 μL, 40 μL, 45 μL, 50 μL, 55 μL, 60 μL, 65 μL, 70 μL, 75 μL, 80 μL, 85 μL, 90 μL, 95 μL, 100 μL, 110 μL, 120 μL, 130 μL, 140 μL, 150 μL, 160 μL, 170 μL, 180 μL, 190 μL, or 200 μL.

[0176] In some embodiments, the SEC technique includes the use of a SEC mobile phase having a concentration of a mobile phase chaotropic agent within a range of a predetermined concentration of the chaotropic agent in the test sample. In some embodiments, the range of the concentration of the mobile phase chaotropic agent in the SEC technique is within about ±40% of the predetermined concentration of the chaotropic agent in the test sample, such as about ±35%, ±30%, ±25%, ±20%, ±15%, ±10%, ±8%, ±6%, ±5%, ±4%, ±3%, ±2%, ±1%. For example, in some embodiments, for a test sample containing 6M guanidine hydrochloride, the SEC mobile phase contains guanidine at ±10% of 6M containing 6M.

[0177] In some embodiments, the mobile phase chaotropic agent of the SEC technique is the same as the chaotropic agent of the liquid fixative. In some embodiments, the mobile phase chaotropic agent of the SEC technique is different from the chaotropic agent of the liquid fixative. In some embodiments, the mobile phase chaotropic agent comprises guanidine or a salt thereof, guanidinium or a salt thereof, potassium or a salt thereof, lithium or a salt thereof, magnesium or a salt thereof, or sodium or a salt thereof. In some embodiments, the mobile phase chaotropic agent is selected from the group consisting of guanidine hydrochloride, guanidinium chloride, guanidinium thiocyanate, lithium perchlorate, lithium acetate, magnesium chloride, potassium acetate, and sodium iodide. In some embodiments, the mobile phase chaotropic agent is a guanidine salt. In some embodiments, the mobile phase chaotropic agent is guanidine hydrochloride.

[0178] In some embodiments, the SEC mobile phase comprises a mobile phase chaotropic agent at a concentration of about 5M to about 8M, such as about 5.5M to about 8M, about 5.5M to about 7M, or about 5.5M to about 6.5M. In some embodiments, the SEC mobile phase comprises a mobile phase chaotropic agent at a concentration of at least about 5.5M, such as at least about 6M, 6.5M, 7M, 7.5M, or 8M. In some embodiments, the SEC mobile phase comprises a mobile phase chaotropic agent at a concentration of about 8M or less, such as about 7.5M or less, 6.5M or less, or 6M or less. In some embodiments, the SEC mobile phase comprises a mobile phase chaotropic agent at a concentration of 5.5M, 6M, 6.5M, 7M, 7.5M, or 8M.

[0179] In some embodiments, the SEC mobile phase comprises a mobile phase viscosity modifier. In some embodiments, the mobile phase viscosity modifier is selected from the group consisting of glycerol, propylene glycol, sorbitol, and polyethylene glycol (PEG). In some embodiments, the mobile phase viscosity modifier is glycerol.

[0180] In some embodiments, the SEC mobile phase comprises a mobile phase viscosity modifier at a concentration of about 5% to about 40%, for example, about 5% to about 20%, about 10% to about 30%, about 20% to about 30%, or about 20% to about 40%. In some embodiments, the SEC mobile phase comprises a mobile phase viscosity modifier at a concentration of at least about 5%, for example, at least about 10%, 15%, 20%, 25%, 30%, 35%, or 40%. In some embodiments, the SEC mobile phase comprises a mobile phase viscosity modifier at a concentration of about 40% or less, for example, about 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less. In some embodiments, the SEC mobile phase comprises a mobile phase viscosity modifier at a concentration of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%. In some embodiments, the amount of viscosity modifier in the mobile phase is based on the desired viscosity of the mobile phase (eg, for processing by aspects of the methods described herein, including SEC microfluidic devices).

[0181] In some embodiments, the SEC mobile phase comprises a mobile phase chaotropic agent (e.g., guanidine hydrochloride) at a concentration of about 5M to about 8M, e.g., about 5.5M to about 7.5M, about 5.5M to about 7M, or about 5.5M to about 6.5M, and a mobile phase viscosity modifier (e.g., glycerol) at a concentration of about 5% to about 40%, e.g., about 5% to about 20%, about 10% to about 30%, about 20% to about 30%, or about 20% to about 40%. In some embodiments, the SEC mobile phase comprises a mobile phase chaotropic agent (e.g., guanidine hydrochloride) at a concentration of at least about 5M, e.g., at least about any of 5.5M, 6M, 6.5M, 7M, 7.5M, or 8M, and a mobile phase viscosity modifier (e.g., glycerol) at a concentration of at least about 5%, e.g., at least about any of 10%, 15%, 20%, 25%, 30%, 35%, or 40%. In some embodiments, the SEC mobile phase comprises a mobile phase chaotropic agent (e.g., guanidine hydrochloride) at a concentration of about 8M or less, e.g., at least about 7.5M or less, 6.5M or less, or 6M or less, and a mobile phase viscosity modifier (e.g., glycerol) at a concentration of about 40% or less, e.g., at most about 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less. In some embodiments, the SEC mobile phase comprises a mobile phase chaotropic agent (such as guanidine hydrochloride) at a concentration of about 5M, 5.5M, 6M, 6.5M, 7M, 7.5M, or 8M, and a mobile phase viscosity modifier (such as glycerol) at a concentration of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%.

[0182] In some embodiments, the SEC mobile phase comprises a mobile phase chaotropic agent (e.g., guanidine hydrochloride) at a concentration of about 5.5 M to about 8 M, e.g., about 6 M or more, and a mobile phase viscosity modifier (e.g., glycerol) at a concentration of about 5% to about 40%, e.g., about 10% to about 30%.

[0183] In some embodiments, the mobile phase viscosity modifier of the SEC technique is the same as the viscosity modifier of the liquid fixing agent. In some embodiments, the mobile phase viscosity modifier of the SEC technique is different from the viscosity modifier of the liquid fixing agent.

[0184] In some embodiments, the SEC technique is an isocratic SEC technique (ie, a single SEC mobile phase is used and no gradient of component concentrations is performed).

[0185] In some embodiments, the SEC technique involves the use of a mobile phase flow rate of about 1 μL / min to about 5 μL / min, e.g., about 1 μL / min, 1.5 μL / min, 2 μL / min, 2.5 μL / min, 3 μL / min, 3.5 μL / min, 4 μL / min, 4.5 μL / min, or 5 μL / min. In some embodiments, the mobile phase can be introduced and the flow rate controlled by systems known in the art, such as a syringe pump or an ultra-high performance liquid chromatography pump.

[0186] In some embodiments, the SEC techniques described herein are performed at ambient temperature (e.g., based on column temperature), such as at or near room temperature. In some embodiments, the SEC techniques are performed at elevated temperatures. In some embodiments, the SEC techniques are performed at temperatures between about 15°C and about 60°C, such as between about 15°C and about 45°C, between about 23°C and about 45°C, between about 30°C and about 50°C, or between about 45°C and about 60°C. In some embodiments, the SEC techniques are performed at temperatures of at least about 15°C, such as at least about 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, or 60°C. In some embodiments, the SEC techniques are performed at temperatures less than about 60°C, such as less than about 55°C, 50°C, 45°C, 40°C, 35°C, 30°C, 25°C, 20°C, or 15°C. In some embodiments, the SEC technique is carried out at about any of 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, or 60°C.

[0187] In some embodiments, the SEC technique is performed at a substantially constant temperature. For example, in some embodiments, the SEC technique is performed at a desired temperature range. In some embodiments, the range is about ±8°C, ±6°C, ±5°C, ±4°C, ±3°C, ±2°C, or ±1°C of the desired temperature. For example, in some embodiments, the SEC technique is performed at a range of 21°C ±5°C.

[0188] In some embodiments, the SEC technique involves the use of a SEC medium that is selected based on the desired separation, hi some embodiments, the SEC technique involves selecting a SEC medium based on its properties, such as compatibility with the components and / or pore size of the SEC microfluidic device.

[0189] ii. Reversed-phase high-pressure liquid chromatography Methods are provided that include a reversed-phase liquid chromatography (RPLC) technique as described herein, e.g., an RPLC technique completed using an RPLC microfluidic device as described herein. In some embodiments, the RPLC technique includes introducing a fluid input to an RPLC microfluidic device. In some embodiments, the fluid input is an RPLC-compatible fluid, such as an RPLC-compatible fraction, obtained from a method as described herein, e.g., an SEC technique completed using an SEC microfluidic device as described herein, and optionally subjected to a proteolytic technique.

[0190] In some embodiments, the fraction subjected to the RPLC technique is conditioned from its source. For example, in some embodiments, the fraction subjected to the RPLC technique comprises at least a portion of a SEC fraction, and the SEC fraction is further processed before being subjected to the RPLC technique. In some embodiments, the fraction subjected to the RPLC technique comprises at least a portion of a fraction subjected to a proteolytic technique, and the fraction subjected to a proteolytic technique is further processed before being subjected to the RPLC technique. In some embodiments, the fraction subjected to the RPLC technique comprises at least a portion of a fraction subjected to a quantitative labeling technique, and the fraction subjected to a quantitative labeling technique is further processed before being subjected to the RPLC technique. In some embodiments, the fraction subjected to the RPLC technique has undergone a desalting step. In some embodiments, the fraction subjected to the RPLC technique has undergone a dilution step, such as dilution with an RPLC-compatible solution.

[0191] In some embodiments, each of the set of fractions or a portion thereof is subjected to an RPLC technique described herein, including an RPLC chromatography technique completed using an RPLC microfluidic device. In some embodiments, the set of fractions comprises fractions obtained from a SEC microfluidic device according to an SEC technique, or a processed derivative thereof. In some embodiments, the set of fractions comprises fractions obtained from a proteolytic technique, or a processed derivative thereof. In some embodiments, the set of fractions comprises a portion of the fractions from the SEC microfluidic device and another portion of the fractions from the SEC microfluidic device that have been subjected to a proteolytic technique.

[0192] In some embodiments, fluid inputs to a RPLC microfluidic device, such as fractions, have a volume of about 1 μL to about 50 μL, such as about 1 μL to about 25 μL or about 5 μL to about 20 μL. In some embodiments, fluid inputs to a μRPLC microfluidic device, such as fractions, have a volume of at least about 1 μL, such as at least about any of 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, 15 μL, 20 μL, 25 μL, 30 μL, 35 μL, 40 μL, 45 μL, or 50 μL. In some embodiments, fluid inputs to an RPLC microfluidic device, such as fractions, have a volume of less than about 50 μL, such as less than about any of 45 μL, 40 μL, 35 μL, 30 μL, 25 μL, 20 μL, 15 μL, 10 μL, 9 μL, 8 μL, 7 μL, 6 μL, 5 μL, 4 μL, 3 μL, 2 μL, or 1 μL. In some embodiments, fluid inputs to an RPLC microfluidic device, such as fractions, have a volume of less than about any of 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, 15 μL, 20 μL, 25 μL, 30 μL, 35 μL, 40 μL, 45 μL, or 50 μL.

[0193] In some embodiments, the RPLC technique includes the use of an RPLC mobile phase. RPLC mobile phases are well known in the art and are compatible with the methods and devices described herein. In some embodiments, the RPLC mobile phase is a dynamic mobile phase that is adjusted during the course of the RPLC technique, such as to facilitate elution of components of the sample or its products. For example, in some embodiments, the RPLC mobile phase includes a concentration of an aqueous solution and a concentration of an organic solution. In some embodiments, the aqueous solution includes water, such as ultrapure water. In some embodiments, the organic solution includes acetonitrile. In some embodiments, the RPLC mobile phase includes additional components useful for the RPLC technique and / or mass spectrometry. For example, in some embodiments, the RPLC mobile phase is adjusted with a weak acid to have an acidic pH. In some embodiments, the RPLC mobile phase includes a weak acid, such as formic acid, trifluoroacetic acid, or acetic acid. In some embodiments, the concentration of the weak acid in the RPLC mobile phase is less than about 0.5%, such as about 0.4%, 0.3%, 0.2%, or 0.1%.

[0194] In some embodiments, the RPLC technique is a gradient RPLC technique (ie, a gradient of mobile phase components, such as increasing amounts of organic phase in the mobile phase, is used for elution).

[0195] In some embodiments, the RPLC technique involves the use of a mobile phase flow rate of about 0.05 μL / min to about 2 μL / min, e.g., about 0.1 μL / min, 0.2 μL / min, 0.3 μL / min, 0.4 μL / min, 0.5 μL / min, 0.6 μL / min, 0.7 μL / min, 0.8 μL / min, 0.9 μL / min, 1 μL / min, 1.1 μL / min, 1.2 μL / min, 1.3 μL / min, 1.4 μL / min, 1.5 μL / min, 1.6 μL / min, 1.7 μL / min, 1.8 μL / min, 1.9 μL / min, or 2 μL / min. In some embodiments, the mobile phase can be introduced and the flow rate controlled by systems known in the art, such as a syringe pump or an ultra-high performance liquid chromatography pump.

[0196] In some embodiments, the RPLC techniques described herein are performed at ambient temperature, such as at or near room temperature (e.g., as assessed by column temperature). In some embodiments, the RPLC techniques are performed at elevated temperatures. In some embodiments, the SEC techniques are performed at temperatures between about 15°C and about 100°C, such as between about 15°C and about 45°C, between about 23°C and about 45°C, between about 30°C and about 50°C, or between about 45°C and about 60°C. In some embodiments, the RPLC techniques are performed at temperatures of at least about 15°C, such as at least about 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, or 100°C. In some embodiments, the SEC technique is carried out at a temperature less than about 100° C., such as less than about any of 95° C., 90° C., 85° C., 80° C., 75° C., 70° C., 65° C., 60° C., 55° C., 50° C., 45° C., 40° C., 35° C., 30° C., 25° C., 20° C., or 15° C. In some embodiments, the RPLC technique is carried out at a temperature less than about any of 15° C., 20° C., 25° C., 30° C., 35° C., 40° C., 45° C., 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., 80° C., 85° C., 90° C., 95° C., or 100° C.

[0197] In some embodiments, the RPLC technique is performed at a substantially constant temperature. For example, in some embodiments, the RPLC technique is performed at a desired temperature range. In some embodiments, the range is about ±8° C., ±6° C., ±5° C., ±4° C., ±3° C., ±2° C., or ±1° C. of the desired temperature. For example, in some embodiments, the RPLC technique is performed at a range of ±5° C. of 21° C.

[0198] C. Fraction Collection In some aspects, provided herein are fraction collection techniques and fraction collection devices useful for capturing fractions (e.g., individual segments) of a sample after some degree of separation using the chromatographic techniques described herein.

[0199] In some embodiments, the fraction characteristics (such as size or collection duration) are based, at least in part, on the desired division of the separation performed by the liquid chromatography techniques described herein. In some embodiments, the method includes selecting fractions based on elution time. For example, in some embodiments, fractions are collected from a liquid chromatography technique, such as a SEC technique using a SEC microfluidic device described herein, for a period of about 30 seconds to about 5 minutes, e.g., about 30 seconds to about 3 minutes, about 1 minute to about 2 minutes, about 1 minute to about 4 minutes, or about 2 minutes to about 5 minutes. In some embodiments, fractions are collected from a liquid chromatography technique, such as a SEC technique using a SEC microfluidic device described herein, for a period of at least about 30 seconds, e.g., at least about 1 minute, 1.5 minutes, 2 minutes, 2.5 minutes, 3 minutes, 3.5 minutes, 4 minutes, 4.5 minutes, or 5 minutes. In some embodiments, fractions are collected from a liquid chromatography technique, such as a SEC technique using a SEC microfluidic device described herein, for a period of about 5 minutes or less, such as less than about any of 4.5 minutes or less, 4 minutes or less, 3.5 minutes or less, 3 minutes, 2.5 minutes, 2 minutes, 1.5 minutes, 1 minute, or 30 seconds. In some embodiments, fractions are collected from a liquid chromatography technique, such as a SEC technique using a SEC microfluidic device described herein, for a period of about 30 seconds, 1 minute, 1.5 minutes, 2 minutes, 2.5 minutes, 3 minutes, 3.5 minutes, 4 minutes, 4.5 minutes, or 5 minutes. In some embodiments, each of the multiple fractions is collected from the SEC microfluidic device over a period of about 1 minute to about 2 minutes.

[0200] In some embodiments, each of the plurality of fractions is collected for a uniform time from a liquid chromatography technique, such as a SEC technique using a SEC microfluidic device as described herein. In some embodiments, one fraction of the plurality of fractions is collected for a different time from another fraction of the plurality of fractions from a liquid chromatography technique, such as a SEC technique using a SEC microfluidic device as described herein.

[0201] In some embodiments, fractions are collected from a liquid chromatography technique, such as a SEC technique using a SEC microfluidic device as described herein, based on the volume of eluate therefrom. In some embodiments, the fractions have a volume of about 1 μL to about 20 μL, such as about 1 μL to about 8 μL, about 5 μL to about 15 μL, or about 10 μL to about 20 μL. In some embodiments, the fractions have a volume of at least about 1 μL, such as at least about 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, 11 μL, 12 μL, 13 μL, 14 μL, 15 μL, 16 μL, 17 μL, 18 μL, 19 μL, or 20 μL. In some embodiments, the fractions have a volume of about 20 μL or less, such as about 19 μL or less, 18 μL or less, 17 μL or less, 16 μL or less, 15 μL or less, 14 μL or less, 13 μL or less, 12 μL or less, 11 μL or less, 10 μL or less, 9 μL or less, 8 μL or less, 7 μL or less, 6 μL or less, 5 μL or less, 4 μL or less, 3 μL or less, 2 μL or less, or 1 μL or less. In some embodiments, the fractions have a volume of about 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, 11 μL, 12 μL, 13 μL, 14 μL, 15 μL, 16 μL, 17 μL, 18 μL, 19 μL, or 20 μL. In some embodiments, each of the multiple fractions collected from a liquid chromatography technique, such as a SEC technique using a SEC microfluidic device as described herein, has a uniform volume. In some embodiments, one fraction of the multiple fractions collected from a liquid chromatography technique, such as a SEC technique using a SEC microfluidic device as described herein, has a different volume from another fraction of the multiple fractions.

[0202] In some embodiments, the method includes collecting a plurality of fractions from a liquid chromatography technique, such as a SEC technique, using a SEC microfluidic device as described herein. In some embodiments, the plurality of fractions is about 5 fractions to about 50 fractions, such as about 5 fractions to about 30 fractions, about 12 fractions to about 24 fractions, or about 30 fractions to about 50 fractions. In some embodiments, the plurality of fractions is at least about 5 fractions, such as at least about 10 fractions, 11 fractions, 12 fractions, 13 fractions, 14 fractions, 15 fractions, 16 fractions, 17 fractions, 18 fractions, 19 fractions, 20 fractions, 21 fractions, 22 fractions, 23 fractions, 24 fractions, 25 fractions, 30 fractions, 35 fractions, 40 fractions, 45 fractions, or 50 fractions. In some embodiments, the plurality of fractions is about 50 or less fractions, e.g., about 45 or less fractions, 40 or less fractions, 35 or less fractions, 30 or less fractions, 25 or less fractions, 24 or less fractions, 23 or less fractions, 22 or less fractions, 21 or less fractions, 20 or less fractions, 19 or less fractions, 18 or less fractions, 17 or less fractions, 16 or less fractions, 15 or less fractions, 14 or less fractions, 13 or less fractions, 12 or less fractions, 11 or less fractions, 10 or less fractions, or 5 or less fractions. In some embodiments, the plurality of fractions is about 5 fractions, 10 fractions, 11 fractions, 12 fractions, 13 fractions, 14 fractions, 15 fractions, 16 fractions, 17 fractions, 18 fractions, 19 fractions, 20 fractions, 21 fractions, 22 fractions, 23 fractions, 24 fractions, 25 fractions, 30 fractions, 35 fractions, 40 fractions, 45 fractions, or 50 fractions. In some embodiments, the plurality of fractions is about 12 fractions to about 24 fractions, including about 12 fractions.

[0203] In some embodiments, the fractions are collected using a fraction collector. In some embodiments, the fraction collector is connected to a liquid chromatography device described herein, such as a SEC microfluidic device. In some embodiments, the fractions are collected via a microfluidic or chip-based feature, such as a compartment of a microfluidic device (e.g., a lab-on-a-chip device). In some embodiments, the fractions eluted from the SEC microfluidic device described herein are collected using a chip-based fraction collector (e.g., a lab-on-a-chip device).

[0204] D. Melting technology In some embodiments, the method includes a lysis technique, such as a proteolytic technique. In some embodiments, the lysis technique results in the separation of some of the components of the sample or their products. For example, in some embodiments, the lysis technique is a proteolytic technique that degrades a polypeptide into two or more products. In some embodiments, the lysis technique separates metabolic products (such as post-translational modifications) from the polypeptide. In some embodiments, the lysis technique separates metabolic products into two or more products.

[0205] Proteolytic techniques for generating polypeptides, such as peptides, products of parent polypeptides of a sample for analysis by mass spectrometry techniques are known in the art. In some embodiments, polypeptide products, such as peptides, of parent polypeptides are obtained by proteolysis (e.g., sample digestion) before subjecting the polypeptide products to a mass spectrometer. In some embodiments, polypeptides, such as peptides, products of parent polypeptides are obtained within a mass spectrometer. In some embodiments, proteolytic techniques are performed on one or more, e.g., all, of a plurality of fractions obtained from the methods described herein. In some embodiments, proteolytic techniques are performed on a portion of a sample or fractions obtained from the methods described herein.

[0206] In some embodiments, the proteolytic techniques include enzyme-based digestion techniques. In some embodiments, the enzyme-based digestion techniques include the use of proteolytic enzymes, such as proteases. In some embodiments, the proteolytic enzymes are trypsin, chymotrypsin, thermolysin, pepsin, elastase, Lys-C, Lys-N, Asp-N, Glu-C, Arg-C, TEV, IdeS, IdeZ, PNGase F, Factor Xa, or combinations thereof.

[0207] In some embodiments, the proteolytic technique is a chemical-based proteolytic technique, hi some embodiments, the chemical-based proteolytic technique comprises the use of an acid, such as a strong acid.

[0208] In some embodiments, the proteolytic technique is a solution phase proteolytic technique. In some embodiments, the proteolytic technique is a solid phase or solid state proteolytic technique. In some embodiments, the proteolytic technique is a gel phase proteolytic technique.

[0209] Techniques for carrying out the lysis techniques or parts thereof included in the present disclosure are well known in the art. In some embodiments, considerations for such techniques include the environment of the reaction, such as the solution and its components, temperature, duration, and ratio of digestion components, such as proteases, to the components of the sample. For example, in some embodiments, a liquid phase trypsin proteolysis technique includes mixing trypsin with the dilution fraction in a ratio of about 1:30 and incubating at about 37° C. for about 8 hours.

[0210] In some embodiments, the lysis technique, such as a proteolysis technique, includes a step of diluting an input to the technique, such as a fraction obtained from a method described herein. In some embodiments, the dilution is performed using water, an organic solvent, a weak buffer, a compatible buffer, or a combination thereof. In some embodiments, the dilution is performed to ensure compatibility of the resulting diluted material with the lysis technique. In some embodiments, the dilution step is based on obtaining a final concentration of chaotropic agent (such as guanidine hydrochloride) of about 0.1 to about 2M, such as about 0.1M to about 0.5M, about 0.5M to about 1.5M, or about 1M to about 2M. In some embodiments, the dilution step is based on obtaining a final concentration of chaotropic agent less than about 1M, such as less than about 0.9M, 0.8M, 0.7M, 0.6M, 0.5M, 0.4M, 0.3M, 0.2M, 0.1M, or 0.05M.

[0211] In some embodiments, the enzyme-based digestion technique does not include a buffer exchange step. In some embodiments, the enzyme-based digestion technique does not include an alkylation step. In some embodiments, the enzyme-based digestion technique does not include a reduction step.

[0212] E. Quantification technology In some embodiments, the methods described herein include quantification techniques. In some embodiments, the quantification methods provide a measure of the abundance of a component or its product in a sample. In some embodiments, the quantification methods are relative quantification methods. In some embodiments, the quantification methods are semi-relative quantification methods. In some embodiments, the quantification methods are absolute quantification methods. In some embodiments, the quantification methods are label-free quantification methods. In some embodiments, the quantification methods are label-based quantification methods, such as those involving the use of isobaric tags, e.g., tandem mass tags. In some embodiments, the quantification methods are spike-in methods, such as those involving the use of one or more standards, e.g., isotopically labeled peptides. In some embodiments, the quantification methods include any combination of quantification methods.

[0213] In some embodiments, the quantification method includes a cleanup step before starting downstream steps of the method, for example, in some embodiments, the quantification method includes a desalting step, such as to remove excess label that is not bound to components of the sample or its products.

[0214] Methods for quantification by mass spectrometry are well known in the art, for example, as described in Bantscheff et al., Anal Bioanal Chem, 389, 2007, which is incorporated herein by reference in its entirety.

[0215] F. Introducing Sample Components or Their Products into a Mass Spectrometer Provided herein are techniques and devices (such as emitters) for introducing components of a sample or products thereof into mass spectrometry. The introduction techniques and devices thereof are well known in the art and are compatible with the methods described herein. In some embodiments, the introduction technique comprises an ionization technique. In some embodiments, the ionization technique is an electrospray ionization technique. In some embodiments, the electrospray ionization technique is based on the flow rate used in the technique. For example, in some embodiments, the ionization technique is a nano-electrospray ionization technique. In some embodiments, the electrospray ionization technique comprises the use of an electrospray ionization source, such as a nano-electrospray ionization source. In some embodiments, the ionization technique is an atmospheric pressure chemical ionization technique. In some embodiments, the ionization method is an atmospheric pressure photoionization technique. In some embodiments, the ionization technique is an offline desorption electrospray ionization (DESI) technique. In some embodiments, the ionization technique is an offline matrix-assisted laser desorption ionization (MALDI) technique.

[0216] In some embodiments, the electrospray ionization source is a heated electrospray ionization source. In some embodiments, the electrospray ionization source is coupled with a gas drying mechanism, such as a nitrogen flow or curtain.

[0217] In some embodiments, the ionization technique, such as an online ionization technique, is coupled with an atmospheric pressure high field asymmetric waveform ion mobility spectrometry (FAIMS) system equipped with a mass spectrometer.

[0218] G. Mass Spectrometry and Data Acquisition Techniques The present application contemplates a diverse array of mass spectrometry techniques suitable for use in the methods and method steps disclosed herein, including determining a mass spectrometry profile. In some embodiments, the methods disclosed herein include analyzing a sample using one or more mass spectrometry techniques. As described herein, in some embodiments, mass spectrometry techniques are used to obtain data that is useful for providing and / or obtaining a vast amount of information regarding components or products of a sample, including any combination of MS ion information (m / z and abundance), peptide and / or protein identification / sequence information, post-translational modification information, identification / sequence information such as metabolite identity, and quantification information.

[0219] In some embodiments, the mass spectrometer technique includes the use of mass spectrometry techniques. Mass spectrometers contemplated by the present application include high resolution mass spectrometers and low resolution mass spectrometers. In some embodiments, the mass spectrometer is a time-of-flight (TOF) mass spectrometer. In some embodiments, the mass spectrometer is a quadrupole time-of-flight (Q-TOF) mass spectrometer. In some embodiments, the mass spectrometer is a single quadrupole. In some embodiments, the mass spectrometer is a triple quadrupole (QQQ). In some embodiments, the mass spectrometer is a quadrupole ion trap time-of-flight (QIT-TOF) mass spectrometer. In some embodiments, the mass spectrometer is a quadrupole linear ion trap (Q-LIT). In some embodiments, the mass spectrometer relies on a Fourier transform orbitrap as one of its constituent ion optical components, such as a hybrid quadrupole orbitrap, a linear ion trap orbitrap, or a tribrid quadrupole linear ion trap orbitrap variant. In some embodiments, the mass spectrometer is a FT ion cyclotron resonance (FT) mass spectrometer. In some embodiments, the mass spectrometer is a quadrupole FT-ion cyclotron resonance (Q-FT) mass spectrometer. In some embodiments, the mass spectrometer is a magnetic sector mass spectrometer.

[0220] In some embodiments, the mass spectrometry technique comprises the use of a positive ion mode. In some embodiments, the mass spectrometry technique comprises a negative ion mode. In some embodiments, the mass spectrometry technique comprises an ion mobility mass spectrometry technique.

[0221] In some embodiments, the mass spectrometry technique comprises a top-down mass spectrometry technique. In some embodiments, the mass spectrometry technique comprises a middle-down mass spectrometry technique. In some embodiments, the mass spectrometry technique comprises a bottom-up mass spectrometry technique. In some embodiments, the mass spectrometry technique comprises a tandem mass spectrometry technique. In some embodiments, the tandem mass spectrometry technique comprises a fragmentation technique. In some embodiments, the methods described herein encompass any combination thereof.

[0222] A variety of mass spectrometry data acquisition techniques are suitable for the methods described herein. For example, in some embodiments, mass spectrometry data acquisition techniques include data-dependent data acquisition, data-independent data acquisition, targeted data acquisition, or a combination thereof.

[0223] In some aspects, provided herein are methods of analyzing a collection of compositions using mass spectrometry, comprising: (a) subjecting each composition of the collection of compositions to a mass spectrometer; and (b) performing mass spectrometry on each composition of the collection of compositions, wherein the collection of compositions is obtained from a processing technique comprising fractionation of a test sample using a SEC technique comprising use of a SEC microfluidic device, followed by applying each fraction or a product thereof to an RPLC technique comprising use of an RPLC microfluidic device. In some embodiments, the SEC fractions are further processed by a proteolytic technique.

[0224] H. Discovery and Target-Based Modes of the Methods Described Herein The methods described herein include discovery mode methods, semi-targeted mode methods, targeted mode methods, and combinations thereof. The use of and selection of the type of mode can be based on the desired information to evaluate in the sample. For example, in some embodiments, it is desirable to study a large number of components of the sample, e.g., in a hypothesis-free evaluation of the sample (e.g., may be more suitable for discovery mode or semi-targeted mode). In some embodiments, it is desirable to study a small selection of components of the sample (e.g., may be more suitable for targeted mode). Based on the purpose and / or desired information, one of skill in the art will readily understand how to design and implement the methods described herein based on the teachings provided herein. For example, the purpose and / or desired information can be used to design how many fractions are generated and obtained from the SEC technique, how many SEC fractions are further analyzed, and what further processing, if any, is performed (such as proteolytic techniques), as well as which mass spectrometer and mass spectrometry technique is used.

[0225] I. Exemplary Methods for Analyzing Components of a Sample In some embodiments, a method of processing components of a biological sample or products thereof for mass spectrometry comprises: (a) subjecting a test sample to a size exclusion chromatography (SEC) technique using a SEC microfluidic device, wherein the test sample comprises a sample mixed with a liquid fixative, the test sample having a predetermined concentration of a chaotropic agent derived from the liquid fixative, the SEC technique comprising use of a SEC mobile phase having a concentration of the mobile phase chaotropic agent within a range of the predetermined concentration of the chaotropic agent in the test sample, the SEC microfluidic device comprising a plurality of interconnected channels, each channel configured in an open tubular format having an inner surface material having an average pore size of about 10 nm to about 500 nm; (b) collecting a plurality of fractions eluted from the SEC microfluidic device; and (c) collecting one or more of the plurality of fractions from the SEC microfluidic device. (d) subjecting each of the set of RPLC-compatible fractions to a proteolytic technique using a reversed-phase liquid chromatography (RPLC) microfluidic device to prepare components of the sample or products thereof for introduction into a mass spectrometer, the set of RPLC-compatible fractions being (i) fractions obtained from zero or more of the plurality of fractions from the SEC microfluidic device, (ii) one or more of the plurality of fractions subjected to the proteolytic technique, the RPLC technique and the RPLC microfluidic device being configured for online desalting, the RPLC microfluidic device comprising a plurality of interconnected channels, each channel configured in an open tubular format having an inner surface material comprising a reversed-phase medium, and the RPLC microfluidic device being coupled to an electrospray ionization source. In some embodiments, the biological sample is a plasma sample from an individual, such as a human. In some embodiments, the chaotropic agent, such as that found in liquid fixatives and SEC mobile phases, is guanidine hydrochloride. In some embodiments, the method further comprises subjecting the eluate from the RPLC microfluidic device to a mass spectrometer. In some embodiments, the individual is a human.

[0226] In some embodiments, provided herein is a method of processing components of a human-derived plasma sample or products thereof for mass spectrometry analysis, the method comprising: (a) subjecting a test plasma sample to a size exclusion chromatography (SEC) technique using a SEC microfluidic device, wherein the test sample comprises a sample mixed with a liquid fixative, the test sample having at least about 5.5 M, e.g., at least about 6 M, guanidine (e.g., from guanidine hydrochloride) from the liquid fixative, the SEC technique comprising use of a SEC mobile phase having at least about 5.5 M, e.g., at least about 6 M, guanidine (e.g., from guanidine hydrochloride), the SEC microfluidic device comprising a plurality of interconnected channels, each channel configured in an open tubular format having an inner surface material having an average pore size of about 10 nm to about 500 nm; and (b) collecting a plurality of fractions eluted from the SEC microfluidic device. (c) subjecting one or more of the plurality of fractions from the SEC microfluidic device to a proteolytic technique using a proteolytic enzyme, such as trypsin; and (d) subjecting each of a set of RPLC-compatible fractions to a reversed-phase liquid chromatography (RPLC) technique using an RPLC microfluidic device to prepare components of the sample or products thereof for introduction into a mass spectrometer, wherein the set of RPLC-compatible fractions includes (i) fractions obtained from zero or more of the plurality of fractions from the SEC microfluidic device; and (ii) one or more of the fractions subjected to the proteolytic technique, wherein the RPLC technique and the RPLC microfluidic device are configured for online desalting, and the RPLC microfluidic device includes a plurality of interconnected channels, each channel configured in an open tubular format having an inner surface material comprising a reversed-phase medium, and the reversed-phase medium is selected from the group consisting of C2, C4, C8, or C9. 18 and wherein the RPLC microfluidic device is coupled to an electrospray ionization source. In some embodiments, the method further comprises generating a plasma sample. In some embodiments, the method further comprises subjecting the eluate from the RPLC microfluidic device to a mass spectrometer.

[0227] In some embodiments, provided herein is a method of processing components of a blood sample from a human or products thereof for mass spectrometry analysis, the method comprising: (a) generating a test plasma sample from the blood sample, the test plasma sample comprising a plasma sample from the blood sample mixed with a liquid fixative, the test plasma sample having at least about 5.5 M, such as at least about 6 M, guanidine (e.g., from guanidine hydrochloride) from the liquid fixative; (b) subjecting the test plasma sample to a size exclusion chromatography (SEC) technique using a SEC microfluidic device, the SEC technique comprising use of a SEC mobile phase having at least about 5.5 M, such as at least about 6 M, guanidine (e.g., from guanidine hydrochloride), the SEC microfluidic device comprising a plurality of interconnected channels, each channel configured in an open tubular format having an inner surface material having an average pore size of about 10 nm to about 500 nm; and (c) subjecting the test plasma sample to a size exclusion chromatography (SEC) technique using a SEC microfluidic device, the SEC technique comprising use of a SEC mobile phase having at least about 5.5 M, such as at least about 6 M, guanidine (e.g., from guanidine hydrochloride). (d) subjecting one or more of the plurality of fractions from the SEC microfluidic device to a proteolytic technique using a proteolytic enzyme such as trypsin; and (e) subjecting each of a set of RPLC-compatible fractions to a reversed-phase liquid chromatography (RPLC) technique using an RPLC microfluidic device to prepare components of the sample or products thereof for introduction into a mass spectrometer, wherein the set of RPLC-compatible fractions comprises (i) zero or more of the plurality of fractions from the SEC microfluidic device and (ii) one or more of the plurality of fractions subjected to the proteolytic technique, wherein the RPLC technique and the RPLC microfluidic device are configured for online desalting, and the RPLC microfluidic device comprises a plurality of interconnected channels, each channel configured in an open tubular format having an inner surface material comprising a reversed-phase medium, and wherein the reversed-phase medium is selected from the group consisting of C2, C4, C8, or C9. 18and wherein the RPLC microfluidic device is coupled to an electrospray ionization source. In some embodiments, the method further comprises subjecting the eluate from the RPLC microfluidic device to a mass spectrometer.

[0228] III. Characteristics of Coronary Artery Disease (CAD) and How to Use It In certain aspects, a coronary artery disease (CAD) signature is provided herein, comprising a plurality of biomarkers identified using the methods and devices described herein based on the evaluation of samples from individuals with coronary artery disease (CAD) compared to healthy individuals.The major disease subtype of cardiovascular disease (CVD) is coronary artery disease (CAD), characterized by narrowing and stiffening of heart arteries, known as atherosclerosis.Atherosclerosis is caused by multiple pathological mechanisms, including endothelial damage and subendothelial apoB-lipoprotein retention, insulin resistance, oxidative stress, DNA damage and aging, autophagy, lipid metabolism dysregulation, inflammation, and thrombosis, making it difficult to identify its signature.

[0229] The discovery of a CAD signature allows for the use of one or more biomarkers thereof, for example, in analytical, diagnostic, and therapeutic methods for detecting the CAD proteomic signature in an individual. In some embodiments, the methods provided herein utilize only a subset of the biomarkers of the identified CAD signature, such as one or more biomarkers of the CAD signature. In some embodiments, provided is a CAD proteomic signature that includes one or more biomarkers of the CAD signature provided in Table 1 (provided below). In some embodiments, the CAD proteomic signature is assessed via polypeptides in the sample, such as using mass spectrometry techniques. In some embodiments, the CAD proteomic signature is assessed by a non-mass spectrometry based technique, such as ELISA.

[0230] In some embodiments, the methods provided herein include analyzing mass spectrometry (MS) data according to a CAD proteomic signature, the CAD proteomic signature comprising one or more biomarkers of Table 1. In some embodiments, each biomarker of the CAD proteomic signature comprises the identity of the protein and the status of increased or decreased expression of the protein (as described in the CAD signature column of Table 1) compared to a reference (the level of the protein in one or more healthy individuals, e.g., individuals without CAD). For example, in some embodiments, the methods provided herein for evaluating a CAD proteomic signature evaluate a sample obtained from an individual, or a derivative thereof, for the presence of one or more biomarkers of the CAD proteomic signature, and whether the one or more biomarkers of the CAD proteomic signature substantially match (e.g., at least about 70% (including at least about any of 75%, 80%, 85%, 90%, or 95% of the one or more biomarkers)) an increased or decreased expression classification of Table 1. In some embodiments, the methods provided herein for evaluating a CAD proteomic signature evaluate a sample obtained from an individual, or a derivative thereof, for the presence of one or more biomarkers of the CAD proteomic signature, and whether the one or more biomarkers of the CAD proteomic signature are consistent with the up- or down-expression classifications of Table 1.

[0231] In some embodiments, each biomarker of a CAD proteomic signature includes the identity of the protein and the level of increased or decreased expression of the protein (e.g., a level above a defined set threshold for increased or decreased expression) compared to a reference (the level of the protein in one or more healthy individuals, e.g., individuals without CAD). In some embodiments, the increased expression of the protein is an average log2 ratio of at least about 0.2, e.g., at least about any of 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, when measured in an individual compared to a reference. In some embodiments, the reduced expression of the protein, when measured in an individual compared to a reference, is a mean log2 ratio of about -0.2 or less, such as about any of -0.3, -0.4, -0.5, -0.6, -0.7, -0.8, -0.9, -1.0, -1.1, -1.2, -1.3, -1.4, -1.5, -1.6, -1.7, -1.8, -1.9, or -2.0 or less. In some embodiments, the increased or decreased expression of one or more biomarkers of the CAD proteomic signature is within about 0.1 or less standard deviations of the mean log2 ratio of Table 1. In some embodiments, the increased or decreased expression of one or more biomarkers of the CAD proteomic signature is within about 0.05 or less standard deviations of the mean log2 ratio of Table 1.

[0232] In some embodiments, the state and / or degree of increased or decreased expression is based on a comparison to a reference, e.g., a healthy individual, e.g., an individual without CAD. In some embodiments, the reference is a literature value, such as one published in a scientific reference work. In some embodiments, the reference is based on a population of healthy individuals, e.g., individuals without CAD. In some embodiments, the reference is the average expression level measured from a population of healthy individuals, e.g., individuals without CAD.

[0233] In some embodiments, the method is based on one or more measurements from one or more samples or derivatives thereof obtained from an individual. In some embodiments, where one or more measurements are taken to assess a biomarker, the method can be based on an average measurement of said biomarker.

[0234] In some embodiments, the CAD proteomic signature comprises one or more biomarkers associated with calcium signaling pathway, histone regulation, HIF-1 signaling pathway, cAMP signaling pathway, β-adrenergic signaling pathway, PI3K-Akt signaling pathway, complement and / or coagulation cascade, sphingolipid signaling pathway, natural killer cell-mediated cytotoxicity, adipocytokine signaling pathway, DNA damage, calcium energy, metabolites, cell adhesion, inflammation, hypoxia, and histone methylation.

[0235] In some embodiments, the one or more biomarkers of the CAD proteomic signature comprises a subset thereof that includes one or more biomarkers associated with transcription factors. In some embodiments, the one or more biomarkers associated with transcription factors are each selected from the group consisting of NF4A, FOXA2, LMO2, RUNX1, FLI1, EGR1, VDR, RCF21, GATA2, TP63, ELK3, FLI1, GATA1, CTNNB1, SIN3B, STAT3, TAP1, AHR, MTF2, and SRY.

[0236] In some embodiments, the one or more biomarkers of the CAD proteomic signature comprises a subset thereof that includes one or more biomarkers associated with a kinase. In some embodiments, the one or more biomarkers associated with a kinase are each selected from the group consisting of HIPK2, MAPK1, MAPK3, GSK3B, MAPK8, TAF1, AKT1, CDK1, MAPK14, CDK9, CSNK2A1, CHUK, NLK, ABL1, CDK6, CDK2, CDK7, CDK4, TRIM24, and PRKCZ.

[0237] In some embodiments, the CAD proteomic signature comprises 10 biomarkers, 15 biomarkers, 20 biomarkers, 25 biomarkers, 30 biomarkers, 35 biomarkers, 40 biomarkers, 45 biomarkers, 50 biomarkers, 55 biomarkers, 60 biomarkers, 65 biomarkers, 70 biomarkers, 75 biomarkers, 80 biomarkers, 85 biomarkers, 90 biomarkers, 95 biomarkers, 10 ... markers, 110 biomarkers, 120 biomarkers, 130 biomarkers, 140 biomarkers, 150 biomarkers, 160 biomarkers, 170 biomarkers, 180 biomarkers, 190 biomarkers, 200 biomarkers, 210 biomarkers, 220 biomarkers, 230 biomarkers, 240 biomarkers, 250 biomarkers, 260 biomarkers, 270 biomarkers, 280 biomarkers, or 290 biomarkers. In some embodiments, the CAD proteomic signature comprises all of the biomarkers in Table 1. In some embodiments, the CAD proteomic signature is analyzed based on the state of increased or decreased expression of the biomarkers therein according to Table 1. In some embodiments, the CAD proteomic signature is analyzed based on the level of increased or decreased expression of the biomarkers therein according to Table 1.

[0238] In some embodiments, the CAD proteomic signature comprises Q969E1 (LEAP2), Q8NF37 (LPCAT1), Q01082 (SPTBN1), Q7Z333 (SETX), and P30481 (HLA-B).

[0239] In some embodiments, the CAD proteomic signature comprises Q969E1 (LEAP2), Q8NF37 (LPCAT1), Q01082 (SPTBN1), Q7Z333 (SETX), P30481 (HLA-B), Q5T8A7 (PPP1R26), Q9NX02 (NLRP2), P02144 (MB), Q9BQS2 (SYT15), and P62805 (HIST1H4A).

[0240] In some embodiments, the CAD proteomic signature comprises Q969E1 (LEAP2), Q8NF37 (LPCAT1), Q01082 (SPTBN1), Q7Z333 (SETX), P30481 (HLA-B), Q5T8A7 (PPP1R26), Q9NX02 (NLRP2), P02144 (MB), Q9BQS2 (SYT15), P62805 (HIST1H4A), P80370 (DLK1), P68366 (TUBA4A), P27797 (CALR), P05164 (MPO), and Q99439 (CNN2).

[0241] In some embodiments, the CAD proteomic signature comprises Q86YI8 (PHF13), Q9Y4D8 (HECTD4), Q9UIW2 (PLXNA1), Q6ZS81 (WDFY4), and Q9H329 (EPB41L4B).

[0242] In some embodiments, the CAD proteomic signature comprises Q86YI8 (PHF13), Q9Y4D8 (HECTD4), Q9UIW2 (PLXNA1), Q6ZS81 (WDFY4), Q9H329 (EPB41L4B), A2RUB1 (C17orf104), O15031 (PLXNB2), Q9NYF3 (FAM53C), O75146 (HIP1R), and P80362 (Ig kappa chain VI region WAT).

[0243] In some embodiments, the CAD proteomic signature comprises Q86YI8 (PHF13), Q9Y4D8 (HECTD4), Q9UIW2 (PLXNA1), Q6ZS81 (WDFY4), Q9H329 (EPB41L4B), A2RUB1 (C17orf104), O15031 (PLXNB2), Q9NYF3 (FAM53C), O75146 (HIP1R), P80362 (Ig kappa chain VI region WAT), P01880 (IGHD), Q9C0K0 (BCL11B), A0AVI2 (FER1L5), Q86XJ1 (GAS2L3), and Q00688 (FKBP3).

[0244] In some embodiments, the CAD proteomic signature comprises Q969E1 (LEAP2), Q8NF37 (LPCAT1), Q01082 (SPTBN1), Q7Z333 (SETX), P30481 (HLA-B), Q86YI8 (PHF13), Q9Y4D8 (HECTD4), Q9UIW2 (PLXNA1), Q6ZS81 (WDFY4), and Q9H329 (EPB41L4B).

[0245] In some embodiments, the CAD proteomic signature is selected from the group consisting of Q969E1 (LEAP2), Q8NF37 (LPCAT1), Q01082 (SPTBN1), Q7Z333 (SETX), P30481 (HLA-B), Q5T8A7 (PPP1R26), Q9NX02 (NLRP2), P02144 (MB), Q9BQS2 (SYT15), P62805 (HIS These include T1H4A), Q86YI8 (PHF13), Q9Y4D8 (HECTD4), Q9UIW2 (PLXNA1), Q6ZS81 (WDFY4), Q9H329 (EPB41L4B), A2RUB1 (C17orf104), O15031 (PLXNB2), Q9NYF3 (FAM53C), O75146 (HIP1R), and P80362 (Ig κ chain VI region WAT).

[0246] In some embodiments, the CAD proteomic signature is selected from the group consisting of Q969E1 (LEAP2), Q8NF37 (LPCAT1), Q01082 (SPTBN1), Q7Z333 (SETX), P30481 (HLA-B), Q5T8A7 (PPP1R26), Q9NX02 (NLRP2), P02144 (MB), Q9BQS2 (SYT15), P62805 (HIST1H4A), P80370 (DLK1), P68366 (TUBA4A), P27797 (CALR), P05164 (MPO), Q99439 (CNN2), Q969E1 (LEAP2), Q8NF37 (LPCAT1), Q0 These include 1082 (SPTBN1), Q7Z333 (SETX), P30481 (HLA-B), Q5T8A7 (PPP1R26), Q9NX02 (NLRP2), P02144 (MB), Q9BQS2 (SYT15), P62805 (HIST1H4A), Q86YI8 (PHF13), Q9Y4D8 (HECTD4), Q9UIW2 (PLXNA1), Q6ZS81 (WDFY4), Q9H329 (EPB41L4B), A2RUB1 (C17orf104), O15031 (PLXNB2), Q9NYF3 (FAM53C), O75146 (HIP1R), and P80362 (Ig κ chain VI region WAT).

[0247] In some embodiments, the one or more biomarkers are indicative of safety, efficacy, diagnosis, prognosis, disease progression, response to treatment, or any combination thereof.

[0248] In some aspects, provided herein are methods of subjecting an individual to a determination of a coronary artery disease (CAD) diagnosis, the methods comprising: (a) obtaining mass spectrometry (MS) data from a sample or a derivative thereof obtained from the individual; (b) analyzing the MS data according to a CAD proteomic signature, the CAD proteomic signature comprising one or more biomarkers in Table 1; and (c) determining whether the individual has the CAD proteomic signature. In some embodiments, if the individual has the CAD proteomic signature, the individual is diagnosed with CAD.

[0249] In some aspects, provided herein is a method of diagnosing an individual as having coronary artery disease (CAD), the method comprising: (a) obtaining mass spectrometry (MS) data from a sample obtained from the individual, or a derivative thereof; (b) analyzing the MS data according to a CAD proteomic signature, wherein the CAD proteomic signature comprises one or more biomarkers in Table 1; and (c) diagnosing the individual as having CAD based on the presence of the CAD proteomic signature.

[0250] In some aspects, provided herein are methods of treating an individual having coronary artery disease (CAD), the method comprising: (a) diagnosing the individual as having CAD according to the presence of a CAD proteomic signature in a sample obtained from the individual, or a derivative thereof, where the CAD proteomic signature comprises one or more biomarkers in Table 1; and (b) administering a CAD treatment to the individual.

[0251] In some aspects, provided herein is a method for detecting a coronary artery disease (CAD) proteomic signature in an individual, the method comprising: (a) obtaining mass spectrometry (MS) data from a sample obtained from the individual, or a derivative thereof; and (b) analyzing the MS data according to a CAD proteomic signature to detect a CAD proteomic signature, the CAD proteomic signature comprising one or more biomarkers in Table 1. In some embodiments, the individual is suspected of having CAD.

[0252] In some embodiments, the presence of a CAD proteomic signature is determined by analyzing the MS data according to the CAD proteomic signature.

[0253] In some embodiments, the method further comprises obtaining MS data from a sample obtained from the individual, or a derivative thereof, such as by performing a mass spectrometry technique as described herein.

[0254] In some embodiments, CAD treatment comprises lifestyle adjustments, hi some embodiments, lifestyle adjustments are dieting, undertaking an exercise routine, quitting smoking, and / or cessation of alcohol consumption.

[0255] In some embodiments, the CAD treatment comprises a pharmaceutical intervention. Medication and drugs for treating CAD are known. It is within the level of ordinary skill in the art to select an appropriate drug for treating a subject. In some embodiments, the pharmaceutical intervention comprises administration of a drug selected from the group consisting of calcium channel blockers, histone deacetylase (HDAC) inhibitors (such as HDAC6), Ca2+ / calmodulin (CaM)-dependent protein kinase II (CaMKII) inhibitors, guanylyl cyclase (sGC) activators, MMP inhibitors, statins, and antihypertensive drugs. In some embodiments, the pharmaceutical intervention comprises a drug selected from the group consisting of amlodipine, tubastatin-a, forskolin, trichostatin A, KN-93, CFM-1571, ilomastat, CAY-10603, and rosuvastatin, or a pharmaceutical salt thereof. In some embodiments, the drug is BRD-K52306726, BRD-K71361154, acetazolamide, rolipram, ruxolitinib, BRD-A59808129-001-01-7, BRD-K76876037, ZM336372, trehalose, SCHEMBL3092652, BMS-387032, BRD-K01425431, 4-hydroxyretinoic acid, CHEMBL585951, CHEMBL1673039, HY-11007, primidone, BRD-K81417919, SPECTRUM_000826, tamoxifen, BRD-K00544996, CID67066889, CX-5 461, BRD-K63944563, SCHEMBL6851809, BRD-A86146706, FR-180204, CHEMBL552425, hexachlorophene, Aggc, SUGA1_008424, BRD-K96640811, anastrozole, wortmannin, vandetanib, AC1NWALF, OTSSP167, WZ3105, dihydroergotamine, BRD-K99839793, SR33805 oxalate, AT-7519, sulfadoxine, SPECTRUM_001319, MLS003329219, trichostatin A, and rotenone, or a pharmaceutical salt thereof. In some embodiments, the drug is 6-mercaptopurine, vincristine, bevacizumab, prednisone, thalidomide, zoledronic acid,Paclitaxel, pemetrexed, topotecan, cabazitaxel, prednisolone, capecitabine, gemcitabine, docetaxel, oxaliplatin, seviparin, colchicine, probenecid, cyclophosphamide, daunorubicin, imatinib, 5-fluorouracil, epirubicin, trastuzumab, vinorelbine, rituximab, etoposide, gemcitabine, mitoxantrone, mitoxantrone, topotecan, vinorelbine, davunetide, dexamethasone, gemcitabine, gemcitabine, gemcitabine , vinorelbine, hydrocortisone, irinotecan, pamidronate, vinorelbine, epothilone B, eribulin, gemcitabine, gemcitabine, gemcitabine, vinorelbine, irinotecan, temozolomide, irinotecan, cytarabine, L-asparaginase, prednisone, larotaxel, mirataxel, topotecan, plinabulin, podophyllotoxin, vinorelbine, vinblastine, vinflunine, vinorelbine, vintafolide, AZD4831, GCS-1, GR-MD-2, BI76563, CC-95251, eculizumab, IFX-1, IgG, ravulizumab, pegcetacoplan, CNGRC peptide-TNFα conjugate, stamulumab, BI836845, MEDI-573, MORAb-4, lavendustin C, alirocumab, BMS-844421, evinacumab, PCSK9 inhibitor, CALAA-1, CX-229, emicizumab, moroctocog alfa, L19-IL2 monoclonal antibody cytokine fusion protein, L19TNFα, ocriplasmin, carotuximab, APO1, ASP8232, hydralazine, hydrochlorothiazide, hydralazine, reserpine, dinitrate Isosorbide, amrinone, anagrelide, cilostazol, dipyridamole, dyphylline, enoximone, medrinone, milrinone, nitroglycerin, pentoxifylline, theophylline, tolbutamide, alvespimycin, cisplatin, luminespib, letaspimycin, TAS-116, bapineuzumab, florbetaben F, florbetapir F18, collagenase clostridium histolyticum, trilostane, activated recombinant human factor VII, apixaban, clopidogrel, rivaroxaban, enoxaparin, aspirin, rivaroxaban,Rivaroxaban, ticlopidine, betrixaban, dalteparin, derigoparin, DPC423, edoxaban, emicizumab, enoxaparin, F8, F9, fondaparinux, warfarin, heparin, idraparinux, nematode anticoagulant protein c2, rivaroxaban, RPR12844, RPR28566, tifacogin, AGN2194, AR-H4718, clarithromycin, dexlansoprazole, diclofenac, esomeprazole magnesium, esomeprazole, naproxen, ilaprazole, lansoprazole, hydroxylated Magnesium, sodium bicarbonate, omeprazole, sodium bicarbonate, pantoprazole, rabeprazole, tenatoprazole, AZD425, baricitinib, methotrexate, brepositinib, erlotinib, ruxolitinib, filgotinib, INCB52793, itacitinib, JAK1 inhibitors, jactinib, methotrexate, tofacitinib, momelotinib, tofacitinib, upadacitinib, cedazuridine, cytidine deaminase inhibitors, and rociptor, or any combination thereof.

[0256] In some embodiments, the method of treatment further comprises monitoring CAD treatment. In some embodiments, the method comprises performing CAD proteomic signature analysis after treatment and evaluating changes that indicate improvement of CAD, such as return to health. In some embodiments, the method comprises monitoring one or more symptoms of CAD.

[0257] In some embodiments, the method further comprises obtaining a sample from the individual. In some embodiments, the sample or derivative thereof is a blood sample or a derivative thereof. In some embodiments, the sample or derivative thereof is a plasma sample. In some embodiments, the sample or derivative thereof comprises a liquid fixative. In some embodiments, the sample is obtained and processed as described in other sections of this application.

[0258] In some embodiments, obtaining MS data from the sample or a derivative thereof comprises performing mass spectrometry of the sample or a derivative thereof using a mass spectrometer. In some embodiments, the mass spectrometry is performed according to the description provided herein.

[0259] In some embodiments, analyzing the MS data according to the CAD proteomic signature comprises subjecting the MS data to the methods described herein. In some embodiments, analyzing the MS data according to the CAD proteomic signature comprises assessing the presence or absence or level of each of one or more biomarkers of the CAD proteomic signature in the MS data. In some embodiments, the match with the CAD proteomic signature is based on whether one or more biomarkers of the CAD proteomic signature substantially match (e.g., at least about 70% (including at least about any of 75%, 80%, 85%, 90%, or 95% of one or more biomarkers)) with the up- or down-expression classification of Table 1.

[0260] In some embodiments, the method further comprises performing one or more of the following factor assessments of the individual: gender, age, body mass index (BMI), systolic blood pressure, diastolic blood pressure, total cholesterol, HDL, LDL, triglycerides, hyperlipidemia, hypertension, diabetes mellitus, insulin resistance, kidney disease, smoking status, physical activity level, sleep level, or nutritional quality.

[0261] In some embodiments, the method further comprises performing a medical procedure on the individual to assess the presence of CAD, such as cardiac catheterization or coronary CT angiography.

[0262] [Table 1] TIFF2024500571000002.tif240141TIFF2024500571000003.tif242133TIFF2024500571000004.tif242142TIF F2024500571000005.tif241142TIFF2024500571000006.tif241141TIFF2024500571000007.tif240147TIFF202 4500571000008.tif241141TIFF2024500571000009.tif241142TIFF2024500571000010.tif240142TIFF2024500 571000011.tif241142TIFF2024500571000012.tif241131TIFF2024500571000013.tif240131TIFF20245005710 00014.tif241142TIFF2024500571000015.tif240135TIFF2024500571000016.tif241137TIFF20245005710000 17.tif241130TIFF2024500571000018.tif241135TIFF2024500571000019.tif240137TIFF2024500571000020.t if240131TIFF2024500571000021.tif241132TIFF2024500571000022.tif242130TIFF2024500571000023.tif24 0129TIFF2024500571000024.tif241140TIFF2024500571000025.tif241144TIFF2024500571000026.tif242123

[0263] IV. Devices and Systems Provided herein are devices and systems useful for the methods described herein. In some embodiments, provided are microfluidic devices for separating components of a sample or products thereof, such as size exclusion chromatography microfluidic devices or reversed phase liquid chromatography microfluidic devices. In some embodiments, provided herein are systems that integrate steps of the methods described herein. In some embodiments, the systems include microfluidic devices for separating components of a sample or products thereof, and other mechanisms useful for completing and / or integrating steps of the methods described herein. In some embodiments, the systems include mechanisms for automation, such as robotics.

[0264] A. Microfluidic Devices for Separating Sample Components In some aspects, provided herein is a microfluidic device configured to separate components of a sample. In some embodiments, the microfluidic device comprises a plurality of interconnected channels that contain a medium (such as a porous medium or a reversed-phase medium) useful for separation. Microfluidic devices that include a plurality of interconnected channels are useful for efficient and effective separation of a diverse array of components of a sample, thus enabling, for example, simultaneous proteomic, peptidomic, and metabolomic analysis of complex biological samples.

[0265] A schematic diagram of an exemplary microfluidic device 300 is provided in FIG. 3. The microfluidic device 300 comprises an input port 305 in fluid communication with an upstream network of connecting channels 310 that connects the input port 305 with a plurality of interconnected channels 315. The microfluidic device 300 is configured to receive fluid via the input port 305 and direct a portion of the fluid to each of the interconnected channels 315 via the upstream network of connecting channels 310. The interconnected channels 315 are also in fluid communication with a downstream network of connecting channels 320 that terminate at an output port 325. The microfluidic device is configured to direct eluate from each of the plurality of interconnected channels to an output mechanism, such as a single output port 325, via the downstream network of connecting channels 320. In some embodiments, the input port is configured to interface with a sample injector and / or a mobile phase source (such as a pump). In some embodiments, the output port is configured to interface with a downstream tool or mechanism useful for the methods described herein. In some embodiments, the output port is configured to interface with a collection device, such as a fraction collector. In some embodiments, the output port is configured to interface with an electrospray ionization source.

[0266] In some embodiments, the microfluidic device includes a plurality of interconnected channels. In some embodiments, the plurality of interconnected channels is configured as a plurality of interconnected parallel channels. In such embodiments, the term "parallel" indicates that a fluid input to the microfluidic device is split and a portion of the fluid passes through different channels or different sections of the interconnected channels simultaneously, and is not intended to be interpreted as a restriction on the shape of the interconnected channels (e.g., the interconnected parallel channels may only be straight lines configured in a geometrically parallel manner, etc.). In some embodiments, the plurality of interconnected channels includes one or more channels that include a substantially linear feature of the channel. In some embodiments, the plurality of interconnected channels includes one or more channels that include a non-linear feature of the channel, such as including a branched, staggered, or wavy shape. In some embodiments, the microfluidic devices described herein may include any number of interconnected channels such that the interconnected channels are in fluid communication with the input port(s) of the microfluidic device, and the interconnected channels are configured to receive a portion of the fluid introduced into the microfluidic device via the input port(s). In some embodiments, the plurality of interconnected channels of the microfluidic device includes eight or more interconnected channels. In some embodiments, the plurality of interconnected channels of the microfluidic device include 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 , 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 interconnected channels.In some embodiments, the plurality of interconnected channels of the microfluidic device comprises 32 interconnected channels. In some embodiments, the plurality of interconnected channels of the microfluidic device comprises 64 interconnected channels.

[0267] In some embodiments, each of the plurality of interconnected channels of the microfluidic device is in fluid communication with an input port of the microfluidic device. In some embodiments, each of the plurality of interconnected channels of the microfluidic device is in fluid communication with an input port of the microfluidic device via an upstream network of connecting channels. In some embodiments, the upstream network of connecting channels is configured to direct and split the flow of fluid introduced through the input port of the microfluidic device such that a portion of the fluid is delivered to each interconnected channel. In some embodiments, the upstream network of connecting channels, or a portion thereof, is connected to a proximal region of each of the plurality of interconnected channels. In some embodiments, the proximal region of the interconnected channel is a region of the interconnected channel that is first exposed to the fluid introduced through the input port of the microfluidic device. The upstream network of connecting channels can be configured in a number of ways and can provide one or more functions, such as, for example, a mixing function and / or a filtering function and / or a dilution function, in addition to directing the fluid to the interconnected channels. In some embodiments, the upstream network of connecting channels includes a series of branching channels configured to split the flow of fluid from the input port of the microfluidic device to each of the plurality of interconnected channels. In some embodiments, the series of branching channels of the upstream network of connecting channels are structured using a 1:2 split (e.g., one channel splits into two channels in an upstream to downstream direction based on the intended fluid flow). For example, in some embodiments, for a microfluidic device having a single input port and 32 interconnected channels, the upstream network of connecting channels includes a single channel that splits into two channels, each of which splits into two channels (now a total of four channels).Each of the four channels is split into two channels (now eight channels total), each of the eight channels is split into two channels (now 16 channels), each of the sixteen channels is split into two channels (now 32 channels), where each of the 32 channels is connected to one of the 32 interconnected channels. In some embodiments, the upstream network of connecting channels includes a 1:2 split, a 1:3 split, a 1:4 split, a 1:5 split, a 1:6 split, a 1:7 split, a 1:8 split, a 1:9 split, a 1:10 split, a 1:11 split, a 1:12 split, or any combination thereof. In some embodiments, the channels of the upstream network of connecting channels after splitting (i.e., the split channels) have smaller cross-sectional dimensions (e.g., height and / or width) compared to the original channels.

[0268] In some embodiments, each of the multiple interconnected channels of the microfluidic device is in fluid communication with an output port of the microfluidic device. In some embodiments, each of the multiple interconnected channels of the microfluidic device is in fluid communication with an output port of the microfluidic device through a downstream network of connecting channels. In some embodiments, the downstream network of connecting channels is configured to couple the fluid flow from each of the multiple interconnected channels toward an output port of the microfluidic device (e.g., including two or more output ports of the microfluidic device). In some embodiments, the downstream network of connecting channels, or a portion thereof, is connected to a distal region of each of the multiple interconnected channels. In some embodiments, the distal region of the interconnected channel is a region of the interconnected channel where the fluid exits the interconnected channel to a downstream feature. The downstream network of connecting channels can be configured in a number of ways and can provide one or more functions, such as, for example, a mixing function and / or a filtering function and / or a dilution function, in addition to directing the fluid to the output port. In some embodiments, the downstream network of connecting channels includes a series of converging channels configured to combine the fluid flow from the multiple interconnected channels of the microfluidic device to the output port. In some embodiments, the series of converging channels of the downstream network of connecting channels are structured using a 2-to-1 split (e.g., two channels converge into one channel in an upstream to downstream direction based on the intended fluid flow). For example, in some embodiments, for a microfluidic device having a single output port and 32 interconnected channels, the downstream network of connecting channels includes 32 channels, each of the 32 channels of the downstream network of connecting channels is connected to 32 interconnected channels, pairs of the 32 channels of the downstream network of connecting channels converge into a single channel (here 16 channels), and pairs of the 16 channels of the downstream network of connecting channels converge into a single channel (here 8 channels).Here, eight pairs of channels of the downstream network of connecting channels converge into a single channel (here 4 channels) and four pairs of channels of the downstream network of connecting channels converge into a single channel (here 2 channels). Here, two channels of the downstream network of connecting channels converge into a single channel that is in fluid communication with the output port. In some embodiments, the downstream network of connecting channels comprises a 2 to 1 convergence, a 3 to 1 convergence, a 4 to 1 convergence, a 5 to 1 convergence, a 6 to 1 convergence, a 7 to 1 convergence, a 8 to 1 convergence, a 9 to 1 convergence, a 10 to 1 convergence, an 11 to 1 convergence, a 12 to 1 convergence, or any combination thereof. In some embodiments, the channels of the downstream network of connecting channels after convergence (i.e., the converging channels) have a larger cross-sectional dimension (such as height and / or width) compared to the channels from which they originate.

[0269] In some embodiments, multiple interconnected channels of a microfluidic device are connected only through an upstream network of connecting channels and / or a downstream network of connecting channels, for example, in some embodiments, one interconnected channel is not connected to another interconnected channel except through an upstream network of connecting channels and / or a downstream network of connecting channels.

[0270] In some embodiments, each of the multiple interconnected channels of the microfluidic device has a length of about 2 cm to about 50 cm, such as about 5 cm to about 20 cm. In some embodiments, the length of the interconnected channels is at least about 5 cm, such as at least about 6 cm, 7 cm, 8 cm, 9 cm, 10 cm, 11 cm, 12 cm, 13 cm, 14 cm, 15 cm, 16 cm, 17 cm, 18 cm, 19 cm, 20 cm, 21 cm, 22 cm, 23 cm, 24 cm, 25 cm, 26 cm, 27 cm, 28 cm, 29 cm, 30 cm, 31 cm, 32 cm, 33 cm, 34 cm, 35 cm, 36 cm, 37 cm, 38 cm, 39 cm, 40 cm, 41 cm, 42 cm, 43 cm, 44 cm, 45 cm, 46 cm, 47 cm, 48 cm, 49 cm, or 50 cm. In some embodiments, the length of the interconnected channels is less than about 50 cm, such as less than about any of 49 cm, 48 cm, 47 cm, 46 cm, 45 cm, 44 cm, 43 cm, 42 cm, 41 cm, 40 cm, 39 cm, 38 cm, 37 cm, 36 cm, 35 cm, 34 cm, 33 cm, 32 cm, 31 cm, 30 cm, 29 cm, 28 cm, 27 cm, 26 cm, 25 cm, 24 cm, 23 cm, 22 cm, 21 cm, 20 cm, 19 cm, 18 cm, 17 cm, 16 cm, 15 cm, 14 cm, 13 cm, 12 cm, 11 cm, 10 cm, 9 cm, 8 cm, 7 cm, 6 cm, 5 cm, 4 cm, 3 cm, or 2 cm. In some embodiments, the length of the interconnected channels is about any of 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, 10 cm, 11 cm, 12 cm, 13 cm, 14 cm, 15 cm, 16 cm, 17 cm, 18 cm, 19 cm, 20 cm, 21 cm, 22 cm, 23 cm, 24 cm, 25 cm, 26 cm, 27 cm, 28 cm, 29 cm, 30 cm, 31 cm, 32 cm, 33 cm, 34 cm, 35 cm, 36 cm, 37 cm, 38 cm, 39 cm, 40 cm, 41 cm, 42 cm, 43 cm, 44 cm, 45 cm, 46 cm, 47 cm, 48 cm, 49 cm, or 50 cm.

[0271] In some embodiments, the total length of the plurality of interconnected channels is between about 20 cm and about 3,200 cm. In some embodiments, the total length of the plurality of interconnected channels is greater than about 20 cm, e.g., greater than about any of 50 cm, 75 cm, 100 cm, 150 cm, 200 cm, 250 cm, 300 cm, 350 cm, 400 cm, 450 cm, 500 cm, 600 cm, 700 cm, 800 cm, 900 cm, 1,000 cm, 1,250 cm, 1,500 cm, 1,750 cm, 2,000 cm, 2,250 cm, 2,500 cm, 2,750 cm, or 3,000 cm.

[0272] The channels of the interconnected channels, upstream network of connecting channels, and downstream network of connecting channels described herein can be formed to have a variety of cross-sectional shapes and sizes. In some embodiments, the cross-sectional shapes and sizes of the channels described herein can vary at different points of the channels. In some embodiments, the channels have cross-sectional shapes that include rectangular, square, or circular.

[0273] In some embodiments, the interconnected channels of the microfluidic device have cross-sectional dimensions of about 1 μm to about 15 μm, such as about 3 μm to about 10 μm. In some embodiments, the interconnected channels of the microfluidic device have cross-sectional dimensions of about 15 μm or less, such as about any of 14 μm or less, 13 μm or less, 12 μm or less, 11 μm or less, 10 μm or less, 9 μm or less, 8 μm or less, 7 μm or less, 6 μm or less, 5 μm or less, 4 μm or less, 3 μm or less, 2 μm or less, or 1 μm or less. In some embodiments, the interconnected channels of the microfluidic device have cross-sectional dimensions of about any of 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 14 μm, or 15 μm.

[0274] In some embodiments, the interconnected channels of the microfluidic device have a maximum cross-sectional dimension of about 1 μm to about 15 μm, e.g., about 3 μm to about 10 μm. In some embodiments, the interconnected channels of the microfluidic device have a maximum cross-sectional dimension of about 15 μm or less, e.g., about 14 μm or less, 13 μm or less, 12 μm or less, 11 μm or less, 10 μm or less, 9 μm or less, 8 μm or less, 7 μm or less, 6 μm or less, 5 μm or less, 4 μm or less, 3 μm or less, 2 μm or less, or 1 μm or less. In some embodiments, the interconnected channels of the microfluidic device have a maximum cross-sectional dimension of about 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 14 μm, or 15 μm. In some embodiments, the interconnected channels of the microfluidic device have a minimum cross-sectional dimension of about 1 μm or more, such as about 2 μm or more, 3 μm or more, 4 μm or more, 5 μm or more, 6 μm or more, 7 μm or more, 8 μm or more, 9 μm or more, or 10 μm or more. In some embodiments, the interconnected channels of the microfluidic device have a minimum cross-sectional dimension of about 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 14 μm, or 15 μm.

[0275] In some embodiments, the interconnected channels of the microfluidic device have a cross-sectional dimension perpendicular to the largest cross-sectional dimension of about 1 μm to about 15 μm, e.g., about 3 μm to about 10 μm. In some embodiments, the interconnected channels of the microfluidic device have a cross-sectional dimension perpendicular to the largest cross-sectional dimension of about 15 μm or less, e.g., about 14 μm or less, 13 μm or less, 12 μm or less, 11 μm or less, 10 μm or less, 9 μm or less, 8 μm or less, 7 μm or less, 6 μm or less, 5 μm or less, 4 μm or less, 3 μm or less, 2 μm or less, or 1 μm or less. In some embodiments, the interconnected channels of the microfluidic device have a cross-sectional dimension perpendicular to the largest cross-sectional dimension of about 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 14 μm, or 15 μm. In some embodiments, the interconnected channels of the microfluidic device have a maximum cross-sectional dimension of about any of 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 14 μm, or 15 μm, and a cross-sectional dimension perpendicular to the maximum cross-sectional dimension of about any of 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 14 μm, or 15 μm.

[0276] In some embodiments, the interconnected channels of the microfluidic device have a width (e.g., measured across a cross-sectional dimension) of about 1 μm to about 15 μm, such as about 1 μm to about 6 μm, about 3 μm to about 10 μm, or about 6 μm to about 12 μm. In some embodiments, the interconnected channels of the microfluidic device have a width (e.g., measured across a cross-sectional dimension) of about 15 μm or less, such as about 14 μm or less, 13 μm or less, 12 μm or less, 11 μm or less, 10 μm or less, 9 μm or less, 8 μm or less, 7 μm or less, 6 μm or less, 5 μm or less, 4 μm or less, 3 μm or less, 2 μm or less, or 1 μm or less. In some embodiments, the interconnected channels of the microfluidic device have a width (e.g., measured across a cross-sectional dimension) of about any of 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 14 μm, or 15 μm.

[0277] In some embodiments, the interconnected channels of the microfluidic device have a height (e.g., measured across a cross-sectional dimension) of about 1 μm to about 15 μm, e.g., about 1 μm to about 6 μm, about 3 μm to about 10 μm, or about 6 μm to about 12 μm. In some embodiments, the interconnected channels of the microfluidic device have a height (e.g., measured across a cross-sectional dimension) of about 15 μm or less, e.g., about 14 μm or less, 13 μm or less, 12 μm or less, 11 μm or less, 10 μm or less, 9 μm or less, 8 μm or less, 7 μm or less, 6 μm or less, 5 μm or less, 4 μm or less, 3 μm or less, 2 μm or less, or 1 μm or less. In some embodiments, the interconnected channels of the microfluidic device have a height (e.g., measured across a cross-sectional dimension) of about any of 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 14 μm, or 15 μm.

[0278] In some embodiments, the plurality of interconnected channels of the microfluidic device are formed by a pillar array. In some embodiments, the pillar array is an amorphous pillar array. In some embodiments, the pillar array is a non-amorphous pillar array. In some embodiments, the pillar array forms an interior surface of each of the plurality of interconnected channels that the microfluidic device comprises.

[0279] In some embodiments, the microfluidic device comprises a quartz substrate. In some embodiments, the microfluidic device comprises a monolithic substrate forming a plurality of interconnected channels. In some embodiments, the microfluidic device comprises a quartz monolithic substrate. In some embodiments, the microfluidic device comprises a three-dimensional (3D) printed substrate.

[0280] In some embodiments, the interconnected channels of the microfluidic device are in an open tubular format. In some embodiments, the channels of the microfluidic device include an inner surface material. In some embodiments, the inner surface material is configured as a separation medium, such as a size exclusion chromatography medium. In some embodiments, the inner surface material has dimensions, such as a thickness, based on the desired separation.

[0281] In some aspects, the present disclosure provides a method for making the microfluidic device described herein. Methods for making microfluidic devices are well known in the art. See, for example, Gale et al., MDPI Inventions, 3, 2018, which is incorporated herein by reference in its entirety. In some embodiments, the method comprises a masking technique. In some embodiments, the method comprises an etching technique. In some embodiments, the method comprises a three-dimensional (3D) printing technique.

[0282] i. Size Exclusion Chromatography (SEC) Microfluidic Device In some embodiments, the microfluidic device configured for separating components of a sample is a size-exclusion chromatography (SEC) microfluidic device. In such embodiments, the size-exclusion chromatography (SEC) microfluidic device includes a SEC medium disposed in at least a plurality of interconnected channels of the SEC chromatography device, e.g., bonded to an inner surface of the channel. In some embodiments, the SEC medium is further disposed in an upstream network of connecting channels. In some embodiments, the SEC medium is further disposed in a downstream network of connecting channels.

[0283] In some embodiments, the SEC medium is the inner surface material of a plurality of interconnected channels of a SEC microfluidic device. In some embodiments, the inner surface comprises an average pore size of about 10 nm to about 500 nm. In some embodiments, the inner surface comprises an average pore size of at least about 10 nm, such as at least about any of 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 125 nm, 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, 325 nm, 350 nm, 375 nm, 400 nm, 425 nm, 450 nm, 475 nm, 500 nm. In some embodiments, the inner surface comprises an average pore size of less than about 500 nm, e.g., less than any of about 475 nm, 450 nm, 425 nm, 400 nm, 375 nm, 350 nm, 325 nm, 300 nm, 275 nm, 250 nm, 225 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm, or 10 nm. In some embodiments, the inner surface comprises an average pore size of any of about 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 125 nm, 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, 325 nm, 350 nm, 375 nm, 400 nm, 425 nm, 450 nm, 475 nm, or 500 nm.

[0284] In some embodiments, the inner surface material is configured to leave an open space in each of the multiple interconnected channels as seen in an open tubular format. In some embodiments, the inner surface material has a thickness of about 0.5 μm to about 2 μm. In some embodiments, the inner surface material has a thickness of at least about 0.5 μm, such as at least about 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, 1.1 μm, 1.2 μm, 1.3 μm, 1.4 μm, 1.5 μm, 1.6 μm, 1.7 μm, 1.8 μm, 1.9 μm, or 2 μm. In some embodiments, the inner surface material has a thickness of less than about 2 μm, such as less than about any of 1.9 μm, 1.8 μm, 1.7 μm, 1.6 μm, 1.5 μm, 1.4 μm, 1.3 μm, 1.2 μm, 1.1 μm, 1 μm, 0.9 μm, 0.8 μm, 0.7 μm, 0.6 μm, or 0.5 μm. In some embodiments, the inner surface material has a thickness of about any of 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, 1.1 μm, 1.2 μm, 1.3 μm, 1.4 μm, 1.5 μm, 1.6 μm, 1.7 μm, 1.8 μm, 1.9 μm, or 2 μm.

[0285] In some embodiments, the inner surface material is fabricated using plasma etching and / or three-dimensional (3D) printing techniques.

[0286] In some embodiments, the SEC microfluidic device comprises a plurality of interconnected channels. In some embodiments, the SEC microfluidic devices described herein may comprise any number of interconnected channels such that the interconnected channels are in fluid communication with an input port of the SEC microfluidic device, and the interconnected channels are configured to receive a portion of a fluid introduced into the SEC microfluidic device via the input port. In some embodiments, the plurality of interconnected channels of the SEC microfluidic device comprises eight or more interconnected channels. In some embodiments, the plurality of interconnected channels of the SEC microfluidic device comprises eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, one, two, two, three, four, five, six, seventeen, twenty-eight, twenty-nine, thirty-one, thirty-one, thirty-two, three, three, four, five, six, seventeen, twenty-eight, twenty-nine ...one, thirty-two, three, three, four, five, six, seventeen, twenty-eight, twenty-nine, twenty-nine, twenty-nine, twenty-nine, twenty-nine, twenty-nine, twenty-nine, twenty-nine, twenty-nine, In some embodiments, the SEC microfluidic device has 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 interconnected channels. In some embodiments, the SEC microfluidic device has 32 interconnected channels. In some embodiments, the SEC microfluidic device has 64 interconnected channels.

[0287] In some embodiments, each of the multiple interconnected channels of the SEC microfluidic device is in fluid communication with an input port of the SEC microfluidic device. In some embodiments, each of the multiple interconnected channels of the SEC microfluidic device is in fluid communication with an input port of the SEC microfluidic device via an upstream network of connecting channels. In some embodiments, the upstream network of connecting channels is configured to direct and split the flow of fluid introduced through the input port of the SEC microfluidic device so that a portion of the fluid is delivered to each interconnected channel. In some embodiments, the upstream network of connecting channels, or a portion thereof, is connected to a proximal region of each of the multiple interconnected channels. In some embodiments, the proximal region of the interconnected channel is a region of the interconnected channel that is first exposed to the fluid introduced through the input port of the SEC microfluidic device. The upstream network of connecting channels can be configured in a number of ways and can provide one or more functions, such as, for example, a mixing function and / or a filtering function and / or a dilution function, in addition to directing the fluid to the interconnected channels. In some embodiments, the upstream network of connecting channels includes a series of branching channels configured to split the flow of fluid from the input port of the SEC microfluidic device to each of the multiple interconnected channels. In some embodiments, a series of branching channels in an upstream network of connecting channels are structured using a 1:2 split (e.g., one channel splits into two channels in an upstream to downstream direction based on the intended fluid flow).For example, in some embodiments, for a SEC microfluidic device having a single input port and 32 interconnected channels, the upstream network of connecting channels includes a single channel that splits into two channels, each of the two channels splits into two channels (now a total of four channels), each of the four channels splits into two channels (now a total of eight channels), each of the eight channels splits into two channels (now 16 channels), each of the 16 channels splits into two channels (now 32 channels), and each of the 32 channels connects to one of the 32 interconnected channels. In some embodiments, the upstream network of connecting channels includes a 1:2 split, a 1:3 split, a 1:4 split, a 1:5 split, a 1:6 split, a 1:7 split, a 1:8 split, a 1:9 split, a 1:10 split, a 1:11 split, a 1:12 split, or any combination thereof. In some embodiments, the channels of the upstream network of connecting channels after splitting (ie, the split channels) have smaller cross-sectional dimensions (eg, height and / or width) compared to the original channels.

[0288] In some embodiments, each of the multiple interconnected channels of the SEC microfluidic device is in fluid communication with an output port of the SEC microfluidic device. In some embodiments, each of the multiple interconnected channels of the SEC microfluidic device is in fluid communication with an output port of the SEC microfluidic device through a downstream network of connecting channels. In some embodiments, the downstream network of connecting channels is configured to direct and combine the flow of fluids from each of the multiple interconnected channels to an output port of the SEC microfluidic device (e.g., including two or more output ports of the microfluidic device). In some embodiments, the downstream network of connecting channels, or a portion thereof, is connected to a distal region of each of the multiple interconnected channels. In some embodiments, the distal region of the interconnected channel is a region of the interconnected channel where the fluid exits the interconnected channel to a downstream feature. The downstream network of connecting channels can be configured in many ways and can provide one or more functions, such as a mixing function and / or a filtering function and / or a dilution function, in addition to directing the fluid to the output port. In some embodiments, the downstream network of connecting channels includes a series of converging channels configured to combine the flow of fluids from the multiple interconnected channels of the SEC microfluidic device to the output port. In some embodiments, a series of converging channels in a downstream network of connecting channels are structured using a two-to-one split (e.g., two channels converge into one channel from upstream to downstream based on the intended fluid flow).For example, in some embodiments, for a SEC microfluidic device having a single output port and 32 interconnected channels, the downstream network of connecting channels includes 32 channels, each of the 32 channels of the downstream network of connecting channels is connected to a channel of the 32 interconnected channels, pairs of the 32 channels of the downstream network of connecting channels converge into a single channel (here 16 channels), pairs of the 16 channels of the downstream network of connecting channels converge into a single channel (here 8 channels), pairs of the 8 channels of the downstream network of connecting channels converge into a single channel (here 4 channels), pairs of the 4 channels of the downstream network of connecting channels converge into a single channel (here 2 channels), and 2 channels of the downstream network of connecting channels converge into a single channel that is fluidly connected to the output port. In some embodiments, the downstream network of connecting channels comprises a 2 to 1 convergence, a 3 to 1 convergence, a 4 to 1 convergence, a 5 to 1 convergence, a 6 to 1 convergence, a 7 to 1 convergence, a 8 to 1 convergence, a 9 to 1 convergence, a 10 to 1 convergence, an 11 to 1 convergence, a 12 to 1 convergence, or any combination thereof. In some embodiments, the channels of the downstream network of connecting channels after convergence (i.e., the converging channels) have a larger cross-sectional dimension (e.g., height and / or width) compared to the channels from which they originate.

[0289] In some embodiments, multiple interconnected channels of a SEC microfluidic device are connected only through an upstream network of connecting channels and / or a downstream network of connecting channels, for example, in some embodiments, one interconnected channel is not connected to another interconnected channel except through an upstream network of connecting channels and / or a downstream network of connecting channels.

[0290] In some embodiments, each of the multiple interconnected channels of the SEC microfluidic device has a length of about 2 cm to about 30 cm, such as about 5 cm to about 20 cm. In some embodiments, the length of the interconnected channels is at least about 5 cm, such as at least about 6 cm, 7 cm, 8 cm, 9 cm, 10 cm, 11 cm, 12 cm, 13 cm, 14 cm, 15 cm, 16 cm, 17 cm, 18 cm, 19 cm, 20 cm, 21 cm, 22 cm, 23 cm, 24 cm, 25 cm, 26 cm, 27 cm, 28 cm, 29 cm, or 30 cm. In some embodiments, the length of the interconnected channels is less than about 30 cm, such as less than about any of 29 cm, 28 cm, 27 cm, 26 cm, 25 cm, 24 cm, 23 cm, 22 cm, 21 cm, 20 cm, 19 cm, 18 cm, 17 cm, 16 cm, 15 cm, 14 cm, 13 cm, 12 cm, 11 cm, 10 cm, 9 cm, 8 cm, 7 cm, 6 cm, 5 cm, 4 cm, 3 cm, or 2 cm. In some embodiments, the length of the interconnected channels is less than about any of 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, 10 cm, 11 cm, 12 cm, 13 cm, 14 cm, 15 cm, 16 cm, 17 cm, 18 cm, 19 cm, 20 cm, 21 cm, 22 cm, 23 cm, 24 cm, 25 cm, 26 cm, 27 cm, 28 cm, 29 cm, or 30 cm.

[0291] In some embodiments, the total length of the plurality of interconnected channels is between about 20 cm and about 3,200 cm. In some embodiments, the total length of the plurality of interconnected channels is greater than about 20 cm, e.g., greater than about any of 50 cm, 75 cm, 100 cm, 150 cm, 200 cm, 250 cm, 300 cm, 350 cm, 400 cm, 450 cm, 500 cm, 600 cm, 700 cm, 800 cm, 900 cm, 1,000 cm, 1,250 cm, 1,500 cm, 1,750 cm, 2,000 cm, 2,250 cm, 2,500 cm, 2,750 cm, or 3,000 cm.

[0292] The channels of the interconnected channels, upstream network of connecting channels, and downstream network of connecting channels described herein may be formed to have a variety of cross-sectional shapes and sizes. In some embodiments, the cross-sectional shapes and sizes of the channels described herein may vary at different points of the channels. In some embodiments, the channels have cross-sectional shapes that include rectangular, square, or circular.

[0293] In some embodiments, the interconnected channels of the SEC microfluidic device have cross-sectional dimensions of about 1 μm to about 15 μm, e.g., about 3 μm to about 10 μm. In some embodiments, the interconnected channels of the SEC microfluidic device have cross-sectional dimensions of about 15 μm or less, e.g., about 14 μm or less, 13 μm or less, 12 μm or less, 11 μm or less, 10 μm or less, 9 μm or less, 8 μm or less, 7 μm or less, 6 μm or less, 5 μm or less, 4 μm or less, 3 μm or less, 2 μm or less, or 1 μm or less. In some embodiments, the interconnected channels of the SEC microfluidic device have cross-sectional dimensions of about 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 14 μm, or 15 μm.

[0294] In some embodiments, the interconnected channels of the SEC microfluidic device have a maximum cross-sectional dimension of about 1 μm to about 15 μm, e.g., about 3 μm to about 10 μm. In some embodiments, the interconnected channels of the SEC microfluidic device have a maximum cross-sectional dimension of about 15 μm or less, e.g., about 14 μm or less, 13 μm or less, 12 μm or less, 11 μm or less, 10 μm or less, 9 μm or less, 8 μm or less, 7 μm or less, 6 μm or less, 5 μm or less, 4 μm or less, 3 μm or less, 2 μm or less, or 1 μm or less. In some embodiments, the interconnected channels of the SEC microfluidic device have a maximum cross-sectional dimension of about 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 14 μm, or 15 μm. In some embodiments, the interconnected channels of a SEC microfluidic device have a minimum cross-sectional dimension of about 1 μm or more, such as about 2 μm or more, 3 μm or more, 4 μm or more, 5 μm or more, 6 μm or more, 7 μm or more, 8 μm or more, 9 μm or more, or 10 μm or more. In some embodiments, the interconnected channels of a SEC microfluidic device have a minimum cross-sectional dimension of about 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 14 μm, or 15 μm.

[0295] In some embodiments, the interconnected channels of the SEC microfluidic device have a cross-sectional dimension perpendicular to the largest cross-sectional dimension of about 1 μm to about 15 μm, e.g., about 3 μm to about 10 μm. In some embodiments, the interconnected channels of the SEC microfluidic device have a cross-sectional dimension perpendicular to the largest cross-sectional dimension of about 15 μm or less, e.g., about 14 μm or less, 13 μm or less, 12 μm or less, 11 μm or less, 10 μm or less, 9 μm or less, 8 μm or less, 7 μm or less, 6 μm or less, 5 μm or less, 4 μm or less, 3 μm or less, 2 μm or less, or 1 μm or less. In some embodiments, the interconnected channels of the SEC microfluidic device have a cross-sectional dimension perpendicular to the largest cross-sectional dimension of about 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 14 μm, or 15 μm. In some embodiments, the interconnected channels of a SEC microfluidic device have a maximum cross-sectional dimension of about any of 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 14 μm, or 15 μm, and a cross-sectional dimension perpendicular to the maximum cross-sectional dimension of about any of 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 14 μm, or 15 μm.

[0296] In some embodiments, the interconnected channels of the SEC microfluidic device have a width (e.g., measured across a cross-sectional dimension) of about 1 μm to about 15 μm, e.g., about 1 μm to about 6 μm, about 3 μm to about 10 μm, or about 6 μm to about 12 μm. In some embodiments, the interconnected channels of the SEC microfluidic device have a width (e.g., measured across a cross-sectional dimension) of about 15 μm or less, e.g., about 14 μm or less, 13 μm or less, 12 μm or less, 11 μm or less, 10 μm or less, 9 μm or less, 8 μm or less, 7 μm or less, 6 μm or less, 5 μm or less, 4 μm or less, 3 μm or less, 2 μm or less, or 1 μm or less. In some embodiments, the interconnected channels of a SEC microfluidic device have a width (e.g., measured across a cross-sectional dimension) of about any of 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 14 μm, or 15 μm.

[0297] In some embodiments, the interconnected channels of the SEC microfluidic device have a height (e.g., measured across a cross-sectional dimension) of about 1 μm to about 15 μm, e.g., about 1 μm to about 6 μm, about 3 μm to about 10 μm, or about 6 μm to about 12 μm. In some embodiments, the interconnected channels of the SEC microfluidic device have a height (e.g., measured across a cross-sectional dimension) of about 15 μm or less, e.g., about 14 μm or less, 13 μm or less, 12 μm or less, 11 μm or less, 10 μm or less, 9 μm or less, 8 μm or less, 7 μm or less, 6 μm or less, 5 μm or less, 4 μm or less, 3 μm or less, 2 μm or less, or 1 μm or less. In some embodiments, the interconnected channels of a SEC microfluidic device have a height (e.g., measured across a cross-sectional dimension) of about any of 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 14 μm, or 15 μm.

[0298] In some embodiments, the plurality of interconnected channels of the SEC microfluidic device are formed by a pillar array. In some embodiments, the pillar array is an amorphous pillar array. In some embodiments, the pillar array is a non-amorphous pillar array. In some embodiments, the pillar array forms an inner surface of each of the plurality of interconnected channels that the SEC microfluidic device comprises.

[0299] In some embodiments, the SEC microfluidic device comprises a quartz substrate. In some embodiments, the SEC microfluidic device comprises a monolithic substrate forming a plurality of interconnected channels. In some embodiments, the SEC microfluidic device comprises a quartz monolithic substrate. In some embodiments, the SEC microfluidic device comprises a three-dimensional (3D) printed substrate.

[0300] In some embodiments, the interconnected channels of the SEC microfluidic device are in an open tubular format.

[0301] ii. Reversed-phase liquid chromatography (RPLC) microfluidic device In some embodiments, the microfluidic device configured to separate components of a sample is a reversed-phase chromatography (RPLC) microfluidic device. In such embodiments, the RPLC microfluidic device includes a size-exclusion chromatography (RPLC) medium disposed at least within a plurality of interconnected channels of the RPLC chromatography device. In some embodiments, the RPLC medium is further disposed in an upstream network of connecting channels. In some embodiments, the RPLC medium is further disposed in a downstream network of connecting channels.

[0302] In some embodiments, the reversed-phase medium comprises an alkyl moiety, such as an alkyl moiety of any carbon chain length. 20In some embodiments, the reverse phase medium comprises an alkyl moiety having a carbon chain length of C2, C4, C8, or C 18 In some embodiments, the reverse phase medium comprises an alkyl moiety having a carbon chain length of any of C2 to C 20 In some embodiments, the reversed-phase medium comprises an RPLC moiety mixture that includes two or more alkyl moieties having the following carbon chain lengths: C2, C4, C8, and C 18 In some embodiments, the reversed-phase medium comprises an RPLC submixture comprising two or more of C2 to C4. 20 In some embodiments, the reversed-phase medium comprises an RPLC moiety mixture that includes three or more of the following alkyl moieties: C2, C4, C8, and C 18 In some embodiments, the RPLC moiety mixture comprises three or more of the following alkyl moieties: C2, C4, C8, and C 18 Includes.

[0303] The alkyl moieties of the reversed-phase medium can be based on the desired separation, hi some embodiments, the alkyl moieties of the RPLC moiety mixture are present in equimolar amounts.

[0304] In some embodiments, an alkyl moiety of a reversed-phase medium, such as an RPLC moiety mixture, is covalently attached to the surface of each of a plurality of interconnected channels of an RPLC microfluidic device. For example, in some embodiments, the inner surface of a plurality of interconnected parallel channels comprises silica (SiO2).

[0305] In some embodiments, the RPLC microfluidic device includes a plurality of interconnected channels. In some embodiments, the RPLC microfluidic devices described herein can include any number of interconnected channels such that the interconnected channels are in fluid communication with an input port of the RPLC microfluidic device, and the interconnected channels are configured to receive a portion of a fluid introduced into the RPLC microfluidic device via the input port. In some embodiments, the plurality of interconnected channels of the RPLC microfluidic device includes eight or more interconnected channels. In some embodiments, the plurality of interconnected channels includes 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, In some embodiments, the plurality of interconnected channels of the RPLC microfluidic device comprises 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 interconnected channels. In some embodiments, the plurality of interconnected channels of the RPLC microfluidic device comprises 32 interconnected channels. In some embodiments, the plurality of interconnected channels of the RPLC microfluidic device comprises 64 interconnected channels.

[0306] In some embodiments, each of the plurality of interconnected channels of the RPLC microfluidic device is in fluid communication with an input port of the RPLC microfluidic device. In some embodiments, each of the plurality of interconnected channels of the RPLC microfluidic device is in fluid communication with an input port of the RPLC microfluidic device via an upstream network of connecting channels. In some embodiments, the upstream network of connecting channels is configured to direct and split the flow of fluid introduced through the input port of the RPLC microfluidic device such that a portion of the fluid is delivered to each interconnected channel. In some embodiments, the upstream network of connecting channels or a portion thereof is connected to a proximal region of each of the plurality of interconnected channels. In some embodiments, the proximal region of the interconnected parallel channels is a region of the interconnected channel that is first exposed to the fluid introduced through the input port of the RPLC microfluidic device. The upstream network of connecting channels can be configured in many ways and can provide one or more functions, such as a mixing function and / or a filtering function and / or a dilution function, in addition to directing the fluid to the interconnected channels. In some embodiments, the upstream network of connecting channels includes a series of branching channels configured to split the fluid flow from an input port of the RPLC microfluidic device to each of the multiple interconnected channels. In some embodiments, the series of branching channels of the upstream network of connecting channels are structured using a 1:2 split (e.g., one channel splits into two channels in an upstream to downstream direction based on the intended fluid flow).For example, in some embodiments, for an RPLC microfluidic device having a single input port and 32 interconnected channels, the upstream network of connecting channels includes a single channel that splits into two channels, each of the two channels splits into two channels (now a total of four channels), each of the four channels splits into two channels (now a total of eight channels), each of the eight channels splits into two channels (now 16 channels), each of the 16 channels splits into two channels (now 32 channels), and each of the 32 channels connects to one of the 32 interconnected channels. In some embodiments, the upstream network of connecting channels includes a 1:2 split, a 1:3 split, a 1:4 split, a 1:5 split, a 1:6 split, a 1:7 split, a 1:8 split, a 1:9 split, a 1:10 split, a 1:11 split, a 1:12 split, or any combination thereof. In some embodiments, the channels of the upstream network of connecting channels after splitting (ie, the split channels) have smaller cross-sectional dimensions (eg, height and / or width) compared to the original channels.

[0307] In some embodiments, each of the plurality of interconnected channels of the RPLC microfluidic device is in fluid communication with an output port of the RPLC microfluidic device. In some embodiments, each of the plurality of interconnected channels of the RPLC microfluidic device is in fluid communication with an output port of the RPLC microfluidic device via a downstream network of connecting channels. In some embodiments, the downstream network of connecting channels is configured to direct and couple the fluid flow from each of the plurality of interconnected channels to an output port of the RPLC microfluidic device (e.g., including two or more output ports of the microfluidic device). In some embodiments, the downstream network of connecting channels, or a portion thereof, is connected to a distal region of each of the plurality of interconnected channels. In some embodiments, the distal region of the interconnected channel is a region of the interconnected channel where the fluid exits the interconnected channel to a downstream feature. The downstream network of connecting channels can be configured in a number of ways and can provide one or more functions, such as a mixing function and / or a filtering function and / or a dilution function, in addition to directing the fluid to the interconnected channel. In some embodiments, the downstream network of connecting channels includes a series of converging channels configured to combine the fluid flow from the plurality of interconnected channels to the output port of the RPLC microfluidic device. In some embodiments, a series of branching channels in an upstream network of connecting channels are structured using a two-to-one split (e.g., two channels converge into one channel in an upstream to downstream direction based on the intended fluid flow).For example, in some embodiments, for an RPLC microfluidic device having a single output port and 32 interconnected channels, the downstream network of connecting channels includes 32 channels, each of the 32 channels of the downstream network of connecting channels is connected to a channel of the 32 interconnected channels, pairs of the 32 channels of the downstream network of connecting channels converge into a single channel (here 16 channels), pairs of the 16 channels of the downstream network of connecting channels converge into a single channel (here 8 channels), pairs of the 8 channels of the downstream network of connecting channels converge into a single channel (here 4 channels), pairs of the 4 channels of the downstream network of connecting channels converge into a single channel (here 2 channels), and 2 channels of the downstream network of connecting channels converge into a single channel that is in fluid communication with the output port. In some embodiments, the downstream network of connecting channels comprises a 2 to 1 convergence, a 3 to 1 convergence, a 4 to 1 convergence, a 5 to 1 convergence, a 6 to 1 convergence, a 7 to 1 convergence, a 8 to 1 convergence, a 9 to 1 convergence, a 10 to 1 convergence, an 11 to 1 convergence, a 12 to 1 convergence, or any combination thereof. In some embodiments, the channels of the downstream network of connecting channels after convergence (i.e., the converging channels) have a larger cross-sectional dimension (e.g., height and / or width) compared to the channels from which they originate.

[0308] In some embodiments, multiple interconnected channels of an RPLC microfluidic device are connected only via an upstream network of connecting channels and / or a downstream network of connecting channels, for example, in some embodiments, one interconnected channel is not connected to another interconnected channel except via an upstream network of connecting channels and / or a downstream network of connecting channels.

[0309] In some embodiments, each of the multiple interconnected channels of the RPLC microfluidic device has a length of about 2 cm to about 30 cm, such as about 5 cm to about 20 cm. In some embodiments, the length of the interconnected channels is at least about 5 cm, such as at least about 6 cm, 7 cm, 8 cm, 9 cm, 10 cm, 11 cm, 12 cm, 13 cm, 14 cm, 15 cm, 16 cm, 17 cm, 18 cm, 19 cm, 20 cm, 21 cm, 22 cm, 23 cm, 24 cm, 25 cm, 26 cm, 27 cm, 28 cm, 29 cm, or 30 cm. In some embodiments, the length of the interconnected channels is less than about 30 cm, such as less than about any of 29 cm, 28 cm, 27 cm, 26 cm, 25 cm, 24 cm, 23 cm, 22 cm, 21 cm, 20 cm, 19 cm, 18 cm, 17 cm, 16 cm, 15 cm, 14 cm, 13 cm, 12 cm, 11 cm, 10 cm, 9 cm, 8 cm, 7 cm, 6 cm, 5 cm, 4 cm, 3 cm, or 2 cm. In some embodiments, the length of the interconnected channels is less than about any of 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, 10 cm, 11 cm, 12 cm, 13 cm, 14 cm, 15 cm, 16 cm, 17 cm, 18 cm, 19 cm, 20 cm, 21 cm, 22 cm, 23 cm, 24 cm, 25 cm, 26 cm, 27 cm, 28 cm, 29 cm, or 30 cm.

[0310] In some embodiments, the total length of the plurality of interconnected channels is between about 20 cm and about 3,200 cm. In some embodiments, the total length of the plurality of interconnected channels is greater than about 20 cm, e.g., greater than about any of 50 cm, 75 cm, 100 cm, 150 cm, 200 cm, 250 cm, 300 cm, 350 cm, 400 cm, 450 cm, 500 cm, 600 cm, 700 cm, 800 cm, 900 cm, 1,000 cm, 1,250 cm, 1,500 cm, 1,750 cm, 2,000 cm, 2,250 cm, 2,500 cm, 2,750 cm, or 3,000 cm.

[0311] The channels of the interconnected channels, upstream network of connecting channels, and downstream network of connecting channels described herein can be formed to have a variety of cross-sectional shapes and sizes. In some embodiments, the cross-sectional shapes and sizes of the channels described herein can vary at different points of the channels. In some embodiments, the channels have cross-sectional shapes that include rectangular, square, or circular.

[0312] In some embodiments, the interconnected channels of the RPLC microfluidic device have cross-sectional dimensions of about 1 μm to about 15 μm, e.g., about 3 μm to about 10 μm. In some embodiments, the interconnected channels of the RPLC microfluidic device have cross-sectional dimensions of about 15 μm or less, e.g., about 14 μm or less, 13 μm or less, 12 μm or less, 11 μm or less, 10 μm or less, 9 μm or less, 8 μm or less, 7 μm or less, 6 μm or less, 5 μm or less, 4 μm or less, 3 μm or less, 2 μm or less, or 1 μm or less. In some embodiments, the interconnected channels of the RPLC microfluidic device have cross-sectional dimensions of about 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 14 μm, or 15 μm.

[0313] In some embodiments, the interconnected channels of the RPLC microfluidic device have a maximum cross-sectional dimension of about 1 μm to about 15 μm, e.g., about 3 μm to about 10 μm. In some embodiments, the interconnected channels of the RPLC microfluidic device have a maximum cross-sectional dimension of about 15 μm or less, e.g., about 14 μm or less, 13 μm or less, 12 μm or less, 11 μm or less, 10 μm or less, 9 μm or less, 8 μm or less, 7 μm or less, 6 μm or less, 5 μm or less, 4 μm or less, 3 μm or less, 2 μm or less, or 1 μm or less. In some embodiments, the interconnected channels of the RPLC microfluidic device have a maximum cross-sectional dimension of about 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 14 μm, or 15 μm. In some embodiments, the interconnected channels of the RPLC microfluidic device have a minimum cross-sectional dimension of about 1 μm or more, such as about 2 μm or more, 3 μm or more, 4 μm or more, 5 μm or more, 6 μm or more, 7 μm or more, 8 μm or more, 9 μm or more, or 10 μm or more. In some embodiments, the interconnected channels of the RPLC microfluidic device have a minimum cross-sectional dimension of about 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 14 μm, or 15 μm.

[0314] In some embodiments, the interconnected channels of the RPLC microfluidic device have a cross-sectional dimension perpendicular to the largest cross-sectional dimension of about 1 μm to about 15 μm, e.g., about 3 μm to about 10 μm. In some embodiments, the interconnected channels of the RPLC microfluidic device have a cross-sectional dimension perpendicular to the largest cross-sectional dimension of about 15 μm or less, e.g., about 14 μm or less, 13 μm or less, 12 μm or less, 11 μm or less, 10 μm or less, 9 μm or less, 8 μm or less, 7 μm or less, 6 μm or less, 5 μm or less, 4 μm or less, 3 μm or less, 2 μm or less, or 1 μm or less. In some embodiments, the interconnected channels of the RPLC microfluidic device have a cross-sectional dimension perpendicular to the largest cross-sectional dimension of about 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 14 μm, or 15 μm. In some embodiments, the interconnected channels of an RPLC microfluidic device have a maximum cross-sectional dimension of about any of 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 14 μm, or 15 μm, and a cross-sectional dimension perpendicular to the maximum cross-sectional dimension of about any of 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 14 μm, or 15 μm.

[0315] In some embodiments, the interconnected channels of the RPLC microfluidic device have a width (e.g., measured across a cross-sectional dimension) of about 1 μm to about 15 μm, e.g., about 1 μm to about 6 μm, about 3 μm to about 10 μm, or about 6 μm to about 12 μm. In some embodiments, the interconnected channels of the RPLC microfluidic device have a width (e.g., measured across a cross-sectional dimension) of about 15 μm or less, e.g., about 14 μm or less, 13 μm or less, 12 μm or less, 11 μm or less, 10 μm or less, 9 μm or less, 8 μm or less, 7 μm or less, 6 μm or less, 5 μm or less, 4 μm or less, 3 μm or less, 2 μm or less, or 1 μm or less. In some embodiments, the interconnected channels of an RPLC microfluidic device have a width (e.g., measured across a cross-sectional dimension) of about any of 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 14 μm, or 15 μm.

[0316] In some embodiments, the interconnected channels of the RPLC microfluidic device have a height (e.g., measured across a cross-sectional dimension) of about 1 μm to about 15 μm, e.g., about 1 μm to about 6 μm, about 3 μm to about 10 μm, or about 6 μm to about 12 μm. In some embodiments, the interconnected channels of the RPLC microfluidic device have a height (e.g., measured across a cross-sectional dimension) of about 15 μm or less, e.g., about 14 μm or less, 13 μm or less, 12 μm or less, 11 μm or less, 10 μm or less, 9 μm or less, 8 μm or less, 7 μm or less, 6 μm or less, 5 μm or less, 4 μm or less, 3 μm or less, 2 μm or less, or 1 μm or less. In some embodiments, the interconnected channels of an RPLC microfluidic device have a height (e.g., measured across a cross-sectional dimension) of about any of 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 14 μm, or 15 μm.

[0317] In some embodiments, the plurality of interconnected channels of the RPLC microfluidic device are formed by a pillar array. In some embodiments, the pillar array is an amorphous pillar array. In some embodiments, the pillar array is a non-amorphous pillar array. In some embodiments, the pillar array forms an interior surface of each of the plurality of interconnected channels that the RPLC microfluidic device comprises.

[0318] In some embodiments, the RPLC microfluidic device comprises a quartz substrate. In some embodiments, the RPLC microfluidic device comprises a monolithic substrate forming a plurality of interconnected channels. In some embodiments, the RPLC microfluidic device comprises a quartz monolithic substrate. In some embodiments, the RPLC microfluidic device comprises a three-dimensional (3D) printed substrate.

[0319] In some embodiments, the interconnected channels of an RPLC microfluidic device are in an open tubular format.

[0320] In some embodiments, the RPLC microfluidic device includes an online shunting mechanism. In some embodiments, the online shunting mechanism is a channel, such as a valve and / or a channel that receives a fluid flow. In some embodiments, the online shunting mechanism is disposed between the RPLC microfluidic device and a plurality of interconnected channels of the electrospray ionization device. In some embodiments, the online shunting mechanism is in fluid communication with a waste, for example, so that a particular portion of the RPLC eluate can be diverted away from the mass spectrometer interface.

[0321] V. Compositions Obtained from the Methods and / or Devices Described Herein Provided herein, in certain aspects, is a collection of compositions obtained from any of the methods and / or devices described herein. In some embodiments, each composition of the collection of compositions is an RPLC microfluidic device eluate.

[0322] In some embodiments, a composition, as used herein, refers to any mixture of two or more products, substances, liquids, and / or ingredients, including proteins, peptides, nucleic acids, metabolites, other biomolecules, and derivatives thereof. In some embodiments, a composition can be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.

[0323] VI. Kits, components, compositions (consumables, etc.) In some aspects, provided herein are kits, components, and compositions (such as consumables) of the methods, devices, and systems described herein. In some embodiments, the kits include a microfluidic liquid chromatography device, such as a SEC microfluidic device and / or a RPLC microfluidic device. In some embodiments, the kits include reagents, such as liquid fixatives, and / or compositions useful for the methods, devices, and systems described herein. In some embodiments, the kits include instructions for use in accordance with the disclosures herein.

[0324] VII. Analysis method In some embodiments, the mass spectrometry technique includes evaluation of a signal associated with the component or a subpopulation thereof, e.g., peak detection. Many suitable techniques for evaluating signals measured by a mass spectrometer are known in the art. In some embodiments, the mass spectrometry technique includes determining an ionization intensity associated with the component or a subpopulation thereof. In some embodiments, the mass spectrometry technique includes determining a peak height associated with the component or a subpopulation thereof. In some embodiments, the mass spectrometry technique includes determining a peak area associated with the component or a subpopulation thereof. In some embodiments, the mass spectrometry technique includes determining a peak volume associated with the component or a subpopulation thereof. In some embodiments, the mass spectrometry technique includes identifying the peptide product by amino acid sequence. In some embodiments, the mass spectrometry technique includes manually interpreting and verifying the assignment of the amino acid sequence of the peptide product. In some embodiments, the mass spectrometry technique includes identifying the first polypeptide by a protein identifier. In some embodiments, the mass spectrometry technique includes identifying one or more of the multiple polypeptides by a protein identifier, which may be identified in a commercially available or in-house generated database (from recombinant proteins or other synthetic standards of peptides or metabolites). Searching or library searching.

[0325] In some embodiments, identification of the polypeptide product is achieved using a spectral library. The use of a spectral library allows the attribution of knowledge gained about the polypeptide system, resulting in faster data analysis and fewer errors.

[0326] Any of the mass spectrometry techniques described can be applied to the methods described herein. In some embodiments, one or more biomolecules and / or components eluted from the RPLC microfluidic device are subjected to a mass spectrometer. In some embodiments, mass spectrometry is performed on one or more biomolecules and / or components of the test sample using a mass spectrometer. In some embodiments, the mass spectrometry includes analysis of fractions subjected to the RPLC technique using the RPLC microfluidic device. In some embodiments, the mass spectrometry includes obtaining one or more data sets including information obtained from the mass spectrometer for each fraction subjected to the RPLC technique using the RPLC microfluidic device. In some embodiments, a single data set includes information obtained from the mass spectrometer from a single fraction subjected to an RPLC technique, such as the RPLC technique described herein, using the RPLC microfluidic device. In some embodiments, each of the one or more data sets includes mass-to-charge ratio (m / z) and abundance information of ions of one or more biomolecules and / or components introduced into the mass spectrometer.

[0327] In some aspects, the methods provided herein can further include analyzing one or more outputs of the mass spectrometry technique. In some embodiments, the methods provided further include analyzing at least one of the one or more data sets comprising information obtained from the mass spectrometer.

[0328] In some embodiments, at least one of the one or more datasets is used to determine the identity of each of the plurality of one or more biomolecules in the test sample. References to "identity" herein refer to the name of the biomolecule in the test sample. For example, in some embodiments, at least one of the one or more datasets is for determining the protein name of any protein from the test sample or its product introduced into the mass spectrometer. In some embodiments, the m / z information in at least one of the one or more datasets is used to determine the identity of each of the plurality of one or more biomolecules in the test sample.

[0329] In some embodiments, at least one of the one or more data sets is used to determine the amount of each of the plurality of one or more biomolecules in the test sample. In some embodiments, the amount of the identified one or more biomolecules is determined. References herein to "identified biomolecules" refer to biomolecules of the test sample whose identity has been determined. In some embodiments, the abundance information of at least one of the one or more data sets is used to determine the amount of each of the plurality of one or more biomolecules in the test sample. It is within the level of ordinary skill in the art to determine a suitable technique for identifying or quantifying biomolecules from the test sample or its products introduced into the mass spectrometer based on the output of the subsequent mass spectrometry technique, e.g., m / z or abundance information.

[0330] In some embodiments, at least one dataset is used to identify or quantify one or more biomolecules of the test sample. For example, a single dataset can include data related to a single fraction (e.g., any of the fractions described in Section II-C), and the single dataset can be used to identify or quantify the biomolecules or products thereof present in that fraction and introduced into the mass spectrometer. In some embodiments, multiple datasets are used to identify or quantify one or more biomolecules of the test sample, for example, to identify or quantify the biomolecules or products thereof present in multiple fractions introduced into the mass spectrometer. Any number of datasets related to any number of fractions introduced into the mass spectrometer can be used to identify or quantify the associated biomolecules or products thereof.

[0331] In some embodiments, the methods provided herein further comprise identifying a signature comprising one or more identified biomolecules from the determined identities. References herein to a "signature" refer to a set of identified biomolecules. A signature can comprise all or a subset of the identified biomolecules in a test sample. In some embodiments, identifying a signature further comprises selecting a subset of the one or more identified biomolecules originally in the signature. In some embodiments, the subset of the one or more identified biomolecules is selected based on the measured amount of the one or more identified biomolecules. For example, the subset of the one or more identified biomolecules can be selected to comprise a biomolecule with high abundance.

[0332] In other embodiments, the method provided herein further comprises identifying a signature by measuring the amount of each of a plurality of one or more biomolecules in a test sample based on at least one of one or more data sets. The method comprises selecting a subset of the plurality of the one or more biomolecules in the test sample based on the measured amount, and determining the identity of each of the subset of the plurality of the one or more biomolecules in the test sample. That is, the amount of the plurality of biomolecules in the test sample can be first determined without identifying the plurality of biomolecules, and a subset of the plurality of biomolecules can be selected based on the measured amount. The identity of the subset of the plurality of biomolecules can then be determined.

[0333] In some embodiments, a subset of one or more identified biomolecules is selected based on a differential measurement of one or more identified biomolecules compared to a reference sample. In some embodiments, a plurality of subsets of one or more biomolecules in a test sample are selected based on a plurality of differential measurements of one or more biomolecules in a test sample compared to a reference sample. In some embodiments, a subset of one or more identified biomolecules (or a plurality of subsets of one or more biomolecules) is selected based on a differential measurement compared to a plurality of reference samples. In some embodiments, the identified biomolecules and associated measurements are determined for a plurality of test samples, and a subset of one or more identified biomolecules (or a subset of a plurality of biomolecules or products thereof) included in the signature is selected based on a differential measurement between a plurality of test samples and a plurality of reference samples. In some embodiments, the signature includes identified biomolecules that are more abundant, identified biomolecules that are less abundant, or both, compared to a reference sample or a plurality of reference samples.

[0334] In some embodiments, the test sample and the reference sample are selected to identify a specific biomolecular feature that is differentially expressed or has a different amount between subjects or groups of subjects with different health or disease states. In some embodiments, the reference sample is a sample from a healthy subject or a control subject. For example, in some embodiments, the test sample is a sample from a diseased subject and the reference sample is a sample from a healthy subject or a control subject. In some embodiments, the test sample is a sample from a subject with a precondition associated with a disease and the reference sample is a sample from a healthy subject or a control subject. References herein to a "control subject" refer to a subject who is healthy or has a disease or precondition unrelated to that of the subject providing the test sample. In some embodiments, both the test sample and the reference sample are samples from diseased subjects, but the diseased subject has a different state of the disease. For example, in some embodiments, the test sample is a sample from a subject with an active state of the disease and the reference sample is a sample from a subject with an inactive state of the disease. In some embodiments, the inactive state is remission. Remission is the reduction or disappearance of signs and symptoms of a disease. The term can also be used to refer to the period during which this reduction occurs. Remission can be considered partial or complete remission. In some embodiments, both the test sample and the reference sample are from diseased subjects, but the diseased subjects have different stages of disease. Patients are classified into specific disease stages based on etiology, pathophysiology, and severity, and patients at the same disease stage may require similar treatment and have similar expected outcomes. In some embodiments, the test sample is from a subject with advanced disease, and the reference sample is from a subject with early disease. Other exemplary disease stages include stage 1 (e.g., disease without complications), stage 2 (e.g., disease with local complications), and stage 3 (e.g., disease involves multiple systems or has systemic complications).

[0335] Thus, in some embodiments, provided herein is a signature that includes a plurality of identified molecules, or a subset thereof, identified using any of the methods provided herein. In some embodiments, provided herein is a signature that includes a subset of identified biomolecules, identified using any of the methods provided herein.

[0336] In some embodiments, the provided method further comprises subjecting all or a subset of the identified biomolecules of the signature to further analysis. In some embodiments, the provided method further comprises providing all or a subset of the identified biomolecules of the signature as input to one or more processes, each configured to analyze the type of data provided. For example, the identified biomolecules can include a protein name, and the protein name can be provided as input to a process configured to analyze aspects or relationships of the provided protein or its products (e.g., to perform protein-protein network analysis).

[0337] In some embodiments, the methods provided further include providing all or a subset of the signature-identified biomolecules as input to one or more processes each configured to perform gene enrichment analysis, one or more processes each configured to perform pathway analysis, and / or one or more processes each configured to perform network analysis. In some embodiments, methods of analyzing biomolecules of a sample are also provided herein, the methods further including providing any of the signature-identified biomolecules as input to one or more processes each configured to perform gene enrichment analysis, one or more processes each configured to perform pathway analysis, and / or one or more processes each configured to perform network analysis. Such processes can be used to identify patterns and relationships across pairs or groups within the identified biomolecules provided as input.

[0338] In some embodiments, the signature identified biomolecules are provided as input to one or more processes each configured to perform gene enrichment analysis, one or more processes each configured to perform pathway analysis, and one or more processes each configured to perform network analysis.

[0339] In some embodiments, identified biomolecules of one or more molecular types of the signature are provided as input. In some embodiments, the one or more molecular types include proteins. In some embodiments, the one or more molecular types include RNA, including coding and / or non-coding RNA. In some embodiments, the one or more molecular types include peptides. In some embodiments, the one or more molecular types include metabolites. In some embodiments, the one or more molecular types include any combination of proteins, RNA (coding and / or non-coding RNA), peptides, and metabolites. In some embodiments, the one or more molecular types consist solely of proteins.

[0340] In some embodiments, the signature-identified biomolecules are provided as input to one or more processes, each configured to perform gene enrichment analysis. Gene enrichment analysis (also called gene set enrichment analysis or functional enrichment analysis) includes methods that can be used to identify groups of biomolecules (such as groups of genes or proteins) that are over-represented in a set of biomolecules provided. These methods can also be used to identify regulators of the provided biomolecules, such as transcription factors or kinases whose activity affects the expression or activity of genes or proteins provided as input. These methods rely on statistical approaches to identify significantly enriched or depleted groups of biomolecules among the biomolecules provided as input. In some cases, biomolecules are grouped based on their involvement in the same biological pathway. These methods also rely on Gene Ontology (GO) to group biomolecules. GO is known in the art and includes human-curated representations of relationships between various biomolecules. GO includes those that describe cellular components, molecular functions, or biological processes. References herein to a particular GO, e.g., Cellular Component GO, also refer to all sub-ontologies contained within the larger ontology (e.g., references to Cellular Component GO include references to sub-ontologies within the Cellular Component GO).

[0341] In some embodiments, the one or more processes configured to perform gene enrichment analysis include a process configured to identify one or more GOs associated with at least one of the identified biomolecules of the signature provided as input or at least one of their products (i.e., at least one of the products of the identified biomolecules provided as input). In some embodiments, the one or more processes configured to perform gene enrichment analysis include a process configured to identify one or more cellular component GOs associated with at least one of the identified biomolecules of the signature provided as input or at least one of their products, a process configured to identify one or more molecular pathway GOs associated with at least one of the identified biomolecules of the signature provided as input or at least one of their products, and / or a process configured to identify one or more biological process GOs associated with at least one of the identified biomolecules of the signature provided as input or at least one of their products. In some embodiments, the one or more processes configured to perform gene enrichment analysis include a process configured to identify one or more cellular component GOs associated with at least one of the identified biomolecules of the signature provided as input, or at least one of its products, and a process configured to identify one or more molecular pathway GOs associated with at least one of the identified biomolecules of the signature provided as input, or at least one of its products.

[0342] In any of the foregoing embodiments, the one or more processes configured to perform gene enrichment analysis identify GOs that are enriched or highly represented in the identified biomolecules or their products provided as input. In some embodiments, the identified GOs are associated with a plurality or majority of the identified biomolecules or their products provided as input. In some embodiments, the number of identified biomolecules or their products associated with the identified GOs is greater than would be expected by chance (e.g., greater than the number associated on average with a randomly selected GO).

[0343] In some embodiments, the one or more processes configured to perform gene enrichment analysis include a process configured to identify one or more regulators of at least one of the identified biomolecules of the signature provided as input or at least one of their products. Regulators include biomolecules that can affect the abundance or activity of a biomolecule in a test sample, including transcription factors, small molecules, small regulatory RNAs (e.g., microRNAs or siRNAs), kinases, and phosphatases. In some embodiments, the one or more processes configured to perform gene enrichment analysis include a process configured to identify one or more transcription factors that regulate at least one of the identified biomolecules of the signature provided as input or at least one of their products. In some embodiments, the one or more processes configured to perform gene enrichment analysis include a process configured to identify one or more kinases that regulate at least one of the identified biomolecules of the signature provided as input or at least one of their products. In some embodiments, the one or more processes configured to perform gene enrichment analysis include a process configured to identify one or more transcription factors that regulate at least one of the identified biomolecules of the signature provided as input, or at least one of their products, and a process configured to identify one or more kinases that regulate at least one of the identified biomolecules of the signature provided as input, or at least one of their products.

[0344] In any of the foregoing embodiments, the one or more processes configured to perform the gene enrichment analysis identify regulators (e.g., transcription factors or kinases) that regulate a plurality or majority of the identified biomolecules provided as input, or their products. In some embodiments, the number of identified biomolecules or their products that are regulated by the identified regulators is greater than would be expected by chance (e.g., greater than the number that would be regulated on average by a randomly selected regulator).

[0345] Exemplary methods for performing gene enrichment analysis include standard gene set enrichment analysis (GSEA) algorithm, simple enrichment analysis (SEA) algorithm, and spectral gene set enrichment (SGSE) algorithm. Exemplary tools for performing gene enrichment analysis include or are provided by Nucleic Acid Sequence Analysis Resource (NASQAR), PlantRegMap, Molecular Signatures database (MSigDB), Broad Institute, WebGestalt (e.g., using hyper-representation analysis (ORA), GSEA, or network topology-based analysis (NSA) algorithm), Enrichr, GeneSCF, DAVID, Metascape, AmiGO2, Genomic region enrichment of annotations tool (GREAT), Functional Enrichment Analysis (FunRich), FuncAssociate, InterMine, ToppGene, Quantitative Set Analysis for Gene Expression (QuSAGE), Blast2GO, and g:Profiler. Exemplary tools for performing gene enrichment analysis also include those that can identify transcription factors or kinases that regulate proteins provided as input, including transcription factor enrichment analysis (TFEA) and kinase enrichment analysis (KEA), respectively.

[0346] In some embodiments, the signature-identified biomolecules are provided as input to one or more processes, each configured to perform pathway analysis. Pathway analysis includes methods that can be used to identify any biological pathways represented or enriched among the provided biomolecules, given a list of biomolecules as input. Biological pathways include metabolic pathways and signaling pathways. These methods can rely on GO as well as human regulatory pathway collections and interaction networks, such as those from resources KEGG, WikiPathways, Reactome, Pathway Studio, and Ingenuity Pathway Analysis. These pathway collections and interaction networks can be compiled from published materials and can include information on genes, proteins, metabolic pathways, molecular interactions, and biochemical reactions associated with a particular organism. They can also map how these various biomolecules and pathways are organized in cellular structures or larger reaction pathways. Pathway analysis also includes methods of pathway-based modeling. Types of pathway-based models and available tools for developing these models include partial differential equations / Boolean models (available tools include CellNetAnalyzer). network flow models (available tools include NetPhorest and NetworKIN); reconstruction methods based on transcriptional regulatory networks (available tools include ARACNe); and probabilistic graph models (PGM, available tools include PARADIGM).

[0347] In some embodiments, the one or more processes configured to perform pathway analysis include a process configured to identify one or more pathways, e.g., molecular, signaling, or metabolic pathways, associated with at least one of the identified biomolecules of the signature provided as input or at least one of its products. In some embodiments, the one or more processes configured to perform pathway analysis include a process configured to identify one or more molecular pathways associated with at least one of the identified biomolecules of the signature provided as input or at least one of its products, a process configured to identify one or more signaling pathways associated with at least one of the identified biomolecules of the signature provided as input or at least one of its products, and / or a process configured to identify one or more metabolic pathways associated with at least one of the identified biomolecules of the signature provided as input or at least one of its products. In some embodiments, the one or more processes configured to perform pathway analysis include a process configured to identify one or more signaling pathways associated with at least one of the identified biomolecules of the signature provided as input or at least one of its products.

[0348] In any of the preceding embodiments, the one or more processes configured to perform pathway analysis identify one or more pathways that are enriched or highly represented in the identified biomolecules or their products provided as input. In some embodiments, one or more identified pathways (e.g., signal transduction pathways) each include a plurality or a majority of the identified biomolecules or their products provided as input. In some embodiments, the number of identified biomolecules or their products included in each of the one or more identified pathways is greater than expected by chance (e.g., greater than the number that would be included, on average, in a randomly selected pathway).

[0349] Exemplary methods for performing pathway analysis include overrepresentation analysis (ORA), functional class scoring (FCS), signaling pathway impact analysis (SPIA), pathway topology analysis (PTA) including EnrichNet, Gene Graph Enrichment Analysis (GGEA), TopoGSA, and network enrichment analysis (NEA). Exemplary tools for performing pathway analysis include those provided via STRING, Cytoscape, Ingenuity, Pathways Studio, Pathways Studio Viewer, PTA:PathwayGuide, MetaCore, Wiki Pathways, CellNetAnalyzer, NetPhorest / NetworKIN, ARACNe, and Paradigm.

[0350] In some embodiments, the signature identified biomolecules are provided as input to one or more processes each configured to perform network analysis. Network analysis includes methods that can be used to identify relationships between the biomolecules provided as input, given a list of biomolecules as input. The relationships include physical or functional interactions. These networks can be constructed, for example, based on predicted co-expression, co-localization, genetic interactions, physical interactions, as well as predicted and shared protein domain data. Nodes or vertices can be used to represent the identified biomolecules provided as input, and edges connecting two nodes (or the nodes themselves), respectively, can be used to represent predicted or identified relationships between the connected nodes. Types of networks include transcriptional regulatory networks, virus-host networks, metabolic networks, protein-protein interaction networks, disease networks, and drug effect networks (e.g., networks of biomolecules whose expression or activity is affected by a particular drug). Networks can be identified with the provided list of biomolecules using interaction databases that can be constructed automatically or via human curation. Human-curated interaction databases include BioGRID and IntAct. Network analysis can be used to analyze the interconnectivity of the provided identified biomolecules (i.e., ), including detecting clusters of nodes (i.e., identified biomolecules) that are similar or part of closely connected groups, for example, groups of nodes that have many edges connecting them to each other. Network analysis can also be used to identify hubs of a network. Hubs of a network are nodes that have more than or more than the average number of edges connecting them to other nodes in the network. In biological networks, these hubs can be the central control devices of their associated pathways. Thus, in some embodiments, identification of drugs that target these hubs can broadly affect the pathways or processes affected by disease.

[0351] In some embodiments, the one or more processes configured to perform network analysis include a process configured to identify one or more networks each associated with at least one of the identified biomolecules of the signature provided as input or at least one of their products. In some embodiments, the one or more processes configured to perform network analysis include a process configured to identify one or more hubs of one or more networks each associated with at least one of the identified biomolecules of the signature provided as input or at least one of their products.

[0352] In some embodiments, the one or more processes configured to perform network analysis include a process configured to identify one or more molecular pathways associated with at least one of the identified biomolecules of the signature provided as input or at least one of their products, respectively, and / or a process configured to identify one or more signaling pathways associated with at least one of the identified biomolecules of the signature provided as input or at least one of their products, respectively. In some embodiments, the one or more processes configured to perform network analysis include a process configured to identify one or more molecular pathways associated with at least one of the identified biomolecules of the signature provided as input or at least one of their products, respectively, and a process configured to identify one or more signaling pathways associated with at least one of the identified biomolecules of the signature provided as input or at least one of their products, respectively.

[0353] In some embodiments, the one or more processes configured to perform network analysis include a process configured to identify one or more protein-protein interaction networks associated with at least one of the identified biomolecules of the signature provided as input or at least one of its products, respectively. In some embodiments, the process is further configured to identify one or more hubs associated with the one or more identified protein-protein interaction networks.

[0354] In some embodiments, the one or more processes configured to perform network analysis include a process configured to identify one or more drugs that target at least one of the identified biomolecules of the signature provided as input or at least one of their products. In some embodiments, the one or more processes configured to perform network analysis include two processes configured to identify one or more drugs that target at least one of the identified biomolecules of the signature provided as input or at least one of their products. In some embodiments, the process or each of the two processes is configured to identify one or more drugs that target at least one hub of a network that includes a plurality of identified biomolecules of the signature provided as input. In some embodiments, the process or each of the two processes is configured to (1) identify one or more networks associated with at least one of the identified biomolecules of the signature provided as input, (2) identify one or more hubs of the one or more identified networks, and (3) identify one or more drugs that target at least one of the identified hubs, respectively. In any of the foregoing embodiments, the network or one or more networks is a protein-protein interaction network.

[0355] In any of the foregoing embodiments, the process is configured to identify one or more networks or hubs thereof associated with a plurality of identified biomolecules, or a plurality of products thereof, of the signature provided as input, respectively. In some embodiments, the number of identified biomolecules, or products thereof, associated with the identified one or more networks is greater than would be expected by chance.

[0356] Exemplary network clustering algorithms include or are available through the Girvin-Newman method, the Markov Cluster algorithm, the HotNet algorithm, the HyperModules Cytoscape App, and Reactome FI Network and ReactomeFIViz. Exemplary tools for performing network analysis include GeneMANIA (which can be used, for example, to identify protein-protein interaction networks), HotNet, HyperModules, and Reactome Cytoscape FI App, as well as the L1000 Fireworks Display (L1000FWD) and iLINCS chemical perturbation type (piNET) algorithms, both of which can be used to identify drugs that target genes or proteins provided as input.

[0357] In some embodiments, the identified biomolecules of the signature are provided as input to one or more processes each configured to perform a gene enrichment analysis (e.g., any of the processes described above configured to perform a gene enrichment analysis), one or more processes each configured to perform a pathway analysis (e.g., any of the processes described above configured to perform a pathway analysis), and one or more processes each configured to perform a network analysis (e.g., any of the processes described above configured to perform a network analysis). In some embodiments, the one or more processes configured to perform a gene enrichment analysis include a process configured to identify one or more cellular component gene ontologies associated with at least one of the identified components provided as input or at least one of their products, respectively. In some embodiments, the one or more processes configured to perform a gene enrichment analysis include a process configured to identify one or more molecular pathway gene ontologies associated with at least one of the identified components provided as input or at least one of their products, respectively. In some embodiments, the one or more processes configured to perform a gene enrichment analysis include a process configured to identify one or more transcription factors that regulate at least one of the identified components provided as input or at least one of their products. In some embodiments, the one or more processes configured to perform gene enrichment analysis include a process configured to identify one or more kinases that regulate at least one of the identified components provided as input or at least one of their products. In some embodiments, the one or more processes configured to perform pathway analysis include a process configured to identify one or more signaling pathways associated with at least one of the identified components provided as input or at least one of their products, respectively.In some embodiments, the one or more processes configured to perform network analysis include a process configured to identify one or more networks associated with at least one of the identified components provided as input, or at least one of the products thereof. In some embodiments, the one or more networks are protein-protein interaction networks. In some embodiments, the one or more processes configured to perform network analysis include one or more processes configured to identify one or more drugs that target at least one of the identified components provided as input, or at least one of the products thereof, respectively. In some embodiments, the one or more processes configured to identify one or more drugs or at least one of the products thereof that target at least one of the identified components provided as input, respectively, are configured to identify one or more drugs that target a hub of a protein-protein interaction network that includes the identified components provided as input, or the products thereof, respectively.

[0358] In some embodiments, a method of analyzing a signature of identified biomolecules is also provided herein, the method comprising providing a plurality of identified biomolecules to each of a plurality of processes each configured to perform a gene enrichment analysis, a pathway analysis, or a network analysis, the providing being performed in any order, the plurality of identified biomolecules comprising a protein set, a transcriptome set, a peptide set, and / or a metabolite set, the plurality of processes including: a process configured to perform a gene enrichment analysis to identify one or more gene ontologies associated with at least one of the plurality of identified biomolecules or at least one of their products provided as input; a process configured to perform a pathway analysis to identify one or more signaling pathways associated with at least one of the plurality of identified biomolecules or at least one of their products provided as input; a process configured to perform a gene enrichment analysis to identify one or more transcription factors that regulate at least one of the plurality of identified biomolecules provided or at least one of their products; a process configured to perform a gene enrichment analysis to identify one or more kinases that regulate gene products of at least one of the plurality of identified biomolecules provided as input or at least one of their products; a process configured to perform a network analysis to identify one or more networks associated with at least one of the plurality of identified biomolecules provided as input or at least one of their products, respectively; and each of two processes configured to perform a network analysis to identify one or more drugs that target at least one of the plurality of identified biomolecules provided as input or at least one of their products, respectively.

[0359] In some embodiments, the plurality of identified biomolecules comprises a set of proteins. In some embodiments, the plurality of identified biomolecules comprises only proteins. In some embodiments, the one or more networks are protein-protein interaction networks. In some embodiments, each of the two processes configured to perform network analysis to identify one or more drugs that target at least one of the plurality of identified biomolecules provided as input or at least one of their products is configured to identify one or more drugs that target hubs of the protein-protein interaction network that includes the plurality of identified biomolecules provided as input or their products.

[0360] In some embodiments, a method of analyzing protein signatures comprises providing a plurality of proteins to each of a plurality of processes, each configured to perform a gene enrichment analysis, a pathway analysis, or a network analysis, the providing being performed in any order, the plurality of processes including: a process configured to perform a gene enrichment analysis to identify one or more gene ontologies associated with at least one of the plurality of proteins provided as input or at least one of their products; a process configured to perform a pathway analysis to identify one or more signaling pathways associated with at least one of the plurality of proteins provided as input or at least one of their products; a process configured to perform a signaling pathway analysis to identify one or more signaling pathways that regulate at least one of the plurality of proteins provided as input or at least one of their products; Also provided herein is a method, comprising: a process configured to perform a gene enrichment analysis to identify one or more transcription factors that regulate at least one of the plurality of proteins provided as input or at least one of their products; a process configured to perform a gene enrichment analysis to identify one or more kinases that regulate a gene product of at least one of the plurality of proteins provided as input or at least one of their products; a process configured to perform a network analysis to identify one or more networks associated with at least one of the plurality of proteins provided as input or at least one of their products, respectively; and each of two processes configured to perform a network analysis to identify one or more drugs that target at least one of the plurality of proteins provided as input or at least one of their products, respectively.

[0361] In some embodiments, the one or more networks are protein-protein interaction networks. In some embodiments, each of the two processes configured to perform network analysis to identify one or more drugs that target at least one of the plurality of identified biomolecules provided as input or at least one of their products is configured to identify one or more drugs that target the hubs of the protein-protein interaction network that includes the plurality of identified biomolecules provided as input or their products.

[0362] VIII. Exemplary Embodiments Embodiments of the subject matter of the present application are provided herein.

[0363] Embodiment 1. 1. A method for processing a test sample for mass spectrometry, comprising the steps of: (a) subjecting the test sample to a size exclusion chromatography (SEC) technique using a SEC microfluidic device, the test sample comprises one or more biomolecules and a chaotropic agent; the SEC microfluidic device comprises a plurality of interconnected channels; the providing step; (b) collecting a plurality of fractions eluted from the SEC microfluidic device; (c) subjecting one or more of the plurality of fractions from the SEC microfluidic device to a proteolytic technique; and (d) individually subjecting one or more fractions from one or both of steps (b) and (c) to a reversed-phase liquid chromatography (RPLC) technique using an RPLC microfluidic device under conditions to prepare each component of said one or more fractions for introduction into a mass spectrometer; the RPLC microfluidic device comprises a plurality of interconnected channels containing a reversed-phase medium; The individually providing step, wherein the RPLC microfluidic device is coupled to an electrospray ionization source.

[0364] Embodiment 2. 2. The method of embodiment 1, wherein the test sample is a biological sample.

[0365] Embodiment 3. 3. The method of embodiment 1 or 2, wherein said test sample is from an individual.

[0366] Embodiment 4. 4. The method of any one of embodiments 1-3, wherein said test sample has a concentration of chaotropic agent of about 5M to about 8M.

[0367] Embodiment 5. The method of any one of embodiments 1-4, wherein the chaotropic agent comprises guanidine or a salt thereof, guanidinium or a salt thereof, potassium or a salt thereof, lithium or a salt thereof, magnesium or a salt thereof, or sodium or a salt thereof.

[0368] Embodiment 6. The method of any one of embodiments 1-3, wherein said chaotropic agent is guanidine hydrochloride or guanidinium chloride.

[0369] Embodiment 7. The method of any one of embodiments 1-6, wherein the chaotropic agent in the test sample is derived from a liquid fixative.

[0370] Embodiment 8. The method of any one of the preceding embodiments, wherein the test sample has a concentration of viscosity modifier of about 5% to about 40%.

[0371] EMBODIMENT 9. The method of embodiment 8, wherein the viscosity modifier is glycerol.

[0372] EMBODIMENT 10. The method of embodiment 8 or 9, wherein the test sample comprises at least about 6M guanidine and about 10% to about 30% glycerol.

[0373] Embodiment 11. 11. The method of any one of embodiments 1 to 10, wherein the test sample subjected to the SEC technique using the SEC microfluidic device has a volume of about 1 μL to about 200 μL.

[0374] Embodiment 12. 12. The method of any one of embodiments 1-11, wherein the range of concentration of the mobile phase chaotropic agent of said SEC technique is within about ±40% of the predetermined concentration of chaotropic agent in said test sample.

[0375] Embodiment 13. 13. The method of any one of embodiments 1-12, wherein said SEC technique comprises the use of a SEC mobile phase having a concentration of a mobile phase chaotropic agent within the range of the chaotropic agent in said test sample.

[0376] Embodiment 14. 14. The method of any one of embodiments 1-13, wherein the mobile phase chaotropic agent of said SEC technique is the same as the chaotropic agent of said test sample.

[0377] EMBODIMENT 15. 14. The method of any one of embodiments 1-13, wherein the mobile phase chaotropic agent of said SEC technique is different from the chaotropic agent of said test sample.

[0378] EMBODIMENT 16. 16. The method of any one of embodiments 1-15, wherein said SEC mobile phase comprises a mobile phase chaotropic agent at a concentration of about 4M to about 8M.

[0379] EMBODIMENT 17. 17. The method of any one of embodiments 1-16, wherein the mobile phase chaotropic agent of the SEC technique comprises guanidine or a salt thereof, guanidinium or a salt thereof, lithium or a salt thereof, magnesium or a salt thereof, or sodium or a salt thereof.

[0380] EMBODIMENT 18. 18. The method of any one of the preceding embodiments, wherein the mobile phase chaotropic agent of the SEC technique is selected from the group consisting of guanidine hydrochloride, guanidinium chloride, guanidinium thiocyanate, lithium perchlorate, lithium acetate, magnesium chloride, potassium acetate and sodium iodide.

[0381] EMBODIMENT 19. The method of any one of the preceding embodiments, wherein the SEC mobile phase comprises a mobile phase viscosity modifier.

[0382] EMBODIMENT 20. The method of embodiment 20, wherein the mobile phase viscosity modifier in the SEC technique has a concentration of about 5% to about 40%.

[0383] EMBODIMENT 21. The method of embodiment 19 or 20, wherein the viscosity modifier is glycerol.

[0384] EMBODIMENT 22. The method of any one of embodiments 19-21, wherein the mobile phase viscosity modifier of the SEC technique is the same as the viscosity modifier of the liquid fixing agent.

[0385] EMBODIMENT 23. The method of any one of embodiments 19-21, wherein the mobile phase viscosity modifier of the SEC technique is different from the viscosity modifier of the liquid fixative.

[0386] EMBODIMENT 24. 22. The method of any one of embodiments 19-21, wherein the test sample comprises at least about 6M guanidine and about 10% to about 30% glycerol.

[0387] EMBODIMENT 25. The method of any one of embodiments 1-24, wherein said SEC technique is an isocratic SEC technique.

[0388] 26. 26. The method of any one of embodiments 1-25, wherein said SEC technique comprises using a mobile phase flow rate of about 1 μL / min to about 5 μL / min.

[0389] EMBODIMENT 27. 27. The method of any one of embodiments 1-26, wherein said SEC technique is carried out at elevated temperature.

[0390] 28. The method of any one of embodiments 1-27, wherein the SEC technique is carried out at a temperature of about 45° C. to about 60° C.

[0391] 29. 29. The method of embodiment 27 or 28, wherein said SEC technique is carried out at a substantially constant temperature.

[0392] EMBODIMENT 30. 30. The method of any one of embodiments 1-29, wherein said SEC microfluidic device comprises a SEC medium.

[0393] EMBODIMENT 31. 31. The method of embodiment 30, wherein said SEC medium is a material having an average pore size of about 10 nm to about 500 nm.

[0394] EMBODIMENT 32. 32. The method of embodiment 30 or 31, wherein the SEC medium is an inner surface of each of the plurality of interconnected channels.

[0395] EMBODIMENT 33. 33. The method of any one of embodiments 1-32, wherein the inner surface material of the plurality of interconnected channels of the SEC microfluidic device has a thickness of about 0.5 μm to about 2 μm.

[0396] EMBODIMENT 34. 34. The method of any one of embodiments 1-33, wherein the plurality of interconnected channels of the SEC microfluidic device are configured in an open tubular format.

[0397] EMBODIMENT 35. The method of any one of embodiments 1-34, wherein the plurality of interconnected channels of the SEC microfluidic device comprises eight or more interconnected channels.

[0398] 36. 36. The method of embodiment 35, wherein the plurality of interconnected channels of the SEC microfluidic device comprises 32 interconnected channels.

[0399] 37. 36. The method of embodiment 35, wherein the plurality of interconnected channels of the SEC microfluidic device comprises 64 interconnected channels.

[0400] 38. The method of any one of embodiments 1 to 37, wherein each of the plurality of interconnected channels of the SEC microfluidic device is in fluid communication with an input port of the SEC microfluidic device via an upstream network of connecting channels.

[0401] 39. 39. The method of embodiment 38, wherein the upstream network of connecting channels or a portion thereof is connected to a proximal region of each of the plurality of interconnected channels.

[0402] EMBODIMENT 40. 40. The method of embodiment 38 or 39, wherein the upstream network of connecting channels comprises a series of branching channels configured to split the flow of fluid from the input port of the SEC microfluidic device to each of the plurality of interconnected channels.

[0403] EMBODIMENT 41. The method of any one of embodiments 1 to 40, wherein each of the plurality of interconnected channels of the SEC microfluidic device is in fluid communication with an output port of the SEC microfluidic device via a downstream network of connecting channels.

[0404] EMBODIMENT 42. 42. The method of embodiment 41, wherein the downstream network of connecting channels or a portion thereof is connected to a distal region of each of the plurality of interconnected channels.

[0405] EMBODIMENT 43. 43. The method of embodiment 41 or 42, wherein the downstream network of connecting channels comprises a series of converging channels configured to combine fluid flow from the plurality of interconnected channels of the SEC microfluidic device to the output port.

[0406] EMBODIMENT 44. The method of any one of embodiments 41 to 43, wherein the multiple interconnected channels of the SEC microfluidic device are connected only via the upstream network of connecting channels or the downstream network of connecting channels.

[0407] EMBODIMENT 45. 45. The method of any one of the preceding embodiments, wherein each of the plurality of interconnected channels of the SEC microfluidic device has a length of about 2 cm to about 50 cm.

[0408] EMBODIMENT 46. 46. ​​The method of any one of embodiments 1-45, wherein each of the plurality of interconnected channels of the SEC microfluidic device has a width of about 1 μm to about 15 μm.

[0409] EMBODIMENT 47. 47. The method of any one of embodiments 1-46, wherein each of the plurality of interconnected channels of the SEC microfluidic device has a depth of about 1 μm to about 15 μm.

[0410] 48. 48. The method of any one of embodiments 1-47, wherein the plurality of interconnected channels of the SEC microfluidic device are formed by a pillar array.

[0411] 49. The method of embodiment 48, wherein the pillar array is an amorphous pillar array.

[0412] EMBODIMENT 50. The method of embodiment 48, wherein said pillar array is a non-amorphous pillar array.

[0413] EMBODIMENT 51. The method of any one of embodiments 32-50, wherein the pillar array forms an inner surface of each of the plurality of interconnected channels of the SEC microfluidic device.

[0414] EMBODIMENT 52. 52. The method of any one of embodiments 1-51, wherein said SEC microfluidic device comprises a quartz substrate.

[0415] EMBODIMENT 53. 43. The method of any one of embodiments 1-42, wherein said SEC microfluidic device comprises a monolithic substrate forming said plurality of interconnected channels.

[0416] EMBODIMENT 54. The method of any one of embodiments 1-53, wherein the SEC microfluidic device comprises a quartz monolithic substrate.

[0417] EMBODIMENT 55. The method of any one of embodiments 1 to 44, wherein the SEC microfluidic device comprises a three-dimensional (3D) printed circuit board.

[0418] EMBODIMENT 56. 56. The method of any one of embodiments 1-55, wherein the step of collecting the multiple fractions eluted from the SEC microfluidic device is carried out using a fraction collector.

[0419] EMBODIMENT 57. 57. The method of any one of embodiments 1-56, wherein each of the plurality of fractions is collected from the SEC microfluidic device based on time.

[0420] EMBODIMENT 58. 58. The method of embodiment 57, wherein each of the plurality of fractions is collected from the SEC microfluidic device over a period of about 30 seconds to about 5 minutes.

[0421] EMBODIMENT 59. 59. The method of embodiment 57 or 58, wherein each of said plurality of fractions is collected from said SEC microfluidic device over a uniform amount of time.

[0422] 60. 59. The method of embodiment 47 or 58, wherein a fraction of said plurality of fractions is collected from said SEC microfluidic device for a different amount of time than another fraction of said plurality of fractions.

[0423] 61. 57. The method of any one of embodiments 1-56, wherein each of the plurality of fractions is collected from the SEC microfluidic device based on the volume of eluate from the SEC microfluidic device.

[0424] 62. 62. The method of embodiment 61, wherein each of the plurality of fractions collected from the SEC microfluidic device has a volume of about 1 μL to about 20 μL.

[0425] 63. 63. The method of embodiment 61 or 62, wherein each of the multiple fractions collected from the SEC microfluidic device has a uniform volume.

[0426] 64. 64. The method of embodiment 62 or 63, wherein a fraction of the plurality of fractions collected from the SEC microfluidic device has a different volume than another fraction of the plurality of fractions.

[0427] 65. 65. The method of any one of embodiments 1-64, wherein said plurality of fractions is about 5 to about 50 fractions.

[0428] 66. 66. The method of embodiment 65, wherein said plurality of fractions is from about 12 to about 24 fractions.

[0429] 67. The method of any one of the preceding embodiments, wherein said proteolytic techniques include enzyme-based digestion techniques.

[0430] 68. 68. The method of embodiment 67, wherein said enzyme-based digestion technique comprises the use of an enzyme selected from the group consisting of trypsin, chymotrypsin, pepsin, LysC, LysN, AspN, GluC and ArgC, or a combination thereof.

[0431] 69. 69. The method of embodiment 67 or 68, wherein said enzyme-based digestion technique comprises a step of diluting the fraction eluted from said SEC microfluidic device.

[0432] EMBODIMENT 70. The dilution step comprises: mixing the fraction eluted from the SEC microfluidic device with water to reach a concentration of the chaotropic agent; 70. The method of embodiment 69, comprising:

[0433] EMBODIMENT 71. 71. The method of embodiment 70, wherein the final concentration of said chaotropic agent for said enzymatic digestion is about 0.5M.

[0434] EMBODIMENT 72. The method of any one of embodiments 67-71, wherein said enzyme-based digestion technique does not include a buffer exchange step.

[0435] EMBODIMENT 73. The method of any one of embodiments 67-72, wherein said enzyme-based digestion technique does not include an alkylation step.

[0436] 74. The method of any one of embodiments 67-72, wherein said enzyme-based digestion technique does not include a reduction step.

[0437] 75. The method of any one of embodiments 1-66, wherein said proteolytic techniques include non-enzyme-based approaches.

[0438] EMBODIMENT 76. subjecting one or more of the plurality of fractions from the SEC microfluidic device and / or one or more of the plurality of fractions subjected to the proteolytic technique to a quantitative labeling technique; The quantitative labeling technique is performed prior to the reverse phase liquid chromatography (RPLC) technique using the RPLC microfluidic device; The method of any one of embodiments 1 to 75.

[0439] EMBODIMENT 77. 77. The method of embodiment 76, wherein said quantitative labeling technique comprises the use of isobaric mass tags.

[0440] 78. 78. The method of embodiment 76 or 77, wherein said quantitative labeling technique comprises the use of tandem mass tags (TMT).

[0441] 79. The method of any one of embodiments 76-78, wherein said quantitative labeling technique includes a desalting step.

[0442] 80. mixing an internal standard with one or more of the plurality of fractions from the SEC microfluidic device and / or with one or more of the plurality of fractions subjected to the proteolytic technique; The mixing step of the internal standard is performed prior to the reverse phase liquid chromatography (RPLC) technique using the RPLC microfluidic device. The method of any one of embodiments 1 to 79.

[0443] 81. 80. The method of embodiment 79, wherein said internal standard is an isotopically labeled peptide.

[0444] 82. The one or more fractions subjected to the RPLC technique are selected from the group consisting of: (i) zero or more fractions obtained from said SEC microfluidic device; and (ii) subjecting one or more of said plurality of fractions to said proteolytic technique. One or more fractions or portions thereof obtained from The method of any one of embodiments 1-81, comprising:

[0445] 83. 83. The method of any one of the preceding embodiments, wherein each of said one or more fractions subjected to said RPLC technique comprises said respective source fraction mixed with an aqueous solution.

[0446] 84. 84. The method of any one of the preceding embodiments, wherein the fraction subjected to the RPLC technique has a volume of about 1 μL to about 50 μL.

[0447] 85. The method of any one of embodiments 1-84, wherein said RPLC technique comprises the use of an RPLC mobile phase.

[0448] 86. 86. The method of embodiment 85, wherein said RPLC technique comprises a mobile phase flow rate of said RPLC mobile phase of about 0.05 μL / min to about 2 μL / min.

[0449] 87. The method of any one of embodiments 1-86, wherein said RPLC technique is a gradient RPLC technique.

[0450] 88. The method of any one of embodiments 1-87, wherein said RPLC technique is carried out at elevated temperature.

[0451] 89. 38. The method of any one of the preceding embodiments, wherein the RPLC technique is carried out at a temperature of about 30° C. to about 100° C.

[0452] EMBODIMENT 90. 90. The method of embodiment 88 or 89, wherein said RPLC technique is carried out at a substantially constant temperature.

[0453] EMBODIMENT 91. The reverse phase medium may comprise the following alkyl moieties: C2, C4, C8, and C 18 91. The method of any one of embodiments 1-90, comprising an RPLC submixture comprising two or more of:

[0454] EMBODIMENT 92. The RPLC moiety mixture comprises the following alkyl moieties: C2, C4, C8, and C 18 92. The method of embodiment 91, comprising three or more of:

[0455] EMBODIMENT 93. The RPLC moiety mixture comprises the following alkyl moieties: C2, C4, C8, and C 18 92. The method of embodiment 91, comprising:

[0456] EMBODIMENT 94. The method of any one of embodiments 91-93, wherein the alkyl moieties of the RPLC moiety mixture are present in equimolar amounts.

[0457] EMBODIMENT 95. 95. The method of any one of embodiments 91-94, wherein the alkyl moieties of the RPLC moiety mixture are covalently attached to a surface of each of the plurality of interconnected channels of the RPLC microfluidic device.

[0458] EMBODIMENT 96. 96. The method of embodiment 95, wherein a surface of each of the plurality of interconnected channels comprises silica (SiO2).

[0459] EMBODIMENT 97. 97. The method of any one of embodiments 1-96, wherein the plurality of interconnected channels of the RPLC microfluidic device comprises eight or more interconnected channels.

[0460] EMBODIMENT 98. 98. The method of embodiment 97, wherein the plurality of interconnected channels of the RPLC microfluidic device comprises 32 interconnected channels.

[0461] EMBODIMENT 99. 98. The method of embodiment 97, wherein the plurality of interconnected channels of the RPLC microfluidic device comprises 64 interconnected channels.

[0462] EMBODIMENT 100. 86. The method of any one of embodiments 1 to 85, wherein each of the plurality of interconnected channels of the RPLC microfluidic device is in fluid communication with an input port of the RPLC microfluidic device via an upstream network of connecting channels.

[0463] EMBODIMENT 101. The method of embodiment 100, wherein the upstream network of connecting channels or a portion thereof is connected to the proximal region of each of the plurality of interconnected channels.

[0464] EMBODIMENT 102. 102. The method of embodiment 100 or 101, wherein the upstream network of connecting channels comprises a series of branching channels configured to split the flow of fluid from the input port of the RPLC microfluidic device to each of the plurality of interconnected channels.

[0465] EMBODIMENT 103. 103. The method of any one of embodiments 1-102, wherein each of the plurality of interconnected channels of the RPLC microfluidic device is in fluid communication with an output port of the RPLC microfluidic device via a downstream network of connecting channels.

[0466] EMBODIMENT 104. The method of embodiment 103, wherein the downstream network of connecting channels or a portion thereof is connected to a distal region of each of the plurality of interconnected channels.

[0467] EMBODIMENT 105. The method of embodiments 103 and 104, wherein the downstream network of connecting channels comprises a series of converging channels configured to combine fluid flow from the multiple interconnected channels of the RPLC microfluidic device to the output port.

[0468] EMBODIMENT 106. The method of any one of embodiments 103 to 105, wherein the plurality of interconnected channels of the RPLC microfluidic device are connected only via the upstream network of connecting channels or the downstream network of connecting channels.

[0469] EMBODIMENT 107. 107. The method of any one of embodiments 1-106, wherein each of the plurality of interconnected channels of the RPLC microfluidic device has a length of about 2 cm to about 50 cm.

[0470] EMBODIMENT 108. 108. The method of any one of embodiments 1-107, wherein each of the plurality of interconnected channels of the RPLC microfluidic device has a width of about 1 μm to about 15 μm.

[0471] EMBODIMENT 109. 109. The method of any one of embodiments 1-108, wherein each of the plurality of interconnected channels of the RPLC microfluidic device has a depth of about 1 μm to about 15 μm.

[0472] EMBODIMENT 110. 110. The method of any one of embodiments 1-109, wherein the plurality of interconnected channels of the RPLC microfluidic device are formed by a pillar array.

[0473] EMBODIMENT 111. 111. The method of embodiment 110, wherein the pillar array is an amorphous pillar array.

[0474] EMBODIMENT 112. 111. The method of embodiment 110, wherein the pillar array is a non-amorphous pillar array.

[0475] EMBODIMENT 113. The method of any one of embodiments 110 to 112, wherein the pillar array forms an inner surface of each of the plurality of interconnected channels that the RPLC microfluidic device comprises.

[0476] EMBODIMENT 114. The method of any one of embodiments 1-113, wherein said RPLC microfluidic device comprises an online flow splitting mechanism.

[0477] EMBODIMENT 115. 115. The method of embodiment 114, wherein the online flow shunting mechanism is a valve and / or a channel.

[0478] EMBODIMENT 116. 116. The method of embodiment 114 or 115, wherein the online shunting mechanism is disposed between the plurality of interconnected channels of the RPLC microfluidic device and the electrospray ionization device.

[0479] EMBODIMENT 117. 117. The method of any one of embodiments 1-116, wherein the RPLC microfluidic device comprises a quartz substrate.

[0480] EMBODIMENT 118. 118. The method of any one of embodiments 1-117, wherein the RPLC microfluidic device comprises a monolithic substrate forming the plurality of interconnected channels.

[0481] EMBODIMENT 119. 119. The method of any one of embodiments 1-118, wherein the RPLC microfluidic device comprises a quartz monolithic substrate.

[0482] EMBODIMENT 120. The method of any one of embodiments 1-119, wherein the RPLC microfluidic device comprises a three-dimensional (3D) printed circuit board.

[0483] EMBODIMENT 121. 121. The method of any one of embodiments 1-120, wherein said RPLC microfluidic device is configured in an open tubular format.

[0484] EMBODIMENT 122. 122. The method of any one of embodiments 1-121, wherein said RPLC microfluidic device is configured for online desalting.

[0485] EMBODIMENT 123. The method of any one of embodiments 1-122, wherein the electrospray ionization source is a nanoelectrospray ionization source.

[0486] EMBODIMENT 124. The method of any one of embodiments 1-123, wherein the electrospray ionization source is a heated electrospray ionization source.

[0487] EMBODIMENT 125. The method of any one of embodiments 1-124, wherein said sample is selected from the group consisting of a blood sample, a cerebrospina...

Claims

1. 1. A method for processing a test sample for mass spectrometry, comprising the steps of: (a) subjecting the test sample to a size exclusion chromatography (SEC) technique using a SEC microfluidic device, the test sample comprises one or more biomolecules and a chaotropic agent; the SEC microfluidic device comprises a plurality of interconnected channels; the providing step; (b) collecting a plurality of fractions eluted from the SEC microfluidic device; (c) subjecting one or more of the plurality of fractions from the SEC microfluidic device to a proteolytic technique; and (d) individually subjecting one or more fractions from one or both of steps (b) and (c) to a reversed-phase liquid chromatography (RPLC) technique using an RPLC microfluidic device under conditions to prepare each component of said one or more fractions for introduction into a mass spectrometer; the RPLC microfluidic device comprises a plurality of interconnected channels containing a reversed-phase medium; The individually providing step, wherein the RPLC microfluidic device is coupled to an electrospray ionization source. (i) the test sample has a concentration of chaotropic agent of about 5M to about 8M; and / or (ii) the chaotropic agent comprises guanidine or a salt thereof, guanidinium or a salt thereof, potassium or a salt thereof, lithium or a salt thereof, magnesium or a salt thereof, or sodium or a salt thereof; The method of claim 1.

3. The method of claim 1 or 2, wherein the chaotropic agent is guanidine hydrochloride or guanidinium chloride.

4. The method of any one of claims 1 to 3, wherein the chaotropic agent in the test sample is derived from a liquid fixative.

5. The method of any one of claims 1 to 4, wherein the test sample subjected to the SEC technique using the SEC microfluidic device has a volume of about 1 μL to about 200 μL.

6. 6. The method of any one of claims 1 to 5, wherein the range of concentrations of the mobile phase chaotropic agent of the SEC technique is within about ±40% of the predetermined concentration of chaotropic agent in the test sample.

7. The method of any one of claims 1 to 6, wherein the SEC microfluidic device comprises an SEC medium comprising a material having an average pore size of about 10 nm to about 500 nm.

8. The method of any one of claims 1 to 7, wherein the SEC microfluidic device comprises a SEC medium that is an inner surface of each of the plurality of interconnected channels.

9. (i) the inner surface material of the plurality of interconnected channels of the SEC microfluidic device has a thickness of about 0.5 μm to about 2 μm; (ii) the plurality of interconnected channels of the SEC microfluidic device are configured in an open tubular format; and / or (iii) the plurality of interconnected channels of the SEC microfluidic device comprises eight or more interconnected channels; The method according to any one of claims 1 to 8.

10. 10. The method of any one of claims 1 to 9, wherein each of the plurality of interconnected channels of the SEC microfluidic device is in fluid communication with an input port of the SEC microfluidic device via an upstream network of connecting channels.

11. 11. The method of any one of claims 1 to 10, wherein each of the plurality of interconnected channels of the SEC microfluidic device is in fluid communication with an output port of the SEC microfluidic device via a downstream network of connecting channels.

12. 12. The method of any one of claims 1 to 11, wherein each of the plurality of interconnected channels of the SEC microfluidic device has a length of about 2 cm to about 50 cm, a width of about 1 μm to about 15 μm, and / or a depth of about 1 μm to about 15 μm.

13. The method of any one of claims 1 to 12, wherein the plurality of interconnected channels of the SEC microfluidic device are formed by a pillar array.

14. The method of any one of claims 1 to 13, wherein the SEC microfluidic device comprises a quartz substrate.

15. The method of claim 1, wherein the plurality of interconnected channels of the SEC microfluidic device include an inner surface having an average pore size of less than about 500 nm.

16. The method according to any one of claims 1 to 15, wherein the proteolytic technique comprises an enzyme-based digestion technique.

17. 17. The method of claim 16, wherein the enzyme-based digestion technique comprises the use of an enzyme selected from the group consisting of trypsin, chymotrypsin, pepsin, LysC, LysN, AspN, GluC and ArgC, or a combination thereof.

18. 18. The method of claim 16 or 17, wherein the enzyme-based digestion technique comprises a step of diluting the fraction eluted from the SEC microfluidic device.

19. The diluting step comprises: mixing the fraction eluted from the SEC microfluidic device with water to reach a concentration of the chaotropic agent; 20. The method of claim 18, comprising:

20. The method according to any one of claims 16 to 19, wherein the enzyme-based digestion technique does not include a buffer exchange step, an alkylation step, and / or a reduction step.

21. The reverse phase medium comprises an alkyl moiety: ２ , C ４ , C ８ , and C １８ 21. The method of any one of claims 1 to 20, comprising an RPLC submixture comprising two or more of:

22. The RPLC moiety mixture comprises the following alkyl moieties: ２ , C ４ , C ８ , and C １８ 22. The method of claim 21 , comprising:

23. A method according to any one of claims 1 to 22, wherein the sample is a blood sample derived from an individual, the method further comprising the step of preparing a plasma sample, and further wherein the step of preparing the plasma sample comprises subjecting the blood sample to a plasma generation technique.

24. The method of any one of claims 1 to 23, further comprising subjecting the components or products thereof eluted from the RPLC microfluidic device to the mass spectrometer.

25. The method of claim 25, further comprising: (i) performing mass spectrometry analysis of said components of said sample or products thereof using said mass spectrometer, said mass spectrometry analysis comprising analysis of each fraction subjected to said RPLC technique using said RPLC microfluidic device; and / or (ii) performing mass spectrometry of the components of the sample or products thereof using the mass spectrometer, the mass spectrometry comprising obtaining one or more data sets comprising information obtained from the mass spectrometer for each fraction subjected to the RPLC technique using the RPLC microfluidic device.

25. The method of claim 24, further comprising:

26. 1. A method for analyzing a collection of compositions using mass spectrometry techniques, comprising: (a) subjecting each composition of the collection of compositions to a mass spectrometer; and (b) performing mass spectrometry analysis of each composition of the collection of compositions. Including, The method, wherein the collection of compositions is obtained from a processing technique comprising fractionation of a test sample using a SEC technique comprising the use of a SEC microfluidic device, and subsequently applying each fraction or a product thereof to an RPLC technique comprising the use of an RPLC microfluidic device.

27. identifying a signature comprising one or more identified biomolecules; The step of identifying determining an amount of each of a plurality of the one or more biomolecules in the test sample based on at least one of the one or more data sets; selecting a plurality of subsets of the one or more biomolecules in the sample based on the measured amounts; determining the identity of each of a subset of a plurality of one or more biomolecules in the test sample; 27. The method of any one of claims 25 or 26, comprising:

28. below: An input port; an upstream network of connecting channels; A plurality of interconnected channels, each channel of the plurality of interconnected channels is in an open tubular format; each channel of the plurality of interconnected channels includes an interior surface that includes an SEC medium; each channel of the plurality of interconnected channels is in fluid communication with the input port through the upstream network of connecting channels; the plurality of interconnected channels; A size exclusion chromatography (SEC) microfluidic device comprising:

29. below: An input port; an upstream network of connecting channels; A plurality of interconnected channels, each channel of the plurality of interconnected channels is in an open tubular format; each channel of the plurality of interconnected channels includes an inner surface that includes an RPLC medium; each channel of the plurality of interconnected channels is in fluid communication with the input port through the upstream network of connecting channels; the plurality of interconnected channels; The reversed phase liquid chromatography (RPLC) microfluidic device comprising:

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