Method for processing biological sample to identify protein therein

Abstract

The present invention belongs to the field of spatial proteomics. Provided are a device for processing a biological sample to identify a protein therein, a kit comprising the device, and a method for processing a biological sample to identify a protein therein by means of using the device. By means of using the device for processing the biological sample, the cost of sample processing is decreased, the loss of peptide fragments during sample processing is reduced, and the processing capability for complex samples is improved. The device can be compatible with an automated device, thereby increasing the detection throughput. The device does not require desalination, and has a reduced missed cleavage rate, no sample loss problem, good detection reproducibility and increased peptide fragment and protein identifications, and can process extremely small tissue.

Description

A method for processing biological samples to identify proteins therein

[0001] The present disclosure is based on and claims priority to an application with CN application number 202310889444.4 and filing date July 18, 2023. The disclosure of the CN application is hereby incorporated into the present disclosure as a whole. Technical Field

[0002] The present disclosure belongs to the field of spatial proteomics, and particularly relates to a device for processing biological samples to identify proteins therein, a kit containing the device, and a method for processing biological samples to identify proteins therein using the device. Background Art

[0003] Spatial proteomics allows the study of the subcellular localization of proteins in cells or tissues that contribute to disease phenotypes. Spatial information is crucial for understanding the complex interactions between these proteins and their subcellular environment, guiding the identification of potential drug targets and the development of more effective therapies. While inter-patient variability in the tumor proteome has been extensively studied, the extent of intra-tumor heterogeneity remains largely unknown. Several studies have utilized mass spectrometry-based proteomics to quantify inter- and intra-tumor heterogeneity across different human tissues. These studies have demonstrated that even in the absence of overt morphological differences or genetic alterations, variations in protein abundance within tumors can influence clinical phenotypes. This suggests that spatial proteomic differences should be considered in the identification and utilization of biomarkers, thereby facilitating cancer research and clinical applications.

[0004] However, spatial proteomics remains challenging due to the limitations of tissue isolation and microsampling from different regions, as well as microsample preparation and analysis. Several methods are currently under development to address these unmet needs, such as imaging mass cytometry (IMC) and matrix-assisted laser desorption / ionization mass spectrometry imaging (MALDI-MSI). These methods require tissue dissociation and rely on computational mapping. Their common drawback is that, compared to optical imaging, resolution is often compromised to accommodate the sensitivity of mass spectrometry instruments for low-abundance proteins from small regions. These methods are subsequently primarily based on mass spectrometry detection. Furthermore, there is a class of methods based on the combination of laser microdissection (LCM) and liquid chromatography-tandem mass spectrometry (LC-MS / MS), which can, to some extent, address the shortcomings of these methods. LCM is a well-established method for isolating regions of interest from tissue slides. The team of Matthias Mann, a leader in the international field of proteomics, was able to spatially dissect 80–100 cells and identify 3,653 proteins. However, LCM instruments are expensive, highly dependent on expertise, and require high consumables for sample preparation, with the risk of sacrificing sample quality and quantity. To address these issues, there is currently a method called hydrogel-based tissue expansion spatial proteomics that maintains the integrity of tissue samples. This involves embedding the tissue in a swellable hydrogel, amplifying the sample while maintaining its original morphology, and then performing micro-sample collection and in-gel enzymatic sample preparation. This technology can currently identify approximately 1,000 proteins from as little as 0.37 nL of tissue, and tissue expansion technology can achieve high spatial resolution without the need for high-configuration equipment (such as LCM).

[0005] However, the existing in-gel enzymatic digestion method based on tissue expansion technology (sample processing, protein hydrolysis and peptide collection) still has some defects. These include the large workload and cumbersomeness caused by the large number of sample processing steps, making it difficult to use it in conjunction with automated devices to achieve automated sample processing; the sample processing process will introduce a large amount of salt, and subsequent commercial desalting column treatment is required, which increases the cost of sample processing and increases the sample loss rate; the sample processing process requires continuous transfer between multiple consumables, which increases the risk of sample loss such as nonspecific adsorption and reduces the repeatability of the test; the sample processing size has a lower limit of about 0.37nL, but it is currently impossible to process samples with sub-nanoliter volumes, which will reduce the technology's ability to resolve sample spatial heterogeneity; at the same time, the number of peptides and proteins identified in samples treated with in-gel enzymatic digestion still has a lot of room for improvement.

[0006] Public content

[0007] The present disclosure primarily addresses one or more of the problems faced by in-gel enzymatic hydrolysis in spatial proteomics techniques based on tissue expansion, including low throughput, low sample processing resolution, cumbersome procedures, low reproducibility, and insufficient peptide / protein identification.

[0008] In view of the above problems, the first aspect of the present disclosure provides a device for processing a biological sample to identify proteins therein, comprising:

[0009] The tube body includes a reaction zone and a guide zone located below the reaction zone, the reaction zone and the guide zone are in liquid communication, and a solid phase extraction adsorption filler is located at the bottom of the reaction zone or the top of the guide zone, and is configured to be able to retain liquid in the reaction zone when the air pressure in the reaction zone and the guide zone is the same, and to allow the liquid in the reaction zone to flow out when the air pressure in the reaction zone is greater than the air pressure in the guide zone.

[0010] wherein the ratio of the thickness to the diameter of the solid phase extraction adsorption filler is less than 1, or

[0011] The ratio of the thickness to the diameter of the solid phase extraction adsorption filler is 1 to 1.7, and the maximum inner diameter of the guide area is 0.1 mm to 0.7 mm.

[0012] In certain embodiments, the ratio of the thickness to the diameter of the solid phase extraction adsorbent material described in the present disclosure is less than 1.

[0013] In certain embodiments, the ratio of the thickness to the diameter of the solid phase extraction adsorption packing described in the present disclosure is less than or equal to 0.8.

[0014] In certain embodiments, the ratio of the thickness to the diameter of the solid phase extraction adsorption packing described in the present disclosure is less than or equal to 0.7.

[0015] In certain embodiments, the ratio of the thickness to the diameter of the solid phase extraction adsorption packing described in the present disclosure is less than or equal to 0.6.

[0016] In certain embodiments, the ratio of the thickness to the diameter of the solid phase extraction adsorption packing described in the present disclosure is less than or equal to 0.5.

[0017] In certain embodiments, the maximum inner diameter of the flow conducting zone of the present disclosure is smaller than the inner diameter of the reaction zone.

[0018] In certain embodiments, the inner diameter of the reaction zone described in the present disclosure is 2 to 4 mm.

[0019] In certain embodiments, the inner diameter of the reaction zone described in the present disclosure is 2 to 3 mm.

[0020] In certain embodiments, the maximum inner diameter of the diversion area described in the present disclosure is 1 to 1.5 mm.

[0021] In certain embodiments, the thickness of the solid phase extraction adsorption packing material described in the present disclosure is less than or equal to 1 mm.

[0022] In certain embodiments, the thickness of the solid phase extraction adsorption packing described in the present disclosure is about 0.9 mm, about 0.8 mm, about 0.7 mm, about 0.6 mm, about 0.5 mm, about 0.4 mm, about 0.3 mm, about 0.2 mm, or about 0.1 mm.

[0023] In certain embodiments, the inner cavity of the reaction zone disclosed herein is a cylindrical hollow structure with equal inner diameters at the top and bottom, and the inner cavity of the guide zone is a conical hollow structure with a cross section that is wide at the top and narrow at the bottom.

[0024] In certain embodiments, the length of the diversion area described in the present disclosure is less than or equal to 10 mm.

[0025] In certain embodiments, the length of the flow-guiding region described herein is less than or equal to 5 mm.

[0026] In certain embodiments, the length of the diverting region described herein is about 4.5 mm, about 4 mm, about 3.5 mm, about 3 mm, about 2.5 mm, about 2 mm, or about 1.5 mm.

[0027] In certain embodiments, the guide zone of the present disclosure is in liquid communication with the reaction zone via a through-hole located at the bottom of the reaction zone.

[0028] In certain embodiments, the solid phase extraction adsorption filler disclosed herein is located at the through-hole.

[0029] In certain embodiments, the interior of the connection between the reaction zone and the guide zone described in the present disclosure is a smooth chamfered structure.

[0030] In certain embodiments, the reaction zone described herein is provided with visible volume markings.

[0031] In certain embodiments, the tube body of the present disclosure further comprises a protruding component protruding from the outer surface of the tube body, which is configured to enable the solid phase extraction device to be hung on a placement rack.

[0032] In certain embodiments, the raised component of the present disclosure is disposed on the upper portion of the tube body.

[0033] In certain embodiments, the placement racks disclosed herein are deep-well plates of different specifications.

[0034] In certain embodiments, the reaction zone described herein has a volume of 2-250 μL.

[0035] In certain embodiments, the reaction zone described herein has a volume of 2-100 μL.

[0036] In certain embodiments, the reaction zone described herein has a volume of 10-100 μL.

[0037] In certain embodiments, the reaction zone described herein has a volume of 20-100 μL.

[0038] In certain embodiments, the reaction zone described herein has a volume of 20-50 μL.

[0039] In certain embodiments, the tube body of the present disclosure further includes a tube cover matched therewith.

[0040] In certain embodiments, the solid phase extraction adsorption filler disclosed herein is made of porous silica gel or porous ceramic particles modified with alkyl groups of different chain lengths, ion exchange resins, or chemically bonded ion exchangers.

[0041] In certain embodiments, the pore size of the solid phase extraction adsorption packing material described in the present disclosure is 1-12 μm.

[0042] In certain embodiments, the pore size of the solid phase extraction adsorption packing material described in the present disclosure is 1-5 μm.

[0043] In certain embodiments, the solid phase extraction adsorption filler disclosed herein is a C8 or C18 solid phase extraction adsorption filler, a strong cation exchange filler, or a strong anion exchange filler.

[0044] In certain embodiments, the solid phase extraction adsorption filler disclosed herein is a C8 or C18 solid phase extraction membrane, a strong cation exchange membrane, or a strong anion exchange membrane, and the membrane is in contact with the inner surface of the guide area.

[0045] In certain embodiments, the solid phase extraction adsorption filler disclosed herein is a C18 solid phase extraction membrane, and the C18 solid phase extraction membrane is in contact with the inner surface of the guide area.

[0046] In certain embodiments, the tubing described herein is a pipette tip.

[0047] In certain embodiments, the tubing disclosed herein is a pipette tip that is cut to meet the aforementioned specifications.

[0048] In certain embodiments, the biological sample described herein is in the form of a sample-gel complex.

[0049] In certain embodiments, the gel in the sample-gel complex described herein is a hydrogel.

[0050] In certain embodiments, the hydrogel described herein is a polyacrylamide gel.

[0051] In certain embodiments, the biological sample described herein is in the form of a swollen sample-gel complex.

[0052] In certain embodiments, the ratio of thickness to diameter of the solid phase extraction adsorption filler disclosed herein is 1 to 1.7, and the maximum inner diameter of the flow guide zone is 0.1 mm to 0.7 mm.

[0053] In certain embodiments, the maximum inner diameter of the flow guide zone of the present disclosure is less than or equal to the minimum inner diameter of the reaction zone.

[0054] In certain embodiments, the minimum inner diameter of the reaction zone described in the present disclosure is 0.2 to 0.6 mm.

[0055] In certain embodiments, the maximum inner diameter of the flow guiding region described in the present disclosure is 0.2 to 0.6 mm.

[0056] In certain embodiments, the maximum inner diameter of the flow deflector described herein is about 0.2 mm, about 0.3 mm, about 0.4 mm, about 0.5 mm, or about 0.6 mm.

[0057] In certain embodiments, the thickness of the solid phase extraction adsorption packing described in the present disclosure is less than or equal to 2 mm.

[0058] In certain embodiments, the thickness of the solid phase extraction adsorption packing described in the present disclosure is less than or equal to 1.5 mm.

[0059] In certain embodiments, the thickness of the solid phase extraction adsorption packing described in the present disclosure is about 1.4 mm, about 1.3 mm, about 1.2 mm, about 1.1 mm, about 1.0 mm, about 0.9 mm, about 0.8 mm, about 0.7 mm, about 0.6 mm, about 0.5 mm, about 0.4 mm, about 0.3 mm, about 0.2 mm or about 0.1 mm.

[0060] In certain embodiments, the inner cavity of the diversion area disclosed herein is a conical hollow structure with a cross section that is wide at the top and narrow at the bottom.

[0061] In certain embodiments, the length of the flow-guiding region described herein is less than or equal to 5 mm.

[0062] In certain embodiments, the length of the flow-guiding region described in the present disclosure is less than or equal to 3 mm.

[0063] In certain embodiments, the length of the diversion area described in the present disclosure is about 2.5 mm, about 2.0 mm, about 1.5 mm, about 1.4 mm, about 1.3 mm, about 1.2 mm, about 1.1 mm, about 1.0 mm, about 0.9 mm, about 0.8 mm, about 0.7 mm, about 0.6 mm, or about 0.5 mm.

[0064] In certain embodiments, the inner cavity of the reaction zone described in the present disclosure is a cylindrical hollow structure with equal inner diameters at the top and bottom, or a conical hollow structure with a cross section that is wide at the top and narrow at the bottom.

[0065] In certain embodiments, the inner cavity of the reaction zone described in the present disclosure is a conical hollow structure with a cross section that is wide at the top and narrow at the bottom.

[0066] In certain embodiments, the guide zone of the present disclosure is in liquid communication with the reaction zone via a through-hole located at the bottom of the reaction zone.

[0067] In certain embodiments, the interior of the connection between the reaction zone and the guide zone described in the present disclosure is a smooth chamfered structure.

[0068] In certain embodiments, the solid phase extraction adsorption filler disclosed herein is located at the through-hole.

[0069] In certain embodiments of the present disclosure, the diversion zone is directly connected to the reaction zone.

[0070] In certain embodiments, the connection between the reaction zone and the guide zone described in the present disclosure is a smooth straight line structure.

[0071] In certain embodiments, the solid phase extraction adsorption packing material disclosed herein is located at the top of the flow-through zone.

[0072] In certain embodiments, the thickness of the solid phase extraction adsorption packing material disclosed herein is less than or equal to the length of the flow-conducting zone.

[0073] In certain embodiments, the reaction zone described herein is provided with visible volume markings.

[0074] In certain embodiments, the tube body of the present disclosure further comprises a protruding component protruding from the outer surface of the tube body, which is configured to enable the solid phase extraction device to be hung on a placement rack.

[0075] In certain embodiments, the raised component of the present disclosure is disposed on the upper portion of the tube body.

[0076] In certain embodiments, the placement racks disclosed herein are deep-well plates of different specifications.

[0077] In certain embodiments, the reaction zone described herein has a volume of 2-250 μL.

[0078] In certain embodiments, the reaction zone described herein has a volume of 2-100 μL.

[0079] In certain embodiments, the reaction zone described herein has a volume of 10-100 μL.

[0080] In certain embodiments, the reaction zone described herein has a volume of 20-100 μL.

[0081] In certain embodiments, the reaction zone described herein has a volume of 20-50 μL.

[0082] In certain embodiments, the reaction zone described herein has a volume of 20 μL, 30 μL, 40 μL, or 50 μL.

[0083] In certain embodiments, the tube body of the present disclosure further includes a tube cover matched therewith.

[0084] In certain embodiments, the solid phase extraction adsorption filler disclosed herein is made of porous silica gel or porous ceramic particles modified with alkyl groups of different chain lengths, ion exchange resins, or chemically bonded ion exchangers.

[0085] In certain embodiments, the pore size of the solid phase extraction adsorption packing material described in the present disclosure is 1-12 μm.

[0086] In certain embodiments, the pore size of the solid phase extraction adsorption packing material described in the present disclosure is 1-5 μm.

[0087] In certain embodiments, the solid phase extraction adsorption filler disclosed herein is a C8 or C18 solid phase extraction adsorption filler, a strong cation exchange filler, or a strong anion exchange filler.

[0088] In certain embodiments, the solid phase extraction adsorption filler disclosed herein is a C8 or C18 solid phase extraction membrane, a strong cation exchange membrane, or a strong anion exchange membrane, and the membrane is in contact with the inner surface of the guide area.

[0089] In certain embodiments, the solid phase extraction adsorption filler disclosed herein is a C18 solid phase extraction membrane, and the C18 solid phase extraction membrane is in contact with the inner surface of the guide area.

[0090] In certain embodiments, the tubing described herein is a pipette tip.

[0091] In certain embodiments, the tubing disclosed herein is a pipette tip that is cut to meet the aforementioned specifications.

[0092] In certain embodiments, the biological sample described herein is in the form of a sample-gel complex.

[0093] In certain embodiments, the gel in the sample-gel complex described herein is a hydrogel.

[0094] In certain embodiments, the hydrogel described herein is a polyacrylamide gel.

[0095] In certain embodiments, the biological sample described herein is in the form of a swollen sample-gel complex.

[0096] A second aspect of the present disclosure provides a kit for processing a biological sample to identify proteins therein, comprising: a device of the present disclosure, wherein the processing of the biological sample is performed in the device.

[0097] In certain embodiments, the kit of the present disclosure further comprises: a stationary phase activator for activating solid phase extraction adsorption filler, a decolorizing agent, a dehydrating agent, a digestion agent, a digestion termination agent, and a peptide eluent.

[0098] In certain embodiments, in the kit of the present disclosure, the stationary phase activator is alcohol or acetonitrile.

[0099] In certain embodiments, in the kits of the present disclosure, the stationary phase activator is acetonitrile.

[0100] In certain embodiments, in the kit of the present disclosure, the stationary phase activator is used to prepare acetonitrile into a 70-90% acetonitrile aqueous solution.

[0101] In certain embodiments, in the kit of the present disclosure, the stationary phase activator is used to prepare acetonitrile as an 80% acetonitrile aqueous solution.

[0102] In certain embodiments, in the kit of the present disclosure, the decolorizing agent is a mixed solution of an organic solvent and an inorganic salt.

[0103] In certain embodiments, in the kit of the present disclosure, the organic solvent in the decolorizing agent is acetonitrile, and the inorganic salt is an aqueous solution of ammonium bicarbonate.

[0104] In certain embodiments, in the kit of the present disclosure, the decolorizing agent is a combination of acetonitrile and ammonium bicarbonate.

[0105] In certain embodiments, in the kit of the present disclosure, the decolorizing agent is present in the form of a solution.

[0106] In certain embodiments, in the kit of the present disclosure, the decolorizing agent in the form of a solution contains 40-60 v / v% acetonitrile, 40-60 mM ammonium bicarbonate and the balance water.

[0107] In certain embodiments, in the kit of the present disclosure, the decolorizing agent in the form of a solution contains 50 v / v% acetonitrile, 50 mM ammonium bicarbonate and the balance water.

[0108] In certain embodiments, in the kit of the present disclosure, the dehydrating agent is alcohol or acetonitrile.

[0109] In certain embodiments, in the kit of the present disclosure, the dehydrating agent is acetonitrile.

[0110] In certain embodiments, in the kits of the present disclosure, the digestion agent comprises trypsin.

[0111] In certain embodiments, in the kit of the present disclosure, the digestion agent is a combination of trypsin, acetic acid, acetonitrile and ammonium bicarbonate.

[0112] In certain embodiments, in the kits of the present disclosure, the digestive agent is present in the form of a solution.

[0113] In certain embodiments, in the kit of the present disclosure, the digestion agent contains 10-25 ng / μL of trypsin and has a pH of 8-9.

[0114] In certain embodiments, in the kit of the present disclosure, the digestion agent contains 10-25 ng / μL trypsin, 0.002-0.005 v / v% acetic acid, 40-60 mM ammonium bicarbonate solution and 10 v / v% acetonitrile.

[0115] In certain embodiments, in the kit of the present disclosure, the digestion agent contains 10-25 ng / μL trypsin, 0.002-0.005 v / v% acetic acid, 45 mM ammonium bicarbonate solution and 10 v / v% acetonitrile.

[0116] In certain embodiments, in the kit of the present disclosure, the digestion termination agent is a mixed solution of an organic solvent and an acid.

[0117] In certain embodiments, in the kit of the present disclosure, the digestion termination agent is a combination of acetonitrile and trifluoroacetic acid.

[0118] In certain embodiments, in the kit of the present disclosure, the digestion termination agent contains an aqueous solution of 1-3 v / v% acetonitrile and 0.05-0.2 v / v% trifluoroacetic acid.

[0119] In certain embodiments, in the kit of the present disclosure, the digestion termination agent contains an aqueous solution of 2 v / v% acetonitrile and 0.1 v / v% trifluoroacetic acid.

[0120] In certain embodiments, in the kit of the present disclosure, the peptide eluent is a mixed solution of an organic solvent and an acid.

[0121] In certain embodiments, in the kit of the present disclosure, the peptide eluent is a combination of acetonitrile or ethanol and trifluoroacetic acid.

[0122] In certain embodiments, in the kit of the present disclosure, the peptide eluent is a combination of acetonitrile and trifluoroacetic acid.

[0123] In certain embodiments, in the kit of the present disclosure, the peptide eluent is an aqueous solution containing 60-80 v / v% acetonitrile and 0.05-0.2 v / v% trifluoroacetic acid.

[0124] In certain embodiments, in the kit of the present disclosure, the peptide eluent is an aqueous solution containing 70 v / v% acetonitrile and 0.1 v / v% trifluoroacetic acid.

[0125] In certain embodiments, the kit of the present disclosure further comprises: acetonitrile or ethanol, ammonium bicarbonate, trypsin, acetic acid, and trifluoroacetic acid.

[0126] In certain embodiments, the kit of the present disclosure further comprises PBS buffer in the form of dry powder or liquid.

[0127] In certain embodiments, the kit of the present disclosure further comprises: a low adsorption centrifuge tube and / or a deep-well plate and / or a mass spectrometry buffer.

[0128] In certain embodiments, in the kit of the present disclosure, the mass spectrometry buffer is a combination of acetonitrile and formic acid.

[0129] In certain embodiments, in the kit of the present disclosure, the mass spectrometry buffer is an aqueous solution containing 1-3 v / v% acetonitrile and 0.05-0.2 v / v% formic acid.

[0130] In certain embodiments, in the kit of the present disclosure, the mass spectrometry buffer is an aqueous solution containing 2% acetonitrile and 0.1% formic acid.

[0131] A third aspect of the present invention provides use of the device of the present disclosure or the kit of the present disclosure for processing a biological sample to identify proteins therein.

[0132] A fourth aspect of the present invention provides a method for processing a biological sample to identify proteins therein, comprising: processing the biological sample using the device or the kit of the present invention.

[0133] In certain embodiments, in the methods of the present disclosure, the processing of the biological sample is performed in a device as described herein.

[0134] In certain embodiments, the methods of the present disclosure comprise:

[0135] (1) activating the solid phase extraction adsorption filler in the device;

[0136] (2) placing the biological sample to be processed in the device after being processed in step (1) to decolorize and dehydrate the biological sample;

[0137] (3) performing enzymatic hydrolysis on the biological sample treated in step (2);

[0138] (4) Recovering the peptide fragment obtained in step (3).

[0139] In certain embodiments, step (1) of the present disclosure includes:

[0140] 1.1 Add 20-100 μL of 70-90% acetonitrile aqueous solution to the device.

[0141] 1.2 Activate by centrifugation at 50g-200g;

[0142] 1.3 Centrifuge until the remaining liquid is discharged from the device.

[0143] In certain embodiments, the centrifugation in step 1.2 of the present disclosure is performed at 80 g to 150 g.

[0144] In certain embodiments, the centrifugation in step 1.2 of the present disclosure is performed at 100 g.

[0145] In certain embodiments, step (2) of the present disclosure includes:

[0146] 2.1 Place the biological sample to be processed in the device after being processed in step (1),

[0147] 2.2 Add a decolorizing agent containing 40-60 v / v% acetonitrile, 40-60 mM ammonium bicarbonate and the remainder of water, incubate until the sample is clear, and centrifuge to remove the solvent.

[0148] 2.3 Add 20-100 μL of acetonitrile, incubate until the sample is dehydrated, and centrifuge to remove the solvent.

[0149] In certain embodiments, the incubation in step 2.2 of the present disclosure is incubation at room temperature.

[0150] In certain embodiments, the incubation in step 2.3 of the present disclosure is incubation at room temperature.

[0151] In certain embodiments, step (3) of the present disclosure includes:

[0152] 3.1 Add digestion agent to the device and incubate;

[0153] 3.2 Add digestion buffer and continue incubation.

[0154] In certain embodiments, the volume of the digestion agent in step 3.1 of the present disclosure is 1 to 3 μL.

[0155] In certain embodiments, in the methods of the present disclosure, the volume of the digestion agent is 2 μL.

[0156] In certain embodiments, in the methods disclosed herein, the digestive agent contains 10-25 ng / μL of trypsin and has a pH of 8-9.

[0157] In certain embodiments, in the methods disclosed herein, the digestive agent contains 10-25 ng / μL trypsin, 0.002-0.005 v / v% acetic acid, 40-60 mM ammonium bicarbonate solution and 10 v / v% acetonitrile.

[0158] In certain embodiments, in the methods disclosed herein, the digestive agent contains 10-25 ng / μL trypsin, 0.002-0.005 v / v% acetic acid, 45 mM ammonium bicarbonate solution and 10 v / v% acetonitrile.

[0159] In certain embodiments, the incubation in step 3.1 of the present disclosure is incubation at 37° C. for 3 to 5 hours.

[0160] In certain embodiments, the incubation in step 3.1 of the present disclosure is incubation at 37° C. for 4 hours.

[0161] In certain embodiments, the volume of the digestion buffer in step 3.2 of the present disclosure is 2 to 15 μL.

[0162] In certain embodiments, in the methods of the present disclosure, the volume of the digestion buffer is 4 μL or 10 μL.

[0163] In certain embodiments, in the methods of the present disclosure, the digestion buffer is an alkaline inorganic salt buffer.

[0164] In certain embodiments, in the methods of the present disclosure, the digestion buffer is an aqueous ammonium bicarbonate solution.

[0165] In certain embodiments, in the methods of the present disclosure, the digestion buffer is an 8-12 mM ammonium bicarbonate aqueous solution.

[0166] In certain embodiments, in the methods of the present disclosure, the digestion buffer is a 10 mM ammonium bicarbonate aqueous solution.

[0167] In certain embodiments, the incubation in step 3.2 of the present disclosure is incubation at room temperature for 7 to 12 hours.

[0168] In certain embodiments, the incubation in step 3.2 of the present disclosure is incubation at room temperature for 8 hours.

[0169] In certain embodiments, step (3) of the present disclosure further comprises:

[0170] 3.3 Add digestion termination agent to terminate digestion.

[0171] In certain embodiments, in the method of the present disclosure, the digestion termination agent is a mixed solution of an organic solvent and an acid.

[0172] In certain embodiments, in the methods of the present disclosure, the terminating digestion agent is a combination of acetonitrile and trifluoroacetic acid.

[0173] In certain embodiments, in the method of the present disclosure, the digestion termination agent is an aqueous solution containing 1-3 v / v% acetonitrile and 0.05-0.2 v / v% trifluoroacetic acid.

[0174] In certain embodiments, in the methods of the present disclosure, the digestion termination agent is an aqueous solution containing 2 v / v% acetonitrile and 0.1 v / v% trifluoroacetic acid.

[0175] In certain embodiments, step (4) of the present disclosure includes:

[0176] 4.1 Centrifuge the product of step (3) and collect the centrifuge liquid;

[0177] 4.2 adding a mixed solution of an organic solvent and an acid to the device, incubating, centrifuging, and collecting the centrifuge;

[0178] 4.3 Repeat step 4.2 1-2 times.

[0179] In certain embodiments, in the method of the present disclosure, the mixed solution of the organic solvent and the acid is a combination of acetonitrile or ethanol and trifluoroacetic acid.

[0180] In certain embodiments, in the method of the present disclosure, the mixed solution of the organic solvent and the acid is a combination of acetonitrile and trifluoroacetic acid.

[0181] In certain embodiments, in the method of the present disclosure, the mixed solution of the organic solvent and the acid is an aqueous solution containing 60-80 v / v% acetonitrile and 0.05-0.2 v / v% trifluoroacetic acid.

[0182] In certain embodiments, in the method of the present disclosure, the mixed solution of the organic solvent and the acid is an aqueous solution containing 70 v / v% acetonitrile and 0.1 v / v% trifluoroacetic acid.

[0183] In certain embodiments, the volume of the mixed solution in step 4.2 of the present disclosure is 20-40 μL.

[0184] In certain embodiments, the incubation time in step 4.2 of the present disclosure is 10-20 minutes.

[0185] In certain embodiments, the incubation time in step 4.2 of the present disclosure is 15 minutes.

[0186] In certain embodiments, step (4) of the present disclosure further comprises:

[0187] 4.4 Add acetonitrile to dehydrate the biological sample, centrifuge, and collect the centrifuge fluid;

[0188] 4.5 Combine the above centrifuged solutions and, optionally, dry them to obtain peptide fragments.

[0189] In certain embodiments, in the methods of the present disclosure, the biological sample to be processed is obtained by laser microdissection.

[0190] A fifth aspect of the present invention provides a method for identifying proteins in a biological sample, comprising:

[0191] 1) using the device or kit disclosed herein to process a biological sample to obtain peptide fragments;

[0192] 2) Detect the peptides using LC-MS / MS.

[0193] In certain embodiments, step 2) of the present disclosure comprises:

[0194] Resuspend the peptides in mass spectrometry buffer;

[0195] The resuspended peptides were centrifuged and the supernatant was used for LC-MS / MS analysis.

[0196] In certain embodiments, in the methods of the present disclosure, the mass spectrometry buffer is a mixed solution of an organic solvent and an acid.

[0197] In certain embodiments, in the methods of the present disclosure, the mass spectrometry buffer is a combination of acetonitrile and formic acid.

[0198] In certain embodiments, in the methods disclosed herein, the mass spectrometry buffer is an aqueous solution containing 1-3 v / v% acetonitrile and 0.05-0.2 v / v% formic acid.

[0199] In certain embodiments, in the methods of the present disclosure, the mass spectrometry buffer is an aqueous solution containing 2 v / v% acetonitrile and 0.1 v / v% formic acid.

[0200] In certain embodiments, in the methods of the present disclosure, the LC-MS / MS detection is performed by an Orbitrap Astral mass spectrometer.

[0201] In certain embodiments, in the methods disclosed herein, the peptide segment in step 1) is obtained by the method of the fourth aspect of the present invention.

[0202] Definition of terms

[0203] In the present invention, unless otherwise specified, the scientific and technical terms used in this disclosure have the meanings commonly understood by those skilled in the art. At the same time, in order to better understand the present invention, the definitions and explanations of relevant terms are provided below.

[0204] In the present disclosure, "solid phase extraction adsorption filler" can be made of porous silica gel or porous ceramic particles modified with alkyl groups of different chain lengths, ion exchange resins or chemically bonded ion exchangers, which are widely used in the separation, purification and analysis of organic compounds, and are usually used as chromatographic column fillers. The average pore size of the solid phase extraction adsorption filler is 1-12 μm, preferably 1-5 μm. In certain embodiments, the solid phase extraction adsorption filler can be a C8 or C18 solid phase extraction adsorption filler. In certain embodiments, the solid phase extraction adsorption filler is made of silica gel modified with chain alkyl groups having a carbon chain length of 8 or 18. For example, the C18 solid phase extraction adsorption filler can be a commonly used octadecyl silane bonded silica gel (Octadecyl silane chemically bonded to porous silica). In certain embodiments, the solid phase extraction adsorption filler can be a strong cation exchange (SCX) or strong anion exchange (SAX) filler. The solid phase extraction adsorption filler can be commercially available or prepared using known methods. The solid phase extraction adsorption filler can be a commercially available C18 solid phase extraction membrane, such as 3M Empore TM C18 47mm Extraction Disks, Resprep-C18 SPE solid phase extraction membrane, Shimadzu SGLC C18 solid phase extraction membrane, etc., can also be commercially available C8 solid phase extraction membrane, strong cation exchange membrane or strong anion exchange membrane, such as "Empore TM SPE disk,C8","Empore TM SPE disk,strong anion exchange"、"Empore TM SPE disk,strong cation exchange”.

[0205] In the present disclosure, "sample-gel complex" refers to the coexistence of tissue and gel. The tissue can be in any combination with the gel, for example, the sample is partially located on the surface of the gel or completely located inside the gel, preferably completely located inside the gel. The sample is a biological sample, including cells, specimens, biopsy samples, organs or parts thereof, or whole organisms. The sample can be living, fixed or preserved, for example, fixed tissue sections, fresh frozen tissues or tissue sections embedded in paraffin. The gel in the sample-gel complex can be a hydrogel, and the hydrogel can also be a polyacrylamide gel. The sample-gel complex can swell in water or aqueous buffer to obtain a visual tissue sample with a linear expansion of about 1-25 times (for example, 1-20 times, 1-10 times, 2-8 times, 2-7 times). During sampling, a microtissue sample with an original diameter of about 10-500 microns can be obtained using a punch (for example, on a millimeter scale) as needed for proteomic identification. The method for preparing the sample-gel complex is well known. For example, it can be prepared by referring to the method disclosed in CN115372527A, or by referring to the method disclosed in Nature Communications, (2022) 13:7242, or by referring to the method of Example 1 of the present disclosure.

[0206] In the present disclosure, "hydrogel" refers to a hydrophilic polymeric material that swells or is capable of swelling with a polar solvent, such as a polymeric material that can rapidly swell in water. Hydrogels can be formed by chemically or physically crosslinking water-soluble or hydrophilic polymers. These polymers include polysaccharides (e.g., starch, cellulose, alginic acid, hyaluronic acid, chitosan, etc.), polypeptides (e.g., collagen, poly-L-lysine, poly-L-glutamine, etc.), alcohols, acrylic acid and its derivatives (e.g., polyacrylic acid, polymethacrylic acid, polyacrylamide, poly-N-polyacrylamide, etc.). Suitable hydrogels include crosslinked hydrogels, swollen hydrogels, and dried or partially dried hydrogels.

[0207] In the present disclosure, "50% acetonitrile / 50% 100mM ammonium bicarbonate solution" means that the proportion of acetonitrile in the solution is 50%, and the proportion of 100mM ammonium bicarbonate solution is 50%. For example, mixing 5mL of acetonitrile with 5mL of 100mM ammonium bicarbonate solution can obtain a 50% acetonitrile / 50% 100mM ammonium bicarbonate solution; similarly, "2% acetonitrile / 0.1% trifluoroacetic acid" means that the proportion of acetonitrile in the solution is 2%, and the proportion of trifluoroacetic acid is 0.1%; other similar definitions can be understood with reference to the above content.

[0208] In the present disclosure, the orientations or positional relationships indicated by “upper”, “lower”, etc. are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present disclosure and simplifying the description, and do not indicate or imply that the device referred to must have a specific orientation, be constructed and operated in a specific orientation. Therefore, it cannot be understood as limiting the scope of protection of the present invention.

[0209] In this disclosure, the term "maximum inner diameter" refers to the inner diameter of the device at its maximum inner diameter. Similarly, in this disclosure, the term "minimum inner diameter" refers to the inner diameter of the device at its minimum inner diameter.

[0210] In the present disclosure, a numerical range includes the endpoint values ​​of the range shown. For example, "the ratio of thickness to diameter is 1 to 1.7" means that the ratio of thickness to diameter is greater than or equal to 1 and less than or equal to 1.7. For example, "add 20-100 μL acetonitrile" means that the volume of acetonitrile is greater than or equal to 20 μL and less than or equal to 100 μL.

[0211] In the present disclosure, the term "about" is understood to mean within + / -10%, + / -9%, + / -8%, + / -7%, + / -6%, + / -5%, + / -4%, + / -3%, + / -2%, + / -1%, + / -0.5%, + / -0.4%, + / -0.3%, + / -0.2%, + / -0.1% of the stated value. Unless otherwise apparent from the context, all numerical values ​​provided herein are modified by the term "about." Beneficial effects

[0212] The present disclosure provides a device and a kit for processing biological samples to identify proteins therein, which can be used for processing biological samples. The present disclosure also provides a method for processing biological samples to identify proteins therein using the device. The device, kit, or method has one or more of the following advantages:

[0213] 1) Sample processing devices can be selected based on sample size and experimental requirements, thereby reducing sample loss, lowering sample processing costs, and improving processing capabilities for complex samples.

[0214] 2) It is compatible with automated devices, allowing multiple samples to be placed in a 96-well plate and processed simultaneously, thereby improving detection throughput and efficiency.

[0215] 3) No desalting is required, thereby reducing the peptide loss rate during the desalting process, reducing sample processing costs, and simplifying operations.

[0216] 4) The missed cutting rate is reduced and there is no sample loss problem.

[0217] 5) The detection has good reproducibility.

[0218] 6) Improved peptide and protein identification.

[0219] 7) It can process extremely small biological samples, such as samples with sub-nanoliter volumes, with the minimum sample volume as low as about 0.042nL. Under the sampling methods of the provided examples and control examples, this is one-eighth of the minimum processing volume of conventional in-gel enzymatic hydrolysis; it can further effectively process samples as small as a single cell nucleus, greatly improving the sample detection resolution and applicable range. BRIEF DESCRIPTION OF THE DRAWINGS

[0220] FIG1 is a schematic flow diagram of the solid phase extraction-assisted in-gel enzymatic hydrolysis method according to an embodiment of the present invention.

[0221] Figure 2 shows the comparison results of the solid phase extraction-assisted in-gel digestion method of an embodiment of the present invention and the conventional in-gel digestion method, including: (A) the number of identified peptides and proteins; (B) the distribution of peptide lengths; (C) the density distribution of GRAVY values; (D) the comparison of missed cleavage rates; (E) the Venn diagram analysis of the two digestion methods; (F) the protein abundance distribution; (G) the coefficient of variation distribution of peptides and proteins; (H) the Pearson correlation distribution of peptides and proteins; (I) the types of proteins identified by the two methods and the proportion of different localizations.

[0222] FIG3 is a comparison of the peptide and protein identification numbers of different tissue volumes processed by the solid phase extraction-assisted in-gel enzymatic digestion method according to an embodiment of the present invention and the conventional in-gel enzymatic digestion method.

[0223] FIG4 is a schematic structural diagram of a solid phase extraction device according to an embodiment of the present invention, wherein: 1-tube body, 11-reaction zone, 12-flow guide zone; 2-solid phase extraction adsorption filler; 3-protruding component.

[0224] FIG5 is a schematic structural diagram of a solid phase extraction device according to an embodiment of the present invention, wherein: 1-tube body, 11-reaction zone, 12-flow guide zone; 2-solid phase extraction adsorption filler; 3-protruding component.

[0225] Figure 6 shows the results of solid-phase extraction-assisted in-gel enzymatic digestion for single cell nuclei in an embodiment of the present invention, including: (A) imaging results of a 5 μm mouse liver FFPE hydrogel sample before laser microdissection, where 5 μm is the actual thickness of the mouse liver sample before expansion; (B) imaging results of a 5 μm mouse liver FFPE hydrogel sample after laser microdissection; (C) results of single mouse liver cell nucleus sample slices collected on the tube cap; (D) comparison of the number of peptides and proteins identified by mass spectrometry for a single cell nucleus and a sample with a volume of 0.042 nL, with green representing the result for the sample with a volume of 0.042 nL and blue representing the result for a single cell nucleus; (E) Pearson correlation distribution of proteins; (F) Venn diagram analysis of the two sample sizes; (G) the ratio of nuclear proteins and transcriptional regulatory factors of the specific proteins in (F); and (H) the result of nuclear-specific pathway enrichment for the specific proteins of Example 5 in (F). DETAILED DESCRIPTION

[0226] The embodiments of the present disclosure will be described in detail below with reference to the examples. However, those skilled in the art will appreciate that the following examples are intended only to illustrate the present disclosure and should not be construed as limiting the scope of the present disclosure. Where specific conditions are not specified in the examples, the experiments were performed under conventional conditions or the conditions recommended by the manufacturer. Where the manufacturer of the reagents or instruments is not specified, all are commercially available conventional products.

[0227] Referring to FIG4 , a preferred embodiment of a device for processing biological samples to identify proteins therein according to the present disclosure is shown, comprising: a tubular body 1, the tubular body 1 including a reaction zone 11 and a flow guide zone 12 located below the reaction zone 11, the reaction zone 11 and the flow guide zone 12 being in liquid communication; and a solid phase extraction adsorption filler 2 located below the reaction zone 11 or above the flow guide zone 12, and configured to retain the liquid in the reaction zone 11 when the air pressures in the reaction zone 11 and the flow guide zone 12 are the same, and to allow the liquid in the reaction zone 11 to flow out when the air pressure in the reaction zone 11 is greater than that in the flow guide zone 12. During use, a biological sample is placed in the reaction zone 11, and various reagents for processing the biological sample are added to the reaction zone 11, so that the biological sample is processed in the reaction zone, thereby obtaining peptides that can be used for protein identification. Various operations on the biological sample are completed in the reaction zone 11. Under normal pressure, the air pressure in the reaction zone 11 and the guide zone 12 is the same, and various reagents can be retained by the solid phase extraction adsorption filler 2. When it is necessary to discharge the liquid reagents during the treatment process, the device is pressurized or depressurized, for example, by centrifugation, so that the air pressure in the reaction zone 11 is greater than the air pressure in the guide zone 12, and the liquid in the reaction zone 11 can be discharged from the guide zone 12 through the solid phase extraction adsorption filler 2.

[0228] Preferably, the ratio of the thickness to the diameter of the solid-phase extraction adsorption filler 2 is less than 1, preferably less than or equal to 0.8, less than or equal to 0.7, less than or equal to 0.6, or less than or equal to 0.5. The amount of the solid-phase extraction adsorption filler 2 used should not be too large, as too much will make it difficult to elute the peptides captured by the filler, resulting in loss of sample volume and a reduction in the types and abundance of identified proteins.

[0229] Preferably, the thickness of the solid phase extraction adsorption filler 2 is less than or equal to 1 mm, for example, about 0.9 mm, about 0.8 mm, about 0.7 mm, about 0.6 mm, about 0.5 mm, about 0.4 mm, about 0.3 mm, about 0.2 mm or about 0.1 mm.

[0230] Preferably, the maximum inner diameter of the guide zone 12 is smaller than the inner diameter of the reaction zone 11, and the inner diameter of the reaction zone 11 is larger than the guide zone 12, which facilitates the addition of reagents. At the same time, the volume of the reaction zone 11 is larger than the volume of the guide zone 12, which is convenient for accommodating various reagents used in biological samples and making various operations easier to perform.

[0231] Preferably, the inner diameter of the reaction zone 11 is 2-4 mm, more preferably 2-3 mm. The volume of the reaction zone 11 is preferably 2-250 μL, for example 2-100 μL, preferably 10-100 μL, more preferably 20-100 μL, and even more preferably 20-50 μL. Preferably, the maximum inner diameter of the flow guide zone 12 is 1-1.5 mm. In practical applications, the dimensions of each component can be designed according to the size of the biological sample to be processed.

[0232] Further preferably, the inner cavity of the reaction zone 11 is a cylindrical hollow structure with equal inner diameters at the top and bottom, and the inner cavity of the guide zone 12 is a conical hollow structure with a cross section that is wide at the top and narrow at the bottom.

[0233] The reaction space provided by the reaction zone is beneficial for biological sample processing, can minimize the amount of enzyme used and increase the effective reaction time, and is superior to conventionally used EP tubes (such as 0.6mL or 1.5mL) and conventionally used V-shaped pipette tips (such as 200μL or 1mL).

[0234] Preferably, the length of the guide area 12 is less than or equal to 10 mm, and more preferably, the length of the guide area 12 is less than or equal to 5 mm, about 4.5 mm, about 4 mm, about 3.5 mm, about 3 mm, about 2.5 mm, about 2 mm, or about 1.5 mm. The guide area 12 should not be too long. If it is too long, liquid residue will be left, affecting the detection accuracy. It is also not suitable for large-scale collection because a deeper well plate is required to accommodate the device.

[0235] Further preferably, the guide area 12 is in liquid communication with the reaction area 11 via a through hole located at the bottom of the reaction area 11. This structure allows the liquid in the reaction area 11 to be discharged through the guide area 12. The solid phase extraction adsorption filler 2 is preferably located at the through hole.

[0236] The connection between the reaction zone 11 and the guide zone 12 may have a smooth chamfered structure, which prevents liquid from being stuck at the connection between the two zones when the liquid in the reaction zone 11 is discharged through the guide zone 12 .

[0237] In order to improve the convenience of operation, the reaction area 11 may also be provided with visible volume scale marks, such as scale lines, to facilitate checking whether there is any error in the sample addition amount.

[0238] Preferably, the tube body 1 may also be provided with a raised component 3 protruding from the outer surface of the tube body. The raised component 3 is configured to enable the solid phase extraction device to be hung on a placement rack, such as a deep-well plate of different specifications. At the same time, the provision of the raised component 3 also makes the device easy to take and place. In certain embodiments, the raised component 3 may be provided on the upper portion of the tube body 1. When processing samples, the device can be placed in batches in 12-well, 24-well, 48-well, or 96-well deep-well plates, preferably 96-well deep-well plates, for batch processing and detection. Preferably, the tube body 1 may also include a tube cap that cooperates with it.

[0239] Preferably, the pore size of the solid phase extraction adsorption filler 2 is 1-12 μm, preferably 1-5 μm. The solid phase extraction adsorption filler 2 is, for example, a C8 or C18 solid phase extraction adsorption filler, a strong cation exchange filler or a strong anion exchange filler, preferably a C18 solid phase extraction adsorption filler. Preferably, the solid phase extraction adsorption filler 2 is a C8 or C18 solid phase extraction membrane, a strong cation exchange membrane or a strong anion exchange membrane, and the membrane is in contact with the inner surface of the guide area 12. Further preferably, the solid phase extraction adsorption filler 2 is a C18 solid phase extraction membrane, and the C18 solid phase extraction membrane is in contact with the inner surface of the guide area 12.

[0240] The tube body 1 can be made by 3D printing, or a commercially available pipette tip can be cut to meet the aforementioned specifications to serve as the tube body 1 .

[0241] In one embodiment, the device for processing a biological sample to identify proteins in the biological sample consists of a pipette tip and a C18 solid-phase extraction membrane sheet, wherein the C18 solid-phase extraction membrane sheet and the pipette tip are cut to a suitable size, and then the cut C18 solid-phase extraction membrane sheet is placed in the pipette tip so that it is in close contact with the pipette tip, thereby obtaining the device, i.e., the solid-phase extraction device described in the embodiment.

[0242] The device can be used to process a biological sample in the form of a sample-gel complex (particularly a biological sample in the form of a swollen sample-gel complex) to obtain peptides that can be used for protein identification. The gel in the sample-gel complex can be a hydrogel, such as a polyacrylamide gel.

[0243] Referring to Figures 4 and 5 , a preferred embodiment of the apparatus disclosed herein for processing biological samples to identify proteins therein is shown, comprising: a tubular body 1, the tubular body 1 including a reaction zone 11 and a flow guide zone 12 located below the reaction zone 11, the reaction zone 11 and the flow guide zone 12 being in liquid communication; and a solid phase extraction adsorption filler 2 located below the reaction zone 11 or above the flow guide zone 12, and configured to retain liquid in the reaction zone 11 when the air pressures in the reaction zone 11 and the flow guide zone 12 are the same, and to allow the liquid in the reaction zone 11 to flow out when the air pressure in the reaction zone 11 is greater than that in the flow guide zone 12. During use, a biological sample is placed in the reaction zone 11, and various reagents for processing the biological sample are added to the reaction zone 11, so that the biological sample is processed in the reaction zone, thereby obtaining peptides that can be used for protein identification. Various operations on the biological sample are completed in the reaction zone 11. Under normal pressure, the air pressure in the reaction zone 11 and the guide zone 12 is the same, and various reagents can be retained by the solid phase extraction adsorption filler 2. When it is necessary to discharge the liquid reagents during the treatment process, the device is pressurized or depressurized, for example, by centrifugation, so that the air pressure in the reaction zone 11 is greater than the air pressure in the guide zone 12, and the liquid in the reaction zone 11 can be discharged from the guide zone 12 through the solid phase extraction adsorption filler 2.

[0244] Preferably, the ratio of the thickness to the diameter of the solid phase extraction adsorption filler 2 is 1 to 1.7 (e.g., 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7), and the maximum inner diameter of the flow guide region 12 is 0.1 mm to 0.7 mm. The amount of the solid phase extraction adsorption filler 2 used should not be too large, as too much will make it difficult to elute the peptides captured by the filler, resulting in loss of sample volume and a reduction in the types and abundance of identified proteins.

[0245] Preferably, the thickness of the solid phase extraction adsorption filler 2 is less than or equal to 2 mm, preferably less than or equal to 1.5 mm, for example, about 1.4 mm, about 1.3 mm, about 1.2 mm, about 1.1 mm, about 1.0 mm, about 0.9 mm, about 0.8 mm, about 0.7 mm, about 0.6 mm, about 0.5 mm, about 0.4 mm, about 0.3 mm, about 0.2 mm or about 0.1 mm.

[0246] Preferably, the maximum inner diameter of the guide area 12 is less than or equal to the minimum inner diameter of the reaction area 11, and the minimum inner diameter of the reaction area 11 is greater than or equal to the maximum inner diameter of the guide area 12, which facilitates the addition of reagents. At the same time, the volume of the reaction area 11 is larger than the volume of the guide area 12, which is convenient for accommodating various reagents used in biological samples and making various operations easier to perform.

[0247] Preferably, the minimum inner diameter of the reaction zone 11 is 0.2 to 0.6 mm. The volume of the reaction zone 11 is preferably 2 to 250 μL, for example, 2 to 100 μL, preferably 10 to 100 μL, more preferably 20 to 100 μL, more preferably 20 to 50 μL, for example, 20 μL, 30 μL, 40 μL, or 50 μL. Preferably, the maximum inner diameter of the flow guide zone 12 is 0.2 to 0.6 mm, for example, about 0.2 mm, about 0.3 mm, about 0.4 mm, about 0.5 mm, or about 0.6 mm. In actual application, the dimensions of each component can be designed according to the size of the biological sample to be processed.

[0248] Further preferably, the inner cavity of the reaction zone 11 is a cylindrical hollow structure with equal upper and lower inner diameters or a conical hollow structure with a wide upper part and a narrow lower part in cross section, preferably a conical hollow structure with a wide upper part and a narrow lower part in cross section, and the inner cavity of the guide zone 12 is a conical hollow structure with a wide upper part and a narrow lower part in cross section.

[0249] Preferably, the length of the flow guide area 12 is less than or equal to 5 mm, preferably less than or equal to 3 mm, such as about 2.5 mm, about 2.0 mm, about 1.5 mm, about 1.4 mm, about 1.3 mm, about 1.2 mm, about 1.1 mm, about 1.0 mm, about 0.9 mm, about 0.8 mm, about 0.7 mm, about 0.6 mm, or about 0.5 mm. The flow guide area 12 should not be too long. If it is too long, liquid residue may be left, affecting detection accuracy. Moreover, it is not suitable for large-scale collection because a deeper well plate is required to accommodate the device.

[0250] Preferably, the guide zone 12 is in liquid communication with the reaction zone 11 via a through-hole located at the bottom of the reaction zone 11. This structure allows the liquid in the reaction zone 11 to be discharged through the guide zone 12. The interior of the connection between the reaction zone 11 and the guide zone 12 can have a smooth chamfered structure. This structure avoids liquid jamming at the connection between the two zones when the liquid in the reaction zone 11 is discharged through the guide zone 12. The solid phase extraction adsorption filler 2 is preferably located at the through-hole.

[0251] Further preferably, the flow guide region 12 is directly connected to the reaction region 11, i.e., the maximum inner diameter of the flow guide region 12 is also the minimum inner diameter of the reaction region 11. Preferably, the connection between the reaction region 11 and the flow guide region 12 is a smooth straight line. The solid phase extraction adsorption packing 2 is preferably located at the top of the flow guide region 12. Preferably, the thickness of the solid phase extraction adsorption packing 2 is less than or equal to the length of the flow guide region 12. If the thickness of the solid phase extraction adsorption packing 2 is equal to the length of the flow guide region 12, the solid phase extraction adsorption packing 2 is filled between the top and bottom of the flow guide region 12.

[0252] In order to improve the convenience of operation, the reaction area 11 may also be provided with visible volume scale marks, such as scale lines, to facilitate checking whether there is any error in the sample addition amount.

[0253] Preferably, the tube body 1 may also be provided with a raised component 3 protruding from the outer surface of the tube body. The raised component 3 is configured to enable the solid phase extraction device to be hung on a placement rack, such as a deep-well plate of different specifications. At the same time, the provision of the raised component 3 also makes the device easy to take and place. In certain embodiments, the raised component 3 may be provided on the upper portion of the tube body 1. When processing samples, the device can be placed in batches in 12-well, 24-well, 48-well, or 96-well deep-well plates, preferably 96-well deep-well plates, for batch processing and detection. Preferably, the tube body 1 may also include a tube cap that cooperates with it.

[0254] Preferably, the pore size of the solid phase extraction adsorption filler 2 is 1-12 μm, preferably 1-5 μm. The solid phase extraction adsorption filler 2 is, for example, a C8 or C18 solid phase extraction adsorption filler, a strong cation exchange filler or a strong anion exchange filler, preferably a C18 solid phase extraction adsorption filler. Preferably, the solid phase extraction adsorption filler 2 is a C8 or C18 solid phase extraction membrane, a strong cation exchange membrane or a strong anion exchange membrane, and the membrane is in contact with the inner surface of the guide area 12. Further preferably, the solid phase extraction adsorption filler 2 is a C18 solid phase extraction membrane, and the C18 solid phase extraction membrane is in contact with the inner surface of the guide area 12.

[0255] The tube body 1 can be made by 3D printing, or a commercially available pipette tip can be cut to meet the aforementioned specifications to serve as the tube body 1 .

[0256] In one embodiment, the device for processing a biological sample to identify proteins in the biological sample consists of a pipette tip and a C18 solid-phase extraction membrane sheet, wherein the C18 solid-phase extraction membrane sheet and the pipette tip are cut to a suitable size, and then the cut C18 solid-phase extraction membrane sheet is placed in the pipette tip so that it is in close contact with the pipette tip, thereby obtaining the device, i.e., the solid-phase extraction device described in the embodiment.

[0257] The device can be used to process a biological sample in the form of a sample-gel complex (particularly a biological sample in the form of a swollen sample-gel complex) to obtain peptides that can be used for protein identification. The gel in the sample-gel complex can be a hydrogel, such as a polyacrylamide gel.

[0258] Without being limited by theory, it is speculated that the solid-phase extraction adsorption filler captures peptides after enzymatic digestion during sample processing, thereby reducing sample loss and increasing the number of peptides and proteins identified in the sample. Furthermore, the device, kit, and method provided by the present invention can reduce the rate of missed excisions, further increasing the number of peptides and proteins identified in the sample. The device, kit, and method provided by the present invention can process extremely small tissue samples, for example, as low as approximately 0.042 nL, which is one-eighth the minimum processing volume of conventional in-gel enzymatic digestion as shown in the control example. Theoretically, the device, kit, and method provided by the present invention can process sample volumes as low as a single cell, such as 0.003 nL. In practice, samples as small as a single cell nucleus can be effectively processed, significantly improving sample detection resolution and the scope of application. Furthermore, sample processing using the device, kit, and method provided by the present invention eliminates the need for desalting, thereby reducing sample processing costs, saving sample processing time, and improving peptide recovery. Furthermore, detection reproducibility is excellent. Sample processing devices of different sizes can also be selected based on sample size and experimental requirements, reducing sample processing costs and improving processing capabilities for complex samples. At the same time, this method is compatible with automated devices, which can improve detection throughput and process multiple samples simultaneously in a shorter sample processing time.

[0259] Example

[0260] The present disclosure will now be described with reference to the following examples which are intended to illustrate the invention rather than to limit it.

[0261] In the embodiments of the present disclosure, the solid phase extraction device is the device for processing biological samples to identify proteins in the biological samples as described in the present disclosure, and the two have the same meaning.

[0262] In the embodiments of the present disclosure, the solid-phase extraction-assisted in-gel enzymatic hydrolysis method is the method for processing biological samples to identify proteins in biological samples described in the present disclosure, and the two have the same meaning. The method of the present disclosure uses the device for processing biological samples to identify proteins in biological samples to process the biological sample, that is, the processing of the biological sample is carried out in the device, thereby obtaining detectable peptides for protein identification in the biological sample. The conventional in-gel enzymatic hydrolysis method is a conventional method for processing biological samples, that is, it does not use the device described in the present disclosure, but uses a conventional container such as an EP tube, that is, the biological sample is processed in a conventional container.

[0263] The main reagents, consumables, and instruments involved in the embodiments of the present disclosure are shown in Table 1.

[0264] Table 1

[0265] Preparation method of main consumables and reagents involved in the embodiment of the present disclosure

[0266] 1. N-Acrylic acid succinimidyl ester stock solution, 10 mg / mL, 10 mL

[0267] 100 mg of N-succinimidyl acrylate was dissolved in anhydrous DMSO to a final volume of 10 mL.

[0268] 2. Protein anchoring buffer (MES solution), 100 mM (pH 6.0), 50 mL

[0269] Mix 25 mL of 0.2 M MES solution with 25 mL of ddH2O to a final volume of 50 mL at a pH of 6.0.

[0270] 3. Protein anchoring solution (0.1-0.2 mg / mL NSA in 100 mM MES)

[0271] Table 2

[0272] 4. Anchoring stop buffer (MOPS solution), 100 mM (pH 7.0), 10 mL

[0273] Mix 2 mL of 0.5 M MOPS stock solution with 8 mL of ddH2O to make a final volume of 10 mL at a pH of 7.0.

[0274] 5. Pentaerythritol allyl ether (PAE) stock solution, 0.115-0.2 g / mL, 0.45 M, 10 mL

[0275] 1.15 g - 2 g of PAE was dissolved in tetrahydrofuran (THF) to a final volume of 10 mL and stored in a refrigerator protected from light.

[0276] 6. Monomer solution (formula see Table 3), pH 6.5

[0277] Table 3

[0278] 7. Ammonium persulfate stock solution, 10%-20% w / v, 1 mL

[0279] Dissolve 0.1 g-0.2 g of ammonium persulfate in ddH2O to a final volume of 1 mL.

[0280] 8.N,N,N',N'-Tetramethylethylenediamine stock solution, 10% w / v, 1 mL

[0281] Dissolve 0.1 g of N,N,N',N'-tetramethylethylenediamine in ddH2O to a final volume of 1 mL.

[0282] 9. Activate the monomer solution (see Table 4 for the formula) and place it on ice before use.

[0283] Table 4

[0284] 10. Protein denaturation buffer (see Table 5 for recipe), pH 7.0, 200 mL

[0285] Table 5

[0286] Mix with ddH2O to make a final volume of 200 mL.

[0287] 11. 80% acetonitrile (formula see Table 6), 10 mL

[0288] Table 6

[0289] 12. 100 mM ammonium bicarbonate solution, 10 mL

[0290] Dissolve 79.06 mg of ammonium bicarbonate in pure water to make a final volume of 10 mL.

[0291] 13. 50 mM ammonium bicarbonate solution: Mix 1 mL of 100 mM ammonium bicarbonate solution and 1 mL of purified water to make a final volume of 2 mL.

[0292] 14. 50% acetonitrile / 50% 100 mM ammonium bicarbonate solution (see Table 7 for the recipe), 10 mL

[0293] Table 7

[0294] 15. Trypsin digestion solution

[0295] Stock solution (0.5 μg / μL): Add 200 μL of 0.1% acetic acid (pH 3) to 100 μg of trypsin, aliquot into 50 μL vials, and store at -80°C. This will keep for 2 months. The stock solution can be thawed multiple times until used up.

[0296] Digestion buffer 1 (45 mM ammonium bicarbonate solution and 10% acetonitrile): Mix 0.5 mL of acetonitrile with 4.5 mL of 50 mM ammonium bicarbonate solution to a final volume of 5 mL.

[0297] Digestion buffer 2 (10 mM ammonium bicarbonate solution): Mix 0.1 mL of 100 mM ammonium bicarbonate with 0.9 mL of purified water to a final volume of 1 mL.

[0298] Trypsin working solution (10-25 ng / μL): Add 4-10 μL of trypsin stock solution to 196-190 μL of digestion buffer 1. The pH should be tested and should be around 8-9. This working solution can be stored in the refrigerator and used within 2-3 days.

[0299] 16. 2% acetonitrile / 0.1% trifluoroacetic acid (see Table 8 for the recipe), 10 mL (should be mass spectrometry grade)

[0300] Table 8

[0301] 17. 70% acetonitrile / 0.1% trifluoroacetic acid (see Table 9 for the recipe), 10 mL (should be mass spectrometry grade)

[0302] Table 9

[0303] 18. Desalination solution

[0304] Buffer A (for activation): 100% methanol

[0305] Buffer B (for equilibration, see Table 10 for the formula):

[0306] Table 10

[0307] Buffer C (for desalting, see Table 11 for the formula):

[0308] Table 11

[0309] Buffer D (for elution, see Table 12 for the formula):

[0310] Table 12

[0311] 19. Mass spectrometry buffer (2% acetonitrile / 0.1% formic acid, both should be mass spectrometry grade reagents, see Table 13 for the formula), 10 mL

[0312] Table 13

[0313] 20. Reducing reagent (20 mM dithiothreitol DTT, formula see Table 14), 25 mL

[0314] Table 14

[0315] 21. Alkylating agent (55 mM iodoacetamide IAA, formula see Table 15), 25 mL

[0316] Table 15

[0317] 22. Test dyeing

[0318] Mix R-250 with ddH2O to a final density of 1 mg / mL. Store at room temperature.

[0319] Example 1, glue making

[0320] 1. Dewax FFPE tissue sections:

[0321] Place mouse liver tissue sections in a dehydration container.

[0322] Heptane was added to the container and discarded after 10 minutes.

[0323] Repeat the heptane dewaxing step once.

[0324] Rehydrate the deparaffinized samples using a gradient of ethanol as follows:

[0325] Add 100% ethanol to the container, keep it for 5 minutes, and discard the liquid.

[0326] Add 90% ethanol to the container, keep it for 5 minutes, and discard the liquid.

[0327] Add 75% ethanol to the container, keep it for 5 minutes, and discard the liquid.

[0328] Add ddH2O to the container, keep it for 5 minutes, and discard the liquid.

[0329] 2. Add 50-100 μL of protein anchoring solution and incubate the sample at room temperature for 1-2 hours.

[0330] 3. Wash the sample three times with 50-100 μL of anchor stop buffer.

[0331] 4. Allow the tissue to air dry completely in a fume hood and add enough activated monomer solution (about 100 μL) to the surface of the tissue. Then place the entire device in a pipette tip box filled with water at the bottom and incubate in a 4°C refrigerator overnight.

[0332] 5. Cover with a glass coverslip and place in a vacuum drying oven preheated to 37°C. Incubate in an oxygen-deficient environment for 3 hours to form a gel.

[0333] 6. After gelation, remove the coverslip and add protein denaturation buffer. Incubate at room temperature for 30 minutes, then perform denaturation treatment, that is, perform it in a sterilizer liquid program at 105-121 degrees Celsius, 1.2-2.0 atmospheres, and continue for 60-90 minutes. The specific conditions vary depending on the tissue type.

[0334] 7. Remove the sample, quickly wash it three times with 1× PBS (pH 7.4), and trim the sample to remove excess gel.

[0335] 8. Reduction and alkylation of samples:

[0336] Add 5 mL of reducing reagent and reduce at 37°C for 30 minutes. Rinse the sample quickly 2-3 times with 1× PBS (pH 7.4). Then, add 5 mL of alkylating reagent and alkylate at room temperature, protected from light, for 30 minutes. Rinse three times with 1× PBS (pH 7.4) at room temperature, 10 minutes each time.

[0337] 9. Add pure water and allow the gel to swell for 30 minutes until it reaches its maximum size.

[0338] 10. Stain with Coumadin staining solution at 37°C for 1 hour.

[0339] 11. Decolorize by replacing the purified water several times until the desired expansion coefficient is achieved. This will yield a swollen sample-hydrogel complex.

[0340] Example 2: Solid Phase Extraction-Assisted In-Gel Enzymatic Hydrolysis (Technical Route as Shown in Figure 1)

[0341] 1. Assembly of solid phase extraction device:

[0342] Depending on the size of the swollen sample-hydrogel complex to be processed, a pipette tip of corresponding size (e.g., a low-retention tip with a volume of 10 μL or 250 μL) is used. The bottom of the tip is shortened to meet the aforementioned specifications to increase the rate of liquid outflow, reduce liquid residue, and allow its overall height to be placed in a deep-well plate. Then, a gasket containing a C18 solid-phase extraction adsorption filler (e.g., a trimmed C18 solid-phase extraction membrane, the gasket has a thickness of 0.5 mm and a diameter that matches the inner diameter of the pipette tip used, for example, a diameter of 1.5 mm that matches a 250 μL low-retention tip) is added to the tip, and the gasket is brought into close contact with the pipette tip by blowing nitrogen through a 1 mL pipette tip, thereby obtaining a solid-phase extraction device (i.e., the device for processing biological samples to identify proteins therein as described in the present disclosure).

[0343] 2. Activation of stationary phase materials:

[0344] 20-100 μL of 80% acetonitrile was added to the solid phase extraction device and centrifuged at 100 g for 1 minute to activate the C18 solid phase extraction adsorption medium, followed by centrifugation to discharge the remaining liquid from the device.

[0345] 3. Sampling and transfer of trace samples:

[0346] The expanded sample-hydrogel complex obtained in Example 1 was sampled using a punch of appropriate size and transferred using forceps to a solid phase extraction device containing 10-50 μL of 50% acetonitrile / 50% 100 mM ammonium bicarbonate solution.

[0347] 4. Decolorization and dehydration of samples:

[0348] Add another 10-50 μL of 50% acetonitrile / 50% 100 mM ammonium bicarbonate solution to the SPE device. Incubate at room temperature until the sample is clear, then centrifuge at 150 g. Then, add 20-100 μL of pure acetonitrile, incubate at room temperature until the gel turns white, and centrifuge at 150 g. Centrifuge further to remove any remaining acetonitrile from the C18 SPE disk.

[0349] 5. In-gel enzymatic hydrolysis assisted by solid phase extraction device:

[0350] Transfer the solid-phase extraction device to a low-adsorption centrifuge tube or 96-well deep-well plate. Add 2 μL of trypsin working solution using a low-adsorption long pipette tip, cover the top with plastic wrap, and incubate at 37°C for 4 hours. Then add an additional 2-4 μL of digestion buffer 2 and continue incubating at 37°C for 8 hours for in-gel digestion. The addition of digestion buffer 2 can keep the solid-phase extraction device moist, preventing the sample from drying out and affecting sample recovery. At the same time, the sample is sealed with liquid to prevent liquid evaporation during subsequent incubations, which may affect the enzymatic reaction.

[0351] 6. Column equilibration and digestion termination:

[0352] Add 6-12 μL of 2% acetonitrile / 0.1% trifluoroacetic acid and keep at room temperature for 15 minutes to equilibrate the column and terminate trypsin digestion. Centrifuge to collect the peptide-containing solution into a low-binding centrifuge tube or 96-well deep-well plate.

[0353] 7. Peptide elution:

[0354] Add 20-40 μL of 70% acetonitrile / 0.1% trifluoroacetic acid, incubate at room temperature for 15 minutes, and centrifuge to collect the peptide solution. Repeat this process once. Then, add 20-40 μL of pure acetonitrile, incubate at room temperature until the gel turns white, and centrifuge at 150 g for 5 minutes. The solution in the low-adsorption centrifuge tube or 96-well deep-well plate is now the recovered peptide solution.

[0355] 8. Peptide drying:

[0356] The peptide solution was concentrated to dryness using a spin dryer at 45°C and 6-7 mbar.

[0357] Control Example 1: Conventional in-gel enzymatic hydrolysis

[0358] Conventional in-gel enzymatic digestion can be performed according to the ProteomEx method (Nature Communications 13(1), 2022: 7242.), which specifically includes the following steps.

[0359] 1. Decolorization and dehydration:

[0360] After sampling the gel using a punch of appropriate size, transfer the sample to a 1.5 mL EP tube using forceps. Add 200 μL of 50% acetonitrile / 50% ddH2O and vortex for 30 minutes (until clear). Centrifuge at 800 rpm at 30°C and discard the supernatant. Then, add 200 μL of 50% acetonitrile / 50% 100 mM ammonium bicarbonate, incubate for 10 minutes, shake at 400 rpm, allow to stand, centrifuge, and discard the supernatant. Repeat this step once more. Dry the sample in a spin dryer for approximately 5 minutes.

[0361] 2. In-gel enzymatic digestion:

[0362] After adding 2 μL of trypsin working solution and incubating at 37°C for 4 hours, add an additional 2-4 μL of digestion buffer 2 and continue incubating at 37°C for 8 hours.

[0363] The amount of trypsin and the enzymatic digestion method used in this step were consistent with those of the solid phase extraction-assisted in-gel enzymatic digestion in Example 2 to obtain more comparable results.

[0364] 3. Peptide collection:

[0365] Centrifuge the enzymatically digested sample obtained in step 2 at 150 g for 2 minutes. Collect the supernatant (sol. A) into a new 1.5 mL centrifuge tube.

[0366] Add 100 μL of 25 mM ammonium bicarbonate and vortex at 600 rpm for 5 minutes at 25°C. Centrifuge at 150 g for 2 minutes. Collect the supernatant (sol. B) in the same centrifuge tube.

[0367] Add 100 μL of 50% acetonitrile / 2.5% formic acid and vortex at 800 rpm for 10 minutes. Centrifuge at 150 g for 2 minutes. Collect the supernatant (sol. C1) into the same centrifuge tube. Repeat three times, collecting the supernatants (sols. C1-C3) into the same centrifuge tube.

[0368] Add 100 μL of pure acetonitrile and vortex at 800 rpm for 5 minutes. Centrifuge at 150 g for 2 minutes. Collect the supernatant (sol. D) into the same centrifuge tube. Repeat this step until the gel becomes white and sticky.

[0369] Centrifuge the tube containing all the mixed solutions at 12,000 g for 10 minutes. Collect the supernatant into a new batch of 1.5 mL centrifuge tubes.

[0370] After spin drying, resuspend in 20 μL of mass spectrometry buffer.

[0371] 4. Sample desalination:

[0372] Desalt using a micropipettor.

[0373] Column activation: Add 20 μL of buffer A and centrifuge at 800 g for 2 minutes. Repeat once.

[0374] First stage column equilibration: Add 20 μL of buffer B, centrifuge at 800 g for 2 minutes, and repeat once.

[0375] Second stage column equilibration: Add 20 μL of buffer C, centrifuge at 800 g for 2 minutes, and repeat once.

[0376] Peptide loading: Load the sample and centrifuge at 600 g for 4 minutes until all the peptide has passed through.

[0377] Desalting: Add 20 μL of buffer C and wash, centrifuge at 800 g for 2 minutes, 10 times.

[0378] Peptide elution: Collect the eluate in a new 1.5 mL centrifuge tube (with a final label). Elute twice with 20 μL of buffer D and centrifuge at 800 g for 2 minutes. Then spin dry the sample.

[0379] Example 3: Mass spectrometry detection

[0380] The samples obtained in Example 2 and Control Example 1 were resuspended in mass spectrometry buffer and centrifuged at 12,000 g at 4°C for 15 minutes. The supernatant was analyzed by mass spectrometry using a 60-minute linear gradient. Using a timsTOF pro, both ddaPASEF (DDA) and diaPASEF (DIA) mass spectrometry acquisition modes were selected.

[0381] Experimental results and conclusions

[0382] The results show (Figure 2) that when sampling and analyzing mouse liver samples, under the same tissue volume (the actual volume of the tissue sample is about 1.55nL) and tissue site, the solid phase extraction-assisted in-gel enzymatic digestion method disclosed in the present invention significantly increased the peptide and protein identification amounts by 97.7% and 30.6%, respectively (Figure 2, A), and was able to better identify longer peptides (Figure 2, B), and the identified peptides and proteins had similar physicochemical properties (Figure 2, C). At the same time, the method disclosed in the present invention has a lower and more stable missed cutting rate, with an average of about 17%, which is significantly lower than the conventional in-gel enzymatic digestion method (Figure 2, D). Furthermore, the method disclosed herein yielded 17,910 and 770 unique peptides and proteins, respectively (Figure 2, E), representing four times and ten times the number of unique peptides and proteins obtained using conventional methods. Furthermore, the method disclosed herein exhibited a higher linear dynamic range for protein abundance and improved reproducibility (Figure 2, FH), while achieving similar results to conventional in-gel enzymatic methods in terms of protein type and subcellular localization (Figure 2, I).

[0383] Example 4: Number of peptides and proteins identified in DDA and DIA modes

[0384] With reference to Examples 1-3 and Control Example 1, the number of peptide and protein identifications using the two methods in different tissue volumes under DDA and DIA modes was explored (Figure 3). For solid-phase extraction-assisted in-gel digestion, using tissue with actual volumes ranging from approximately 0.042 nL to approximately 3.054 nL (the actual volume before expansion was converted after sampling with a puncher with a hole diameter between 0.35 and 3 mm on the expanded sample-hydrogel complex), 5519 / 19499 (lowest identification amount) to 28931 / 48330 peptides (highest identification amount) and 1013 / 3421 (lowest identification amount) to 3258 / 5356 proteins (highest identification amount) were identified under DDA- / DIA-MS, respectively. Due to technical limitations, conventional in-gel enzymatic digestion methods can only process tissue volumes as low as approximately 0.339 nL (the sample volume processing capacity is approximately 8 times lower than that of the method disclosed herein), and for tissues with actual volumes ranging from approximately 0.339 nL to approximately 3.054 nL, only 1994 / 4853 to 23659 / 39488 peptides and 834 / 1898 to 3239 / 5136 proteins were identified under DDA- / DIA-MS.

[0385] Example 5: Solid Phase Extraction-Assisted In-Gel Enzymatic Hydrolysis of Single Cell Nuclei (Technical Route as Shown in Figure 1)

[0386] 1. Assembly of solid phase extraction device:

[0387] A low-retention pipette tip with a volume of 10 μL was used, and then a gasket containing a C18 solid-phase extraction adsorption filler (cut to a diameter and thickness of 0.5 mm) was added to the pipette tip. The gasket was brought into close contact with the pipette tip by blowing nitrogen through a 1 mL pipette tip, thereby obtaining a solid-phase extraction device (i.e., the device for processing biological samples to identify proteins therein as described in the present disclosure).

[0388] 2. Activation of stationary phase materials:

[0389] 20 μL of 80% acetonitrile was added to the solid phase extraction device and centrifuged at 100 g for 1 minute to activate the C18 solid phase extraction adsorption medium, followed by centrifugation until the remaining liquid was discharged from the device.

[0390] 3. Sampling and transfer of trace samples:

[0391] A polylysine solution was applied to the metal diaphragm of a laser microdissection instrument, and the expanded sample-hydrogel complex obtained in Example 1 was then placed on the metal diaphragm. The sample was allowed to stand for 24 hours. After the sample dried, a single cell nucleus sample of rat liver was obtained by laser microdissection and placed in the cap of a 0.2 mL collection tube. 10-50 μL of 50% acetonitrile / 50% 100 mM ammonium bicarbonate solution was added to the cap of the tube and incubated for a period of time to allow the sample to swell. The sample was then transferred to a solid phase extraction device by centrifugation.

[0392] 4. Alkalization and dehydration of samples:

[0393] Add another 20 μL of 50% acetonitrile / 50% 100 mM ammonium bicarbonate solution to the SPE device. Incubate at room temperature for 5-15 minutes and centrifuge at 150 g. Then, add 20 μL of pure acetonitrile, incubate at room temperature until the gel turns white, and centrifuge at 150 g. Centrifuge further to remove any remaining acetonitrile from the C18 SPE disk.

[0394] 5. In-gel enzymatic hydrolysis assisted by solid phase extraction device:

[0395] Transfer the solid phase extraction device to a low-adsorption centrifuge tube or a 96-well deep-well plate, add 1 μL of trypsin working solution using a low-adsorption long pipette tip, cover the top with plastic wrap, and incubate overnight at 37°C for 12 hours for in-gel enzymatic digestion.

[0396] 6. Column equilibration and digestion termination:

[0397] Add 6 μL of 2% acetonitrile / 0.1% trifluoroacetic acid and keep at room temperature for 15 minutes to equilibrate the column and terminate trypsin digestion. Alternatively, centrifuge the solution containing the peptide fragments into a low-binding centrifuge tube or 96-well deep-well plate.

[0398] 7. Peptide elution:

[0399] Add 6 μL of 70% acetonitrile / 0.1% trifluoroacetic acid and incubate at room temperature for 15 minutes. Repeat one or two times and recover by centrifugation. Then add 6 μL of pure acetonitrile and incubate at room temperature until the gel turns white. Centrifuge at 150g for 5 minutes. The solution in the low-adsorption centrifuge tube or 96-well deep-well plate is the recovered peptide solution.

[0400] 8. Peptide drying:

[0401] The peptide solution was concentrated to a dry state using a spin dryer at 45°C and 6-7 mbar to obtain an enzymatically hydrolyzed peptide sample.

[0402] 9. Mass spectrometry detection

[0403] The sample was resuspended in mass spectrometry buffer and all resuspended solutions were directly subjected to mass spectrometry analysis using an 8-minute linear gradient in the liquid phase using the Orbitrap Astral in DIA mass spectrometry acquisition mode.

[0404] The results show (Figure 6) that the method of this embodiment can effectively process samples as small as a single cell nucleus (Figure 6, AC), detecting an average of approximately 550 proteins (Figure 6, D). Under the same mass spectrometry acquisition mode (DIA), compared with the detection results of the tissue volume of approximately 0.042nL in Example 4, the method of this embodiment has a lower Pearson correlation coefficient (Figure 6, E), which can reflect the heterogeneity of single-nuclear samples and has higher resolution; 258 specific proteins were identified (Figure 6, F), and the proportion of nuclear proteins and transcription regulators was significantly higher, increasing by 4.26 times and 3.59 times, respectively (Figure 6, G). In addition, the 258 specific proteins in Example 5 were subjected to nuclear-specific pathway enrichment analysis, and the results showed that the enrichment level of nucleic acid activity-related pathways (such as mRNA processing and splicing-related pathways) was high (Figure 6, H). The above results demonstrate that the method of this embodiment can effectively separate, collect, prepare and detect samples as small as a single cell nucleus at the tissue spatial level, greatly improving the resolution and applicable range of trace sample detection.

[0405] In summary, the solid phase extraction-assisted in-gel enzymatic hydrolysis method provided by the present invention has better performance in identification quality (missed cutting rate and repeatability, etc.) and identification quantity (peptides and proteins) than the conventional in-gel enzymatic hydrolysis method, and its cost is lower and higher throughput can be achieved. At the same time, the new method is particularly suitable for the processing of extremely trace samples. Under the same conditions, the number of identifications is much higher than that of conventional methods, and it can process samples as low as close to the single-cell level. These indicate that the method disclosed in the present invention has great application potential in extremely trace spatial proteomics.

[0406] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present disclosure and not to limit it. Although the present disclosure has been described in detail with reference to the preferred embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present disclosure can still be modified or some technical features can be replaced by equivalents without departing from the spirit of the technical solutions of the present disclosure, which should all be included in the scope of the technical solutions requested for protection in the present disclosure.

Claims

1. A device for processing a biological sample to identify a protein therein, comprising: A tube body (1), the tube body (1) comprising a reaction zone (11) and a flow guide zone (12) located below the reaction zone (11), the reaction zone (11) and the flow guide zone (12) being in liquid communication, and The solid phase extraction adsorption filler (2) is located at the bottom of the reaction zone (11) or the top of the guide zone (12), and is configured to be able to retain the liquid in the reaction zone (11) when the gas pressures in the reaction zone (11) and the guide zone (12) are the same, and to allow the liquid in the reaction zone (11) to flow out when the gas pressure in the reaction zone (11) is greater than the gas pressure in the guide zone (12). The ratio of the thickness to the diameter of the solid phase extraction adsorption filler (2) is less than 1, preferably less than or equal to 0.8, less than or equal to 0.7, less than or equal to 0.6, less than or equal to 0.5, or The ratio of the thickness to the diameter of the solid phase extraction adsorption filler (2) is 1 to 1.7 (for example, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7), and the maximum inner diameter of the guide area (12) is 0.1 mm to 0.7 mm.

2. The device of claim 1, wherein the ratio of the thickness to the diameter of the solid phase extraction adsorption filler (2) is less than 1, preferably less than or equal to 0.8, less than or equal to 0.7, less than or equal to 0.6, or less than or equal to 0.

5.

3. The device of claim 2, wherein the maximum inner diameter of the flow guide zone (12) is smaller than the inner diameter of the reaction zone (11), Preferably, the inner diameter of the reaction zone (11) is 2 to 4 mm, preferably 2 to 3 mm. Preferably, the maximum inner diameter of the guide area (12) is 1 to 1.5 mm. Preferably, the thickness of the solid phase extraction adsorption filler (2) is less than or equal to 1 mm, for example, about 0.9 mm, about 0.8 mm, about 0.7 mm, about 0.6 mm, about 0.5 mm, about 0.4 mm, about 0.3 mm, about 0.2 mm or about 0.1 mm.

4. The device according to claim 2 or 3, wherein the inner cavity of the reaction zone (11) is a cylindrical hollow structure with equal inner diameters at the top and bottom, and the inner cavity of the guide zone (12) is a conical hollow structure with a cross section that is wide at the top and narrow at the bottom, Preferably, the length of the guide area (12) is less than or equal to 10 mm. Further preferably, the length of the guide area (12) is less than or equal to 5 mm, for example, about 4.5 mm, about 4 mm, about 3.5 mm, about 3 mm, about 2.5 mm, about 2 mm or about 1.5 mm.

5. The device according to any one of claims 2 to 4, wherein the guide zone (12) is in liquid communication with the reaction zone (11) via a through hole located at the bottom of the reaction zone (11), Preferably, the solid phase extraction adsorption filler (2) is located at the through hole.

6. The device according to any one of claims 2 to 5, wherein the interior of the connection between the reaction zone (11) and the guide zone (12) is a smooth chamfered structure.

7. The device according to any one of claims 2 to 6, wherein the reaction zone (11) is provided with visible volume scale markings.

8. The device according to any one of claims 2 to 7, wherein the tube body (1) is further provided with a protruding part (3) protruding from the outer surface of the tube body, and is configured to enable the solid phase extraction device to be hung on a placement rack (e.g., deep well plates of different specifications), Preferably, the raised component (3) is arranged on the upper part of the tube body (1).

9. The device according to any one of claims 2 to 8, wherein the volume of the reaction zone (11) is 2-250 μL, such as 2-100 μL, preferably 10-100 μL, more preferably 20-100 μL, more preferably 20-50 μL.

10. The device according to any one of claims 2 to 9, wherein the tube body (1) further comprises a tube cover matched therewith.

11. The device according to any one of claims 2 to 10, wherein the solid phase extraction adsorption filler (2) is made of porous silica gel or porous ceramic particles modified with alkyl groups of different chain lengths, ion exchange resins or chemically bonded ion exchangers, Preferably, the pore size of the solid phase extraction adsorption filler (2) is 1-12 μm, preferably 1-5 μm. Preferably, the solid phase extraction adsorption filler (2) is a C8 or C18 solid phase extraction adsorption filler, a strong cation exchange filler or a strong anion exchange filler. The solid phase extraction adsorption filler (2) is preferably a C8 or C18 solid phase extraction membrane, a strong cation exchange membrane or a strong anion exchange membrane, and the membrane is in contact with the inner surface of the guide area (12). The solid phase extraction adsorption filler (2) is preferably a C18 solid phase extraction membrane, and the C18 solid phase extraction membrane is in contact with the inner surface of the guide area (12).

12. The device according to any one of claims 2 to 11, wherein the tube (1) is a pipette tip.

13. The device according to any one of claims 2 to 12, wherein the biological sample is present in the form of a sample-gel complex, Preferably, the gel in the sample-gel complex is a hydrogel, such as a polyacrylamide gel. Preferably, the biological sample is in the form of a swollen sample-gel complex.

14. The device of claim 1, wherein the ratio of the thickness to the diameter of the solid phase extraction adsorption filler (2) is 1 to 1.7 (e.g., 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7), and the maximum inner diameter of the guide zone (12) is 0.1 mm to 0.7 mm.

15. The device of claim 14, wherein the maximum inner diameter of the flow guide zone (12) is less than or equal to the minimum inner diameter of the reaction zone (11), Preferably, the minimum inner diameter of the reaction zone (11) is 0.2 to 0.6 mm. Preferably, the maximum inner diameter of the guide area (12) is 0.2-0.6 mm, for example, about 0.2 mm, about 0.3 mm, about 0.4 mm, about 0.5 mm or about 0.6 mm. Preferably, the thickness of the solid phase extraction adsorption filler (2) is less than or equal to 2 mm, preferably less than or equal to 1.5 mm, for example, about 1.4 mm, about 1.3 mm, about 1.2 mm, about 1.1 mm, about 1.0 mm, about 0.9 mm, about 0.8 mm, about 0.7 mm, about 0.6 mm, about 0.5 mm, about 0.4 mm, about 0.3 mm, about 0.2 mm or about 0.1 mm.

16. The device of claim 1, 14 or 15, wherein the inner cavity of the flow guide area (12) is a conical hollow structure with a cross section that is wide at the top and narrow at the bottom, Preferably, the length of the flow guide area (12) is less than or equal to 5 mm, preferably less than or equal to 3 mm, for example, about 2.5 mm, about 2.0 mm, about 1.5 mm, about 1.4 mm, about 1.3 mm, about 1.2 mm, about 1.1 mm, about 1.0 mm, about 0.9 mm, about 0.8 mm, about 0.7 mm, about 0.6 mm, about 0.5 mm, Preferably, the inner cavity of the reaction zone (11) is a cylindrical hollow structure with equal inner diameters at the top and bottom, or a conical hollow structure with a wide upper portion and a narrow lower portion in cross section, preferably a conical hollow structure with a wide upper portion and a narrow lower portion in cross section.

17. The device of any one of claims 1, 14-16, wherein The guide zone (12) is in liquid communication with the reaction zone (11) via a through hole located at the bottom of the reaction zone (11). Preferably, the connection between the reaction zone (11) and the flow guide zone (12) is a smooth chamfered structure. Preferably, the solid phase extraction adsorption filler (2) is located at the through hole.

18. The device of any one of claims 1, 14-17, wherein The flow guide zone (12) is directly connected to the reaction zone (11). Preferably, the connection between the reaction zone (11) and the flow guide zone (12) is a smooth straight line structure. Preferably, the solid phase extraction adsorption filler (2) is located at the top of the flow guide area (12). Preferably, the thickness of the solid phase extraction adsorption filler (2) is less than or equal to the length of the flow guide area (12).

19. The device according to any one of claims 1, 14 to 18, wherein the reaction zone (11) is provided with visible volume scale markings.

20. The device according to any one of claims 1, 14 to 19, wherein the tube body (1) is further provided with a protruding part (3) protruding from the outer surface of the tube body, and is configured to enable the solid phase extraction device to be hung on a placement rack (e.g., deep well plates of different specifications), Preferably, the raised component (3) is arranged on the upper part of the tube body (1).

21. The device according to any one of claims 1, 14-20, wherein the volume of the reaction zone (11) is 2-250 μL, such as 2-100 μL, preferably 10-100 μL, further preferably 20-100 μL, further preferably 20-50 μL, such as 20 μL, 30 μL, 40 μL or 50 μL.

22. The device according to any one of claims 1, 14-21, wherein the tube body (1) further comprises a tube cover matched therewith.

23. The device according to any one of claims 1, 14 to 22, wherein the solid phase extraction adsorption filler (2) is made of porous silica gel or porous ceramic particles modified with alkyl groups of different chain lengths, ion exchange resins or chemically bonded ion exchangers, Preferably, the pore size of the solid phase extraction adsorption filler (2) is 1-12 μm, preferably 1-5 μm. Preferably, the solid phase extraction adsorption filler (2) is a C8 or C18 solid phase extraction adsorption filler, a strong cation exchange filler or a strong anion exchange filler. The solid phase extraction adsorption filler (2) is preferably a C8 or C18 solid phase extraction membrane, a strong cation exchange membrane or a strong anion exchange membrane, and the membrane is in contact with the inner surface of the guide area (12). The solid phase extraction adsorption filler (2) is preferably a C18 solid phase extraction membrane, and the C18 solid phase extraction membrane is in contact with the inner surface of the guide area (12).

24. The device according to any one of claims 1, 14 to 23, wherein the tube (1) is a pipette tip.

25. The device of any one of claims 1, 14-24, wherein the biological sample is in the form of a sample-gel complex, Preferably, the gel in the sample-gel complex is a hydrogel, such as a polyacrylamide gel. Preferably, the biological sample is in the form of a swollen sample-gel complex.

26. A kit for processing a biological sample to identify a protein therein, comprising: The device of any one of claims 1 to 25, wherein processing of the biological sample is performed in the device.

27. The kit of claim 26, further comprising: Stationary phase activators, decolorizers, dehydrators, digestion agents, digestion termination agents, and peptide eluents for activating solid phase extraction adsorption media; Preferably, the stationary phase activator is an alcohol or acetonitrile, preferably acetonitrile, and preferably, the acetonitrile is prepared as a 70-90% acetonitrile aqueous solution, such as an 80% acetonitrile aqueous solution when used; Preferably, the decolorizing agent is a mixed solution of an organic solvent and an inorganic salt; preferably, the organic solvent is acetonitrile, and the inorganic salt is an aqueous solution of ammonium bicarbonate; further preferably, the decolorizing agent is a combination of acetonitrile and ammonium bicarbonate, and preferably the decolorizing agent exists in the form of a solution; preferably, the decolorizing agent in the form of a solution contains 40-60 v / v% acetonitrile, 40-60 mM ammonium bicarbonate and the balance water, and further preferably contains 50 v / v% acetonitrile, 50 mM ammonium bicarbonate and the balance water; Preferably, the dehydrating agent is alcohol or acetonitrile, more preferably acetonitrile; Preferably, the digestive agent comprises trypsin, and more preferably, the digestive agent is a combination of trypsin, acetic acid, acetonitrile and ammonium bicarbonate; more preferably, the digestive agent is in the form of a solution; preferably, the digestive agent contains 10-25 ng / μL trypsin, and the pH is 8-9; more preferably, the digestive agent contains 10-25 ng / μL trypsin, 0.002-0.005 v / v% acetic acid, 40-60 mM (e.g. 45 mM) ammonium bicarbonate solution and 10 v / v% acetonitrile; Preferably, the digestion termination agent is a mixed solution of an organic solvent and an acid, more preferably a combination of acetonitrile and trifluoroacetic acid, more preferably an aqueous solution containing 1 to 3 v / v% acetonitrile and 0.05 to 0.2 v / v% trifluoroacetic acid, for example, an aqueous solution containing 2 v / v% acetonitrile and 0.1 v / v% trifluoroacetic acid; Preferably, the peptide eluent is a mixed solution of an organic solvent and an acid; preferably, the peptide eluent is a combination of acetonitrile or ethanol and trifluoroacetic acid, preferably a combination of acetonitrile and trifluoroacetic acid; more preferably, the peptide eluent is an aqueous solution containing 60-80 v / v% acetonitrile and 0.05-0.2 v / v% trifluoroacetic acid, for example, an aqueous solution containing 70 v / v% acetonitrile and 0.1 v / v% trifluoroacetic acid.

28. The kit of claim 26 or 27, further comprising: Acetonitrile or ethanol, ammonium bicarbonate, trypsin, acetic acid, and trifluoroacetic acid, Optionally, PBS buffer is also included in the form of dry powder or liquid.

29. The kit of any one of claims 26-28, further comprising: Low-binding centrifuge tubes and / or deep-well plates and / or mass spectrometry buffer, Preferably, the mass spectrometry buffer is a combination of acetonitrile and formic acid, preferably an aqueous solution containing 1-3 v / v% acetonitrile and 0.05-0.2 v / v% formic acid, and more preferably an aqueous solution containing 2% acetonitrile and 0.1% formic acid.

30. Use of the device of any one of claims 1 to 25 or the kit of claims 26 to 29 for processing a biological sample to identify a protein therein.

31. A method of processing a biological sample to identify a protein therein, comprising: Processing a biological sample using the device according to any one of claims 1 to 25 or the kit according to claims 26 to 29, Preferably, the processing of the biological sample is performed in a device according to any one of claims 1-25.

32. The method of claim 31, comprising: (1) activating the solid phase extraction adsorption filler in the device; (2) placing the biological sample to be processed in the device after being processed in step (1) to decolorize and dehydrate the biological sample; (3) performing enzymatic hydrolysis on the biological sample treated in step (2); (4) Recovering the peptide fragments obtained in step (3).

33. The method of claim 32, wherein Step (1) include: 1.1 Add 20-100 μL of 70-90% acetonitrile aqueous solution to the device. 1.2 Activate by centrifugation at 50 g-200 g (preferably 80 g-150 g, for example 100 g); 1.3 Centrifuge until the remaining liquid is discharged from the device.

34. The method of any one of claims 31 to 33, wherein step (2) include: 2.1 placing the biological sample to be processed in the device after being processed in step (1), 2.2 Add a decolorizing agent containing 40-60 v / v% acetonitrile, 40-60 mM ammonium bicarbonate and the remainder of water, incubate until the sample is clear, and centrifuge to remove the solvent. Preferably, incubation is at room temperature; 2.3 Add 20-100 μL acetonitrile, incubate until the sample is dehydrated, and centrifuge to remove the solvent. Preferably, incubation is at room temperature.

35. The method of any one of claims 32 to 34, wherein step (3) include: 3.1 Add a digestion agent (e.g., 1 to 3 μL, preferably 2 μL) to the device and incubate; preferably incubate at 37° C. for 3 to 5 hours (e.g., 4 hours); 3.2 Add digestion buffer (e.g., 2 to 15 μL, preferably 4 μL or 10 μL) and continue incubation; preferably incubate at room temperature for 7 to 12 hours (e.g., 8 hours); Optionally, it also includes: 3.3 Add digestion termination agent to terminate digestion; Preferably, the digestive agent contains 10-25 ng / μL trypsin and a pH of 8-9; further preferably, the digestive agent contains 10-25 ng / μL trypsin, 0.002-0.005 v / v% acetic acid, 40-60 mM (e.g. 45 mM) ammonium bicarbonate solution and 10 v / v% acetonitrile; Preferably, the digestion buffer is an alkaline inorganic salt buffer, more preferably an aqueous solution of ammonium bicarbonate, and further preferably an aqueous solution of 8 to 12 mM (e.g., 10 mM) ammonium bicarbonate; Preferably, the digestion termination agent is a mixed solution of an organic solvent and an acid, more preferably a combination of acetonitrile and trifluoroacetic acid, more preferably an aqueous solution containing 1-3 v / v% acetonitrile and 0.05-0.2 v / v% trifluoroacetic acid, for example, an aqueous solution containing 2 v / v% acetonitrile and 0.1 v / v% trifluoroacetic acid.

36. The method of any one of claims 32-35, wherein step (4) comprises: 4.1 Centrifuge the product of step (3) and collect the centrifuge liquid; 4.2 adding a mixed solution of an organic solvent and an acid (e.g., 20-40 μL) to the device, incubating (e.g., 10-20 minutes or 15 minutes), centrifuging, and collecting the centrifuge; 4.3 Repeat step 4.2 1-2 times; Optionally, it also includes: 4.4 Add acetonitrile to dehydrate the biological sample, centrifuge, and collect the centrifuge liquid; 4.5 combining the above centrifuge solutions, and optionally drying them to obtain peptide fragments; Preferably, the mixed solution of the organic solvent and the acid is a combination of acetonitrile or ethanol and trifluoroacetic acid, preferably a combination of acetonitrile and trifluoroacetic acid; more preferably, the mixed solution of the organic solvent and the acid is an aqueous solution containing 60-80 v / v% acetonitrile and 0.05-0.2 v / v% trifluoroacetic acid, for example, an aqueous solution containing 70 v / v% acetonitrile and 0.1 v / v% trifluoroacetic acid.

37. A method for identifying a protein in a biological sample, comprising: 1) using the device of any one of claims 1 to 25 or the kit of claims 26 to 29 to perform Processing to obtain peptide fragments; 2) Detect peptides using LC-MS / MS; Further preferably, step 2) comprises: Resuspending the peptide fragments with a mass spectrometry buffer, preferably, the mass spectrometry buffer is a mixed solution of an organic solvent and an acid, more preferably a combination of acetonitrile and formic acid, more preferably an aqueous solution containing 1 to 3 v / v% acetonitrile and 0.05 to 0.2 v / v% formic acid, for example, an aqueous solution containing 2 v / v% acetonitrile and 0.1 v / v% formic acid; The resuspended peptides were centrifuged and the supernatant was taken for LC-MS / MS detection.

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