New method for expanded proteomic analysis of biological samples

Abstract

An improved method for proteomic analysis of biological samples by physical expansion of the sample through hydrogel swelling is provided. A kit and related system for preparing sample-gel complexes for protein identification are also provided.

Description

Invention Field

[0001] This disclosure relates to proteomics, particularly methods for proteomic analysis of tissue samples using hydrogel swelling techniques and secondary gel embedding, specific formulations used in such methods and their applications, and kits and systems for such methods. Background Technology

[0002] Traditional proteomics applications focus on protein expression levels in whole tissues or isolated cells, losing information about intercellular differences and spatial location. However, the types and levels of protein expression in different cells within a tissue are highly specific. Spatial proteomics atlases of tissue protein expression contribute to a better understanding of the mechanisms underlying the tissue microenvironment and tumor heterogeneity. Spatial proteomics also plays a crucial role in refined disease diagnosis, drug selection, and prognostic assessment.

[0003] In the past decade, a number of space proteomics technologies have emerged, such as laser capture microdissection (LCM), matrix-assisted laser desorption / ionization (MALDI), and liquid extraction surface analysis (LESA). All of these technologies strive to achieve higher resolution spatial proteomics analysis of minute samples using advanced equipment.

[0004] On one hand, the main approach for spatial proteomics studies of biological tissue samples is to use laser capture microdissection (LCM) to obtain tiny tissue samples. LCM uses a microscope combined with a laser aiming system to extract single-cell samples from a heterogeneous environment. The laser is used to perforate and remove the target area, separating it from adjacent tissues. Peptide fragments are obtained through laser sampling and traditional in-solution trypsin digestion, followed by proteomics analysis. However, LCM sampling can only obtain samples of about 3-5 millimeters, and laser microdissection sampling is very expensive, with a complete set of equipment costing millions of yuan, making large-scale application impossible. Furthermore, the in-solution trypsin digestion method results in the loss of many low-abundance proteins during the processing.

[0005] On the other hand, methods for spatial analysis of proteomics using gel swelling have been proposed. Expanded proteomics combines expansion microscopy with proteomics, using expansion to magnify samples and achieve higher spatial resolution and preserve high-level proteomics data. However, expanded proteomics has some limitations, such as limited spatial resolution (160 μm), cumbersome and time-consuming procedures, and the need for further improvement in protein preservation.

[0006] In summary, there is a need in this field to improve the sampling and analysis methods for spatial proteomics identification. Summary of the Invention

[0007] This application further improves the expanded proteomics analysis method for protein identification. Compared with existing expanded proteomics analysis methods (referred to as ProteomEx2.0, such as those disclosed in CN115372527A, which is incorporated herein by reference in its entirety), the method of this invention (referred to as ProteomEx2.0) optimizes the experimental process and shortens the working time; applies reductive alkylation to the entire sample-hydrogel composite, improving the efficiency of the entire workflow; incorporates a secondary gel embedding step, making it compatible with laser capture microdissection sampling; enables proteomics processing of micron-sized samples without the need for high-precision instrumentation; can effectively handle hard tissue samples such as heart and kidney; optimizes staining conditions, improving sample processing efficiency, and mass spectrometry identification results show that more proteins and peptides can be identified with a lower rate of missed cleavage.

[0008] In one aspect, this disclosure provides a method for proteomics identification of biological samples, comprising:

[0009] (a) The biological sample is treated with an anchoring agent;

[0010] (b) Add hydrogel monomer solution to form sample-hydrogel composite through polymerization of hydrogel monomer solution;

[0011] (c) Denature the protein in the sample-hydrogel complex;

[0012] (d) The sample-hydrogel composite was subjected to reduction and alkylation treatment;

[0013] (e) The sample-hydrogel complex is stained with proteins and then swelled.

[0014] (f) Cutting samples from the expanded sample-hydrogel composite, embedding the cut samples with a secondary gel solution to form a secondary gel-embedded sample; and

[0015] (g) Peptides are extracted from the secondary gel-embedded samples for protein identification.

[0016] In some embodiments, the biological sample is a specimen, a biopsy sample, an organ or part thereof, or a whole organism. The sample may be alive, fixed, or preserved, such as a fixed tissue section, fresh frozen tissue, or a tissue section embedded in paraffin. In some embodiments, the biological sample is a liver tissue section, a heart tissue section, a kidney tissue section, a brain section, or a cancer tissue section.

[0017] In some embodiments, the sample to be expanded is a tissue section with a thickness of 50 μm or less, i.e., a thin tissue sample. In some other embodiments, the sample to be expanded is a tissue sample with a thickness greater than 400 μm. In some still embodiments, the sample to be expanded is an entire organ.

[0018] In some embodiments, adding the hydrogel monomer solution in step (b) includes perfusing the biological sample with the hydrogel monomer solution or embedding the biological sample in the hydrogel monomer solution.

[0019] The hydrogel monomer solution may contain one or more monomers capable of polymerizing to form a hydrogel. In some embodiments, the monomer is selected from sodium acrylate (SA), sodium methacrylate (SMA), itaconic acid (IA), trans-aconitic acid (TAA), ethyl-2-(hydroxymethyl)-acrylate (EHA), N,N-methylenebisacrylamide (MBAA, also known as BIS), N,N-dimethylacrylamide (DMAA), pentaerythritol tetraacrylate (PT), propoxylated trimethylolpropane triacrylate (TPT), pentaerythritol triacrylate (PA), dipentaerythritol pentaacrylate, or dipentaerythritol hexaacrylate. One or more of the following: acrylate (DPHA), trimethylolpropane triacrylate (TTA), bis(trimethylolpropane)-tetraacrylate (DiTA), trimethylolpropane trimethacrylate (TTMA), glycerol propoxylated (1PO / OH) triacrylate (GPT), ethoxylated trimethylolpropane triacrylate (TET), pentaerythritol allyl ether (PAE), sodium 4-hydroxy-2-methylenebutyrate (SHMB), N,N-dimethylaminopropylacrylamide (DMPAA), and acrylamide (AA).

[0020] In some embodiments, the monomer comprises sodium methacrylate (SMA), N,N-dimethylacrylamide (DMAA), and pentaerythritol allyl ether (PAE). In some embodiments, the molar ratio of DMAA to SMA in the precursor is in the range of 30:1 to 3:1, for example, in the range of 20:1 to 3:1, in the range of 15:1 to 3:1, in the range of 10:1 to 3:1, in the range of 9:1 to 3:1, in the range of 8:1 to 3:1, in the range of 7:1 to 3:1, in the range of 6:1 to 3:1, in the range of 5:1 to 3:1, and in the range of 4:1 to 3:1. Within the range or any subrange thereof, more specifically, the molar ratio of DMAA:SMA can be about 4:1 or 3:1; and wherein the molar ratio of SMA:PAE is in the range of 10:0.004 to 10:3, for example in 10:0.008 to 10:3, 10:0.01 to 10:3, 10:0.02 to 10:3, 10:0.03 to 10:3, 10:0.04 to 10:3, 10:0.05 to 10: 3. 10:0.06 to 10:3, 10:0.07 to 10:3, 10:0.08 to 10:3, 10:0.09 to 10:3, 10:0.1 to 10:3, 10:0.4 to 10:3, 10:0.6 to 10:3, 10:0.8 to 10:3, 10:1 to 10:3, 10:1.1 to 10:3, 10:1.2 to 10:3, 10:1.3 to 10:3, 10:1.4 to 10:3 The SMA:PAE molar ratio can be within the range of 10:1.5 to 10:3, 10:2 to 10:3, or any subrange thereof. More specifically, the SMA:PAE molar ratio can be approximately 10:0.005, 10:0.008, 10:0.1, 10:0.5, 10:0.8, 10:1, 10:1.1, 10:1.2, 10:1.3, 10:1.4, 10:1.5, 10:1.6, 10:1.7, or 10:1.8. The PAE ratio can be adjusted according to the required mechanical strength of the gel.

[0021] In some embodiments, the molar ratio of DMAA to SMA is in the range of 30:1 to 3:1, and the molar ratio of SMA to PAE is in the range of 10:0.004 to 10:3. Preferably, the molar ratio of DMAA:SMA:PAE is (30-50):(5-15):(0.005-2), that is, in molar parts, if DMAA is 30-50 parts, then SMA is 5 to 15 parts, and PAE is 0.005 to 2 parts. In some embodiments, the molar ratio of DMAA:SMA:PAE is about 40:10:0.004 to 40:10:0.2, and more preferably about 40:10:0.02 to 40:10:0.08.

[0022] In some embodiments, a polymerization activator and / or accelerator, such as VA-044, V50, ammonium persulfate, potassium persulfate, and TEMED, is added to the hydrogel monomer solution before use.

[0023] In some embodiments, steps (a) and (b) are performed simultaneously, for example, by adding an anchoring agent to the hydrogel monomer solution, thereby simultaneously anchoring and polymerization. Preferably, the anchoring agent comprises biomolecular reactive chemical groups and hydrogel reactive chemical groups. In some embodiments, the anchoring agent may be selected from N-succinimidyl acrylate (NSA), N-(allyloxycarbonyloxy)-succinimidyl (NAS), and combinations thereof.

[0024] In some embodiments, the secondary adhesive solution is a solution of 30% acrylamide / methylenebisacrylamide diluted in water, for example, by mixing 30% acrylamide / methylenebisacrylamide solution with water at a volume ratio ranging from 10:5.5 to 12:4.5, for example, at a volume ratio of 11:5.

[0025] In some embodiments, a polymerization activator and / or accelerator, such as ammonium persulfate, potassium persulfate, and TEMED, is added to the secondary adhesive solution before use.

[0026] In some embodiments, the method further includes perfusion fixation of the biological sample prior to step (a), optionally using a fixation solution comprising 1% paraformaldehyde and 0.1 mg / ml of N-succinimide acrylate (NSA).

[0027] In some embodiments, homogenization (also known as denaturation) of the sample-gel complex is performed, preferably by mild denaturation. In some embodiments, protein denaturation is performed by treatment with a detergent (e.g., SDS or sodium dodecylbenzenesulfonate). In some embodiments, in step (c), protein denaturation of the sample-hydrogel complex includes treatment with two protein denaturing buffers, wherein the first protein denaturing buffer is acidic and the second protein denaturing buffer is neutral. For example, both the first and second protein denaturing buffers may contain a detergent (e.g., SDS or sodium dodecylbenzenesulfonate), wherein the first protein denaturing buffer is acid-treated to be acidic.

[0028] In some embodiments, a reduction alkylation of the sample-hydrogel composite is further included between steps (c) and (d), optionally by treatment with a disulfide reducing agent selected from TCEP, DTT, and BME.

[0029] In some implementations, deanchoring is also performed after protein denaturation by increasing the solution pH to alkaline (e.g., by contacting with Tris buffer) to break the amide bonds.

[0030] In some embodiments, the staining is selected from Coomassie blue staining, fluorescent staining, Coomassie brilliant blue staining, immunostaining, silver staining, and isotope staining.

[0031] In some embodiments, the sample-hydrogel complex is swelled by incubating it with water or an aqueous buffer such as Tris buffer. The linear expansion factor after the sample-hydrogel complex swells can range from 2 to 7, for example, greater than 5.

[0032] In some embodiments, the sampling is local sampling, such as microsampling, microsampling performed by a punch, or LCM sampling. The diameter of the sample obtained by local sampling can be less than 3 mm, and in particular, the diameter of the sample obtained by LCM sampling can be less than 1 mm.

[0033] In some embodiments, step (f) further includes destaining the secondary gel-embedded sample, optionally by eluting the gel block with a 50% / 50% volume ratio of acetonitrile / 100mM ammonium bicarbonate buffer or a 50% / 50% volume ratio of methanol / 100mM ammonium bicarbonate buffer.

[0034] In some implementations, protein identification is performed via mass spectrometry, including enzymatic digestion of the sample-gel complex, peptide extraction (optionally desalting), and mass spectrometry detection. Mass spectrometry detection can be performed using HPLC-MS or MALDI-TOF.

[0035] In one aspect, this disclosure provides a kit comprising DMAA, SMA, and PAE in one or more containers, and optionally a secondary gel solution such as an acrylamide / methylenebisacrylamide solution for secondary gel embedding.

[0036] In some embodiments, DMAA, SMA, and PAE are each in separate containers. Optionally, the molar ratio of DMAA:SMA is in the range of 30:1 to 3:1, and the molar ratio of SMA:PAE is in the range of 10:0.005 to 10:3. Alternatively, at least two of DMAA, SMA, and PAE are mixed in one container. The molar ratios of DMAA, SMA, and PAE can be as described above.

[0037] In some embodiments, DMAA, SMA, and TPT are each in separate containers. Optionally, the molar ratio of DMAA:SMA is in the range of 30:1 to 3:1, and the molar ratio of SMA:TPT is in the range of 10:0.005 to 10:3. Alternatively, at least two of DMAA, SMA, and TPT are mixed in one container. The molar ratios of DMAA, SMA, and TPT can be as described above.

[0038] In some embodiments, the kit is suitable for storage at a temperature of 0°C-4°C or at room temperature.

[0039] In some embodiments, the kit further comprises one or more containers containing NAS and / or NSA as anchoring agents.

[0040] In some embodiments, the kit further comprises one or more of the following:

[0041] Containers containing disulfide bond reducing agents (e.g., DTT); containers containing one or more polymerization activators or promoters selected from VA-044, V50, TEMED, ammonium persulfate, and potassium persulfate; containers containing fixatives (e.g., paraformaldehyde); containers containing protein denaturing buffers (e.g., protein denaturing buffers containing SDS); containers containing protein staining solutions (e.g., Coomassie Brilliant Blue staining solution); containers containing enzymes for digesting proteins in a sample; and containers containing one or more buffer solutions.

[0042] In some embodiments, the kit further includes one or more of the following: a gel chamber configured to contain a sample prior to gelation, such as a dish with a cavity or groove; a staining chamber for staining the sample-gel complex, such as Coomassie brilliant blue staining; gel handling tools, such as tweezers or a soft brush; a punch; and a cooling device, such as an ice pack.

[0043] This disclosure also provides kits as described above for the physical expansion of biological samples.

[0044] In one aspect, this disclosure provides a system for spatial proteomics analysis of biological samples, comprising:

[0045] Anchoring module, which performs the anchoring of proteins in biological samples;

[0046] A gel reaction module in which hydrogels are polymerized to form sample-gel complexes;

[0047] The protein denaturation module denatures proteins in the sample-gel complex individually.

[0048] The reduction alkylation module performs reduction and alkylation treatment on the sample-gel complex.

[0049] The staining module allows for the staining of proteins and, optionally, local sampling.

[0050] A secondary gelation module, in which the acquired biological samples are subjected to secondary gel embedding; and

[0051] The mass spectrometry analysis module includes pre-processing of locally sampled samples for mass spectrometry and connection to the mass spectrometry detection device.

[0052] In some implementations, the system further includes a sample pretreatment module in which perfusion and / or fixation of biological samples are performed.

[0053] In some implementations, one or more of the modules described above can be integrated together. In some implementations, the anchoring module and the gel reaction module can be integrated into a single module, thereby enabling simultaneous anchoring and gel polymerization.

[0054] The foregoing and other features and advantages of this disclosure will become more apparent from the following detailed description of several embodiments with reference to the accompanying drawings. Attached Figure Description

[0055] Figure 1 The graph shows a comparison of the sample processing and identification time of second-generation micro-space imaging expanded proteomics with that of first-generation expanded proteomics.

[0056] Figure 2 A schematic diagram of the second-generation microspace imaging expanded proteomics method is shown.

[0057] Figure 3 The results show the number of peptides and proteins identified in 5.9 μL of mouse liver tissue under identical conditions using first-generation expanded proteomics and second-generation micro-space imaging expanded proteomics.

[0058] Figure 4 The secondary embedding technique is illustrated. (a) Experimental procedure for secondary embedding; (b) Comparison of particle size between unembedded and secondary embedded particles; (c) Number of peptides and proteins identified in 3.8 μL of liver tissue under PulseDDA mode analysis (6 sampling points per layer), data are expressed as mean ± SD; (d) Figure 4 (c) Total ion chromatography (TIC) identification chromatogram of the peptide, which shows that the obtained peptide has high density, low impurities, and good distribution.

[0059] Figure 5This demonstrates the high resolution and high protein yield achieved through laser microdissection technology. Above: 200×200μm mouse liver swelling gel. 2 Images before and after the cut region; Below: PulseDDA mode analysis 28*28*30μm 3 57*57*30μm 3 The number of identified proteins, the number of identified peptides, and the missed cleavage rate in mouse liver tissue of different sizes.

[0060] Figure 6 The effects of different fixation methods on protein retention are shown. (a) Cellular structure images of mouse liver after expansion under different fixation conditions. (b) Number of peptides and proteins identified in 5.9 μL of mouse liver tissue analyzed in PulseDDA mode (n = 4 wells), expressed as mean ± SD.

[0061] Figure 7 The number of proteins and peptides identified under different conditions is shown. (a) Effect of destaining treatment conditions on protein and peptide identification; (b) Effect of protein anchoring treatment time on protein and peptide identification; (c) Effect of denaturation temperature on protein and peptide identification.

[0062] Figure 8 The validation of the micro-spatial imaging expansion proteomics analysis method in mouse liver is shown. (a) Overlay comparison of bright-field images of mouse liver tissue sections before expansion (left), after expansion (middle), and before expansion (green pseudocolor) with post-expansion staining (right). Arrows represent deformation vector fields (n=3 tissue sections from one mouse); (b) Root mean square length error of mouse liver sections before and after expansion, experimental images are shown in (a) (n=3 tissue sections from one mouse); mean LEF of liver sections are 5.8, 6.5, and 6.2, respectively; blue line represents the mean; shaded area represents the standard deviation (SD).

[0063] Figure 9 The validation of microspatial imaging expansion proteomics in different tissue types is shown. (a) Mouse heart tissue sections before (left) and after (right) expansion (left, bright field image; right, syprored staining). (b) Mouse kidney tissue sections before (left) and after (right) expansion (left, YFP; right, syprored staining). (c) Paraffin sections of clinical colorectal cancer tissue before (left) and after (right) expansion (left, HE staining; right, syprored staining).

[0064] Figure 10Images of the expanded mouse brain with fluorescent staining are shown. (a) Image of mouse brain slices stained with the fluorescent dye Syprored after expansion; (be) Confocal images of mouse brain regions (anterior commissure, corpus callosum, cortex, and striatum).

[0065] Figure 11 The image shows a fluorescently stained confocal image of the inflated mouse heart. Detailed Implementation

[0066] The expanded proteomics method of this invention enables proteomics processing of samples at the micrometer level. Specifically, the method of this invention has one or more of the following advantages: compatibility with laser microdissection; the use of secondary gel embedding reduces the possibility of sample loss and damage after gel extraction, increases the size of gel particles after extraction, and facilitates the positioning of gel particles during sample processing; and the use of two protein denaturing buffers for sequential treatment achieves expansion of tissues with smaller deformation coefficients, such as the heart, kidneys, and intestines. As shown in the examples, the method of this invention optimizes the processing time, operation steps, proteomics retention effect, and staining effect of each step.

[0067] The disclosures and embodiments described herein should be interpreted as exemplary only and not as limiting the scope of the invention. While specific terminology is used herein, it is used in a general and descriptive sense and not for limiting purposes, unless otherwise stated. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0068] definition

[0069] As used herein, the singular forms “a,” “an,” and “the / that” are intended to also include the plural forms, unless the context clearly indicates otherwise.

[0070] As used herein, the terms “swelling” and “expansion” are used interchangeably and generally refer to expansion or amplification that occurs upon contact with a liquid such as water or other solvents, for example, uniform expansion in three dimensions. Expandable materials are preferably transparent, allowing light to pass through the sample during expansion. As exemplified herein, expandable materials can be hydrogels. In some embodiments, expandable materials are formed by polymerization of their precursors, monomers, or oligomers. For example, monomers may contain water-soluble groups containing polymerizable vinyl-bonded unsaturated groups. Monomers or oligomers may comprise one or more of the following: substituted or unsubstituted methacrylates, acrylates, acrylamides, methacrylamides, vinyl alcohols, vinylamines, allylamines, allyl alcohols, including their diethylene crosslinking agents (e.g., N,N-alkylenebisacrylamide). In some embodiments, expandable materials are polyacrylates and copolymers or crosslinked copolymers thereof. Alternatively, expandable materials may be formed in situ by chemically crosslinking water-soluble oligomers or polymers. In this invention, the addition of a monomer solution of an expandable material to a sample is included. These monomers can undergo in-situ polymerization to form a cross-linked network between the biological sample and the gel, i.e., to form a sample-gel complex.

[0071] As used in this article, the term "expanded proteomics" refers to a method in which biological samples (usually tissue samples) are reversibly anchored into a hydrogel that can expand uniformly, followed by protein denaturation, staining visualization, expansion, sampling, and enzymatic digestion to obtain peptides, for use in proteomics analysis (especially spatial proteomics analysis).

[0072] As used herein, the terms "gel monomer solution" or "gel precursor solution" refer to a solution containing one or more monomers capable of polymerizing to form a gel. The monomer solution is typically followed immediately by the addition of a polymerization activator and / or accelerator, such as VA-044, V50, ammonium persulfate, potassium persulfate, and TEMED, which rapidly polymerizes under the action of the polymerization activator and / or accelerator.

[0073] Expanded proteomics analysis of biological samples

[0074] This invention provides a method for spatial omics analysis of proteins from different spatial regions of a biological sample (e.g., tissue sections). In some embodiments, the method includes adding a hydrogel precursor solution to the sample, forming a sample-gel complex through polymerization, reducing and alkylating the proteins in the sample-gel complex, staining the sample-gel complex, swelling the sample-gel complex, sampling from the swollen sample-gel complex, performing secondary gel embedding on the obtained sample, extracting peptides from the colloidal particles formed by secondary gel embedding, and identifying the proteins. Further, the protein identification includes identifying the peptides extracted from the colloidal particles formed by secondary gel embedding using mass spectrometry (e.g., LC-MS / MS).

[0075] In one aspect, this disclosure provides a method for expanding a protein-containing tissue sample to obtain a micro-tissue sample, wherein a sample-gel complex is formed in a manner that preserves the relative positions of biomolecules (e.g., proteins) in their original functional state. Because the relative positions of the proteins are preserved, spatial information about the proteins at different locations within the sample can be obtained by sampling the gel from different regions and identifying the proteins.

[0076] In some specific implementations, the method of the present invention includes one or more of the following steps:

[0077] 1) Optionally, pretreatment of biological samples may be performed;

[0078] 2) The biological sample is treated with an anchoring agent, a hydrogel monomer solution is added and polymerization occurs to form a sample-gel complex, wherein anchoring and polymerization can be carried out simultaneously or sequentially;

[0079] 3) Denature the proteins in the sample-gel complex;

[0080] 4) Preferably, the sample-gel composite is subjected to reduction and alkylation treatment;

[0081] 5) Stain the sample-gel complex;

[0082] 6) Swell the sample-gel complex;

[0083] 7) Take samples from the expanded sample-gel complex;

[0084] 8) Preferably, the sample is subjected to secondary gel embedding to form a secondary gel network; and

[0085] 9) Extract proteins from samples after secondary gel embedding for proteomics identification.

[0086] The biological sample can be a fixed or unfixed sample, a live sample, or a paraffin-embedded sample. In some embodiments, the sample is an organ or tissue section, and the pretreatment preferably includes perfusion and fixation. In some embodiments, perfusion is performed using a solution containing 1% PFA and 0.1 mg / ml NSA. In some embodiments, fixation is performed using a solution containing 1% PFA and 0.1 mg / ml NSA. In some embodiments, the fixation time is about 1-5 hours. In some embodiments, the sample is a paraffin-embedded section, and the pretreatment includes soaking the paraffin section in xylene or pentane.

[0087] In some embodiments, the staining is Coomassie blue staining or Coomassie brilliant blue staining. In some embodiments, the staining is fluorescent staining.

[0088] In some embodiments, for sample sizes of 1 mm and above, samples can be taken from the expanded sample-gel complex using a punch / sampler. In other embodiments, for sample sizes less than 1 mm, samples can be taken from the expanded sample-gel complex using an LCM.

[0089] For samples obtained by LCM cutting, preferably, the sample is embedded in a secondary gel solution, and a secondary gel network is formed through secondary polymerization. The secondary gel solution is preferably different from the previously used gel monomer solution, and the resulting secondary gel network is different from the sample-gel complex network formed in the previous gelation.

[0090] Optionally, the invention also covers deswelling the sample-gel complex by immersion in a high-salt buffer solution, for example, returning it to its original size. Therefore, the method of the present invention is capable of reversibly adjusting the swelling factor of the sample-gel complex.

[0091] This invention also illustrates chemical anchoring agents or combinations thereof that can be used for protein anchoring, enabling faster penetration of larger biological samples to anchor biomolecules. The methods described herein are applicable not only to cells and thin samples, but also to large or dense samples.

[0092] The hydrogel obtained by the method of the present invention exhibits high mechanical stability after expansion, thereby allowing its application in expanding large tissue blocks, tissue sections, entire organs, and even entire organisms without deformation or mechanical decomposition under its own weight, unlike other hydrogels. In some embodiments, the hydrogel monomer solution or hydrogel formulation forming the hydrogel contains DMAA, SMA, and PAE.

[0093] The method of this invention can be used for proteomics studies (especially spatial proteomics) of different organs and tissues, such as brain mapping, connectomics studies, and new diagnostics, personalized medicine, histopathology, and other medical applications.

[0094] Biological samples used for hydrogel swelling

[0095] Tissue samples used for hydrogel anchoring and swelling provided in this disclosure can be fresh, frozen, or fixed (i.e., preserved). The source of the tissue sample can be solid tissue, such as from fresh, frozen, and / or preserved organs, or tissue samples, biopsy samples, or puncture samples, including preserved tumor samples, such as formalin-fixed, paraffin-embedded, or frozen tumor samples. The tissue sample can be a sample routinely prepared and preserved in daily clinical practice. Fixed samples are those that have undergone histological preservation using fixatives (e.g., formaldehyde, paraformaldehyde). Samples can also be embedded in solid and typically rigid media such as paraffin, wax, collodion, or resin, allowing them to be cut into thin sections for processing.

[0096] Fixation can be performed using conventional methods. Those skilled in the art will understand that the choice of fixative is determined based on the purpose of histological staining or other analysis of the sample. They will also understand that the fixation time depends on the size of the tissue sample and the fixative used. For example, neutral buffered formalin, Bouin, or paraformaldehyde can be used to fix the sample. In some embodiments, perfusion fixation of organ or tissue samples is performed using a solution containing 1% PFA and 0.1 mg / ml NSA.

[0097] In some embodiments, the tissue sample is a thin tissue section (e.g., 5 μm thick), such as a brain section, liver section, or tumor section. In other embodiments, the sample used for expansion is a tissue sample thicker than 400 μm or even an entire organ or part thereof, including but not limited to the brain, spinal cord, heart, lungs, liver, kidneys, stomach, colon, bones, muscles, skin, lymph nodes, genitals, breast, pancreas, prostate, bladder, thyroid, and eyes.

[0098] Anchoring of biological samples

[0099] To form an expandable sample-gel complex, the sample is brought into contact with a chemical anchoring agent or the tissue sample is perfused with an anchoring agent solution and incubated for a sufficient time to allow for a chemical reaction.

[0100] The terms "anchoring agent" and "crosslinking agent" are used interchangeably herein. Chemical anchoring agents are preferably bifunctional connectors comprising reactive chemical groups of biomolecules and reactive chemical groups of the hydrogel. In some embodiments, chemical anchoring agents containing one or more functional groups attach reactive groups to functional groups (e.g., primary amines or thiol groups) of biomolecules within a biological sample, said biomolecules may be proteins, nucleic acids, lipids, proteoglycans, lipopolysaccharides, etc. Since the purpose of this study is proteomics, the anchoring agents used are protein-biased anchoring agents. These anchoring agents functionalize biomolecules, such as proteins, in the sample, thereby covalently anchoring the functionalized biomolecules to the hydrogel network during hydrogel polymerization in a chain reaction with the growth of the hydrogel polymer.

[0101] Chemical anchoring agents are typically provided in the form of anchoring agent solutions. In some embodiments, the anchoring agent solution is prepared by dissolving or diluting the chemical anchoring agent in an aqueous buffer, such as PBS or MES buffer. The anchoring agent solution may also contain a fixative to immobilize the sample simultaneously with anchoring. Those skilled in the art can readily determine the buffer solution containing the anchoring agent solution based on the selected anchoring agent.

[0102] In some embodiments, the chemical anchoring agent comprises two distinct functional groups: one for the functionalization of biomolecules, particularly protein molecules, and a second functional group for reaction with the growing polymer chains. The functionalization of biomolecules can occur during chemical fixation, i.e., on a living or freshly frozen sample, or immediately after chemical fixation on a fixed and preserved sample. As those skilled in the art will understand, the two distinct functional groups of a bifunctional chemical anchoring agent can be separated by chemical spacers (including branched spacers) of various lengths and structures.

[0103] In some embodiments, the biomolecular reactive chemical groups include, but are not limited to, N-hydroxysuccinimide (NHS) esters, epoxy groups, aldehyde groups, and formamide groups, which can react with amino or carboxylic acid groups on proteins, peptides, and / or nucleic acids and / or lipids. Hydrogel reactive groups include, but are not limited to, vinyl, allyl, acrylate, methacrylate, acrylonitrile, and acrylamide groups.

[0104] In some preferred embodiments, the chemical anchoring agent used to directly crosslink the protein with any hydrogel polymer chain is selected from N-succinimidyl acrylate (NSA), N-(allyloxycarbonyloxy)-succinimidyl (NAS), or combinations thereof. Treatment of the amine on the protein with NSA or NAS, which is modified with acrylamide or allyloxycarbonyloxyamide functional groups respectively, allows the functionalized protein to anchor to the hydrogel during polymerization.

[0105] In some embodiments, the chemical anchoring agent comprises NSA. In other embodiments, the anchoring agent comprises NAS. In still other embodiments, the anchoring agent comprises both NAS and NSA, for example, NAS and NSA in a 1:1 molar ratio. The anchoring agent can be diluted in the anchoring buffer at different ratios, such as about 1:10, 1:20, 1:30, 1:40, 1:50, 1:100, 1:150, 1:200, depending on the selected buffer and anchoring agent, the desired reaction rate, and the anchoring effect, etc.

[0106] Hydrogel polymerization / gelation reaction

[0107] As disclosed above, a sample is infiltrated (e.g., perfused, injected, impregnated, added, or otherwise mixed) with a hydrogel monomer solution, or the sample is embedded in the hydrogel monomer solution, and then polymerization occurs to form an expandable sample-gel complex. This complex comprises an expandable polymer network in which protein molecules within the sample (already functionalized by or in the process of being functionalized by an anchoring agent) are covalently bonded to the polymer chains of the hydrogel.

[0108] Hydrogel monomer solutions typically contain one or more hydrogel monomers, oligomers, or precursors dissolved in an aqueous solution (e.g., water or an aqueous solution with added acid to adjust the pH). The terms “monomer” and “precursor” used in relation to hydrogels may be used interchangeably herein to include monomers, comonomers, and crosslinking agents that form the small units of the hydrogel polymer chain. In addition to hydrogel precursors, polymerization activators or accelerators, such as VA-044, V50, APA, potassium persulfate, and TEMED, may be added to the hydrogel monomer solution prior to use. Water or an aqueous solution is typically used as the dispersion medium in the formation of the hydrogel monomer solution, but other solvents may also be used.

[0109] The hydrogel precursor will be polymerized and / or crosslinked with the sample to form a hydrogel-sample complex. In some embodiments, the hydrogel precursor includes, but is not limited to, sodium acrylate (SA), sodium methacrylate (SMA), itaconic acid (IA), trans-aconitic acid (TAA), ethyl-2-(hydroxymethyl)-acrylate (EHA), N,N-methylenebisacrylamide (MBAA, also known as BIS), N,N-dimethylacrylamide (DMAA), pentaerythritol tetraacrylate (PT), propoxylated trimethylolpropane triacrylate (TPT), pentaerythritol triacrylate (PA), dipentaerythritol pentaacrylate, or dipentaerythritol tetraacrylate. One or more of the following reagents are used: DPHA (di(trimethylolpropane)-1,3-di(trimethylolpropane)-1,3-tetraacrylate (DiTA),3-methylolpropane-1,3-methacrylate (TTMA),3-glycerol propoxylated (1PO / OH)-1,3-trimethylolpropane-1,3-ethoxylated (TET),3-pentaerythritol allyl ether (PAE),3-hydroxy-2-methylenebutyrate sodium (SHMB),3-dimethylaminopropylacrylamide (DMPAA), and / or3-acrylamide (AA). All reagents are commercially available, chemically stable, and relatively safe (suitable for routine biological laboratories).

[0110] For example, if a target sample is to be embedded in a sodium polyacrylate hydrogel, a solution containing the monomers sodium acrylate and acrylamide, as well as the anchoring agent N,N-methylenebisacrylamide, is infused throughout the sample. A polymerization accelerator or initiator is added to the solution before infusion, thereby activating the solution to form the polymer of the hydrogel during sample permeation. Preferably, such monomer-containing solutions are aqueous.

[0111] It is desirable for the hydrogel to maintain high mechanical stability in its expanded state to achieve adjustable and reversible expansion. For this purpose, the precursor of the hydrogel may be selected from N,N-dimethylacrylamide (DMAA), sodium methacrylate (SMA), and pentaerythritol allyl ether (PAE), preferably containing all three. Alternatively, the precursor of the hydrogel may be selected from N,N-dimethylacrylamide (DMAA), sodium methacrylate (SMA), and propoxylated trimethylolpropane triacrylate (TPT), preferably containing all three. In some embodiments, the precursor comprises DMAA, SMA, and PAE, wherein the molar ratio of DMAA to SMA is in the range of 30:1 to 3:1, for example, in the range of 20:1 to 3:1, in the range of 15:1 to 3:1, in the range of 10:1 to 3:1, in the range of 9:1 to 3:1, in the range of 8:1 to 3:1, in the range of 7:1 to 3:1, in the range of 6:1 to 3:1, in the range of 5:1 to 3:1, in the range of 4:1 to 3:1, and more specifically, the molar ratio of DMAA:SMA may be about 4:1 or 3:1; and wherein the molar ratio of SMA:PAE is in the range of 10:0.004 to 10:3, for example, in the range of 10:0.05 to 10:3, 10:0.08 to 10:3, 10:0.1 to 10:3, or 10:0.004. 2 to 10:3, 10:0.3 to 10:3, 10:0.4 to 10:3, 10:0.5 to 10:3, 10:0.6 to 10:3, 10:0.7 to 10:3, 10:0.8 to 10:3, 10:0.9 to 10:3, 10:1 to 10:3, 10:1.1 to 10:3, 10:1.2 to 10:3, 10:1.3 to 10:3, 10:1.4 The molar ratio of SMA:PAE can be within the range of 10:3, 10:1.5 to 10:3, 10:2 to 10:3, and more specifically, the molar ratio of SMA:PAE can be approximately 10:0.05, 10:0.08, 10:0.1, 10:0.5, 10:1, 10:1.1, 10:1.2, 10:1.3, 10:1.4, 10:1.5, 10:1.6, 10:1.7, or 10:1.8. In some implementations, PAE can be replaced by TPT.

[0112] Further, in some embodiments, the precursor comprises DMAA, SMA, and PAE, with a DMAA:SMA:PAE molar ratio in the range of (40±20):(10±5):(0.005-2), such as about (40±15):(10±3.5):(0.4±0.2), about (40±10):(10±2.5):(0.4±0.2), about (40±5):(10±2):(0.4±0.2), about (40±5):(10±1.2):(0.4±0.2), and about (40±5):(10±1):(0.4±0.1). In other embodiments, the precursor comprises DMAA, SMA, and PAE, wherein, in molar amounts, if DMAA is 20-60 parts, then SMA is 5 to 15 parts, and PAE is 0.005 to 1.5 parts. Specifically, the molar ratio of DMAA:SMA:PAE can be approximately 30:10:1, 40:10:0.008, 40:10:0.4, 40:10:1, 40:10:1.2, or 40:10:1.5.

[0113] A precursor solution can be prepared by adding water or an aqueous solution (e.g., adding an aqueous solution with an acid to adjust the pH) to the hydrogel precursor or by dissolving it in water or an aqueous solution. For example, the concentration of the hydrogel precursor in the precursor solution can be in the range of 30-60% by mass fraction (w / w), such as 30%, 40%, 50%, or 60%. Stock solutions of the hydrogel monomer solution can be prepared and diluted before use. Stock solutions can have high concentrations (w / w) of the precursor, such as about 50% or higher, about 75% or higher, about 80% or higher, or about 90% or higher. When applied to a sample, the stock solution can be diluted to a concentration of about 30-50%.

[0114] As illustrated in the examples, using DMAA, SMA, and PAE as precursors yields hydrogels exhibiting superior mechanical strength and expansion factor properties. The precursors can be mixed in various proportions to form hydrogels that can tunably and reversibly expand in aqueous buffers to up to 7 times their original size in the linear dimension. The inventors have discovered that DMAA:SMA:PAE can be mixed together in a wide range of proportions and can also form the desired hydrogels.

[0115] The proportion of PAE can be appropriately adjusted according to the mechanical force required for the obtained gel; for example, considering the high temperature and / or high pressure treatment during subsequent protein denaturation, the proportion of PAE may need to be appropriately increased. In one embodiment, DMAA:SMA:PAE is mixed in a molar ratio of 40:10:0.02. In another embodiment, DMAA:SMA:PAE is mixed in a molar ratio of 40:10:0.08. In yet another embodiment, DMAA:SMA:PAE is mixed in a molar ratio of 40:10:0.144. In some embodiments, the molar ratio of DMAA:SMA:PAE is in the range of about 40:10:0.004 to 40:10:0.2. In some embodiments, the molar ratio of DMAA:SMA:PAE is in the range of about 40:10:0.02 to 40:10:0.08. The hydrogel monomer solution can be prepared by mixing DMAA, SMA, and PAE in water. The pH range of the hydrogel monomer solution is preferably 6-7, for example, about 6.5. Hydrogel monomer solutions can be freshly prepared before use, or prepared as a stock solution and diluted before use.

[0116] Before polymerization, polymerization activators and / or accelerators can be added to the hydrogel monomer solution to induce polymerization or gelation. These activators and / or accelerators are, for example, but not limited to, ammonium persulfate, potassium persulfate, TEMED, VA-044, or combinations thereof.

[0117] In some embodiments, the tissue sample is perfused with a solution containing the chemical anchoring agent, hydrogel precursor, and polymerization activator / accelerator described above, i.e., the anchoring and gelation reactions occur simultaneously. Alternatively, the sample to be gelled is treated with the anchoring agent before contact with the hydrogel monomer solution, i.e., anchoring occurs first, followed by the gelation reaction.

[0118] protein denaturation

[0119] In protein molecules, amino acids are linked together by peptide bonds and disulfide bonds to form peptide chains with a certain order. Hydrogen bonds can be formed between amino and acyl groups in the polypeptide chain or between peptide chains, giving the main chain of the polypeptide chain a certain regular conformation, including α-helix, β-sheet, β-turn and Ω-ring, etc. Based on the secondary structure, it further coils and folds to form a complete spatial conformation. Multiple peptide chains then aggregate through non-covalent bonds to form a spatial structure.

[0120] Preferably, the sample-gel complex formed by the gelation reaction is subjected to protein denaturation treatment (also referred to herein as "homogenization"), thereby disrupting the spatial structure of the protein to facilitate subsequent expansion. The spatial structure of native proteins is maintained by secondary bonds such as hydrogen bonds. After denaturation, these secondary bonds are broken, and the protein molecules change from their original ordered, tightly coiled structure to a disordered, loosely extended structure (but the primary structure remains unchanged). This transformation of the protein into its primary structure allows it to expand along with the sample-gel complex during expansion.

[0121] In some embodiments, denaturation or homogenization is achieved by treating the sample with an SDS-based buffer, thereby removing protein-protein interactions through denaturation and mechanically homogenizing the tissue-hydrogel complex. In some embodiments of the invention, after the gelation reaction, the sample-gel complex is subjected to protein denaturation treatment with a surfactant such as sodium dodecyl sulfate (SDS) or sodium dodecylbenzene sulfonate (SDBS).

[0122] In some preferred embodiments, two protein denaturing buffers are used sequentially to denature the sample-gel complex. The inventors discovered that by first using an acidic denaturing buffer to inhibit hydrogel swelling, and then replacing it with a neutral buffer for denaturation, the morphology of the tissue sample can be successfully maintained, achieving swelling with a small deformation coefficient in tissues such as the heart, kidneys, and intestines. The two protein denaturing buffers can use the same or different surfactants, for example, both can be SDS or both can be SDBS. In some embodiments, the first protein denaturing buffer is adjusted to an acidic pH with acid, and the second protein denaturing buffer is adjusted to a neutral pH with alkali.

[0123] In some embodiments, the sample-hydrogel complex is denatured by immersing it in a second protein denaturation buffer at approximately 90°C–110°C, for example, at approximately 95°C, 100°C, or 105°C. The denaturation time varies depending on the sample. For example, for softer tissues such as the brain, liver, and kidneys, denaturation may be performed at 105°C. Further, the denaturation can also be carried out under high pressure, for example, at 105°C for 1–2 hours. High temperature and high pressure conditions have some effect on the mechanical strength of the gel; adjusting the DMAA:SMA:PAE concentration to 4:1:0.0144 increases the mechanical strength of the gel. In other embodiments, the sample-hydrogel complex is denatured by immersing it in a second protein denaturation buffer at 95°C under atmospheric pressure for 12–48 hours. The denaturation time can be adjusted according to the stiffness of the tissue sample.

[0124] This invention also covers deanchoring treatment after protein denaturation, primarily by disrupting / breaking the amide bonds anchoring the protein to the hydrogel network, thereby deanchoring the protein from the sample-gel complex. Deanchoring can be performed by increasing the solution pH to alkaline (e.g., by adding Tris solution), which breaks the amide bonds.

[0125] Reductive alkylation of sample-gel complex

[0126] Pretreatment before enzymatic digestion generally requires reducing disulfide bonds in the protein to disrupt its structure and enhance digestion efficiency. Commonly used reducing agents include dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP). To prevent the free thiol groups after reduction from reforming disulfide bonds, alkylating agents can be used to block the thiol groups. Commonly used alkylating agents include iodoacetamide (IAA), chloroacetamide, iodoacetic acid, and N-ethylmaleimide.

[0127] In some implementations, the sample-gel complex is reductively alkylated by first reducing it with DTT and then treating it with IAA, before denaturation and staining. The reducing agent is not limited to DTT, but can also be, for example, TCEP or β-thioethanol (β-ME). Unlike β-ME and DTT, TCEP is odorless, selective, can operate in both alkaline and acidic environments (unlike DTT), is more hydrophilic, and resistant to oxidation. Compared to TCEP, DTT treatment has less impact on the sample hydrogel complex, resulting in a smoother gel and better protein identification.

[0128] In some embodiments, the biosample-gel complex is incubated with a reducing agent for a sufficient time to perform disulfide bond reduction, wherein the reducing agent is selected from TCEP, β-ME, and DTT. Further, the biosample-gel complex is incubated with an alkylating agent for a period of time to block thiol groups, wherein the alkylating agent is selected from iodoacetamide (IAA), chloroacetamide, iodoacetic acid, and N-ethylmaleimide.

[0129] Swelling of sample-gel complex

[0130] In some implementations, after forming the sample-gel complex, the proteins are denatured and then visualized by protein staining (typically Coomassie blue or fluorescent staining). A solvent or liquid is added to the sample-gel complex, which is then absorbed and causes swelling. When the sample-gel complex is water-swellable, an aqueous solution can be used. Because the polymer embeds the entire tissue sample, it expands the sample as the polymer swells (grows). Therefore, the tissue sample itself becomes larger. For example, the volume of the complex can expand approximately 10-fold after incubation in pure water or an aqueous buffer, greatly improving the ability to use micro-sampling for protein identification. Different sample swelling factors can be obtained as needed by adjusting the incubation time, the molar osmotic pressure concentration of the aqueous buffer, temperature, etc.

[0131] The expansion of the molecular chains of the expandable polymer that runs through the sample separates the biomolecules, making the tissue sample itself larger. Importantly, the relative positions of the biomolecules remain unchanged after expansion. As the material expands isotropically, the staining markers of proteins maintain their relative spatial relationships. Once expanded, the expression and / or location of one or more proteins in the tissue can be detected. As shown in the examples, visualized tissue samples with a linear expansion of approximately 6 times are obtained, and tiny tissue samples with an original diameter of 500 micrometers can be obtained using a millimeter-scale punch, thus greatly expanding the precision of tissue sampling.

[0132] In some embodiments, the methods disclosed herein may include different combinations of anchoring, gelation, and staining processes. In some embodiments, the addition of pure water or other aqueous buffers allows the sample-gel complex to expand linearly from the initial sample size by up to approximately 5-8 times while maintaining high mechanical stability and elasticity. Both mechanical stability and elasticity allow for easy handling of the expanded sample without mechanically deforming the sample hydrogel complex, thus preserving its integrity.

[0133] Secondary encapsulation

[0134] The inventors have discovered that re-gel polymerization of small samples cut from sample-gel complexes to form secondary gel networks offers several advantages. Traditional sample processing methods involve in-gel digestion and peptide extraction of the gel cut from the sampler. However, firstly, the addition of acetonitrile causes gel particles to dehydrate and shrink to a size imperceptible to the naked eye, potentially leading to sample loss during experimental processing. Secondly, the mechanical nature of primary gels often results in breakage during sample handling. Broken gel fragments can clog NanoLC columns when loaded into an LC-MS instrument. Finally, for high-resolution (less than 1 mm) micro-samples, visual processing of these small samples is difficult. Secondary gel embedding effectively addresses these drawbacks and brings several improvements.

[0135] In some embodiments, the secondary gel embedding step includes embedding the sample slit from the sample-hydrogel complex into a second gel solution. In some embodiments, the second gel solution is an acrylamide / methylenebisacrylamide solution, such as 30%, 25%, 20%, or 15% acrylamide / methylenebisacrylamide solution. In some embodiments, the second gel solution is prepared by mixing 30% acrylamide / methylenebisacrylamide solution with water at a volume ratio ranging from 10:5.5 to 12:4.5, for example, at a volume ratio of 11:5. In some embodiments, the resulting secondary gel is a polyacrylamide gel. The second gel has better mechanical properties, preventing breakage of the gel mass during sample processing. By using secondary embedding, the volume of the gel particles increases to a visible size, approximately 5 mm in diameter, which facilitates the positioning of the gel particles during sample processing. This secondary gel embedding technique enables successful processing of micron-sized samples, is cost-effective, and allows for proteomics processing of micron-sized samples without requiring high-precision instrumentation.

[0136] Furthermore, this invention tested the compatibility of the micro-space imaging expanded proteomics technique with laser microdissection. For sampling of samples smaller than 1 mm, laser microdissection can be used for precise measurement. Combining secondary embedding gel technology and laser microdissection, micro-scale visualized expanded proteomics can be achieved at a size of 57*57*30 μm. 3 909 specific proteins were identified in mouse liver tissue. Figure 5 The protein identification results demonstrate the applicability and effectiveness of this method for trace samples.

[0137] Mass spectrometry analysis

[0138] Traditional proteomics identification methods involve one of the following steps: microextraction from the tissue surface using aqueous or organic solvents, tissue homogenization of multiple adjacent tissue regions, laser capture microdissection (LCM) collection of the target region, or high-performance liquid chromatography (HPLC) fractionation of a large column, followed by MS analysis of the fractions to determine which fractions contain the target protein.

[0139] In some implementations, protein identification is performed using high-performance liquid chromatography-mass spectrometry (HPLC-MS). Liquid chromatography (LC) effectively separates organic components in a sample, while mass spectrometry (MS) analyzes each separated organic compound individually, providing information on its molecular weight, structure, and concentration. HPLC-MS is a commonly used technique in the art for analyzing and determining high molecular weight compounds, such as proteins and polymers.

[0140] Matching mass spectrometry data with protein databases is central to proteomics identification. Currently, most proteomics studies utilize trypsin, which specifically cleaves the carboxyl termini of lysine and arginine residues. Although trypsin exhibits high cleavage specificity, missed cleavage is not uncommon in protein identification. Therefore, analyzing the missed cleavage rate can assess the effectiveness of different biological sample processing conditions.

[0141] Reagent test kit

[0142] This disclosure also provides a kit for practicing the methods described above. The kit may contain, in one or more containers, a precursor for forming a hydrogel as described above, a gel solution for secondary gel embedding, and / or an anchoring agent, and optionally, a protein disulfide bond reducing agent, a protein denaturing agent, a staining solution, various buffers, etc. Preferably, the precursors are DMAA, SMA, and PAE / TPT. The precursors may be present in separate containers, for example, DMAA in a first container (e.g., in solid or solution form), SMA in a second container (e.g., in solid or solution form), and PAE / TPT in a third container (e.g., in solid or solution form), and are mixed and prepared prior to use.

[0143] Alternatively, any of the following configurations may be used: DMAA and SMA are mixed in a suitable ratio in a first container, while PAE / TPT is present in a second container; DMAA and PAE / TPT are mixed in a suitable ratio in a first container, while SMA is present in a second container; or SMA and PAE / TPT are mixed in a suitable ratio in a first container, while DMAA is present in a second container. These containers may be combined or completely separate.

[0144] In some other embodiments, the kit may comprise a container containing a mixture of DMAA, SMA, and PAE / TPT, wherein the molar ratio of DMAA to SMA is in the range of 30:1 to 3:1, for example, in the range of 20:1 to 3:1, in the range of 15:1 to 3:1, in the range of 10:1 to 3:1, in the range of 9:1 to 3:1, in the range of 8:1 to 3:1, in the range of 7:1 to 3:1, in the range of 6:1 to 3:1, in the range of 5:1 to 3:1, in the range of 4:1 to 3:1, and more specifically, the molar ratio of DMAA:SMA may be about 4:1 or 3:1; and wherein the molar ratio of SMA:PAE / TPT is in the range of 10:0.005 to 10:3, for example, in the range of 10:0.05 to 10:3, 10:0.08 to 10:3, 10:0.1 to... 10:3, 10:0.2 to 10:3, 10:0.3 to 10:3, 10:0.4 to 10:3, 10:0.5 to 10:3, 10:0.6 to 10:3, 10:0.7 to 10:3, 10:0.8 to 10:3, 10:0.9 to 10:3, 10:1 to 10:3, 10:1.1 to 10:3, 10:1.2 to 10:3, 10:1.3 to 10:3, 10: The molar ratio of SMA:PAE / TPT can be in the range of 1.4 to 10:3, 10:1.5 to 10:3, 10:2 to 10:3, and more specifically, the molar ratio of SMA:PAE / TPT can be approximately 10:0.05, 10:0.08, 10:0.1, 10:0.5, 10:1, 10:1.1, 10:1.2, 10:1.3, 10:1.4, 10:1.5, 10:1.6, 10:1.7, or 10:1.8.

[0145] Furthermore, in some embodiments, the precursor comprises DMAA, SMA, and PAE / TPT, wherein the molar ratio of DMAA:SMA:PAE / TPT is in the range of (40±20):(10±5):(0.005-2), such as about (40±15):(10±3.5):(0.4±0.2), about (40±10):(10±2.5):(0.4±0.2), about (40±5):(10±2):(0.4±0.2), about (40±5):(10±1.2):(0.4±0.2), and about (40±5):(10±1):(0.4±0.1). In other embodiments, the precursor comprises DMAA, SMA, and PAE / TPT, wherein, in molar quantities, if DMAA is 30-50 parts, then SMA is 5 to 15 parts, and PAE / TPT is 0.005 to 1.5 parts. Specifically, the molar ratio of DMAA:SMA:PAE / TPT can be approximately 30:10:1, 40:10:0.08, 40:10:0.4, 40:10:1, 40:10:1.2, or 40:10:1.5.

[0146] The kit may also include a container with an anchoring agent, which may be NAS or NSA. The kit may also include a container with a polymerization activator or accelerator for addition to the hydrogel precursor prior to polymerization. The polymerization activator or accelerator may be selected from, for example, VA-044, TEMED, potassium persulfate, and APS.

[0147] The kit may also contain one or more of the following: a container containing a disulfide bond reducing agent (e.g., DTT); a container containing an alkylating agent (e.g., iodoacetamide); a container containing a fixative (e.g., paraformaldehyde); a container containing a protein denaturing buffer (e.g., SDS or SDBS); a container containing a staining agent (e.g., Coomassie blue staining solution or fluorescent staining agent); and a container containing an enzyme for digesting proteins in the sample. Optionally, the kit may also contain several common buffers to facilitate the execution of the methods of the present invention.

[0148] The term "container" should be understood to refer to any structure that can contain or surround components of a hydrogel formulation, anchoring agent, etc.; exemplary containers include bottles (e.g., plastic or glass bottles), syringes, vials, pouches, capsules, ampoules, cartridges, etc. Containers can be protected from visible light, ultraviolet, or infrared radiation by using other components (e.g., an aluminum foil pouch surrounding a vial) or by selecting the material properties of the container itself (e.g., an amber glass vial or an opaque plastic bottle).

[0149] The kit may also include a mixing device for mixing precursors together to produce the hydrogel formulation of the present invention. The kit may also include gel handling devices, such as soft brushes, tweezers, and / or delivery devices (which may or may not include a mixing element), for infusing the hydrogel solution onto a sample or transferring the gel.

[0150] In some implementations, the kit includes one or more of the following components: a gel chamber configured to hold a sample prior to gelation, such as a dish with a cavity or groove; gel manipulation tools, such as tweezers or a soft brush; a punch; a cooling device, such as an ice pack; multiple Eppendorf tubes, a centrifuge column, etc.

[0151] The kit may also include other components, such as desiccants or other means of maintaining water content control in the kit, indicators to indicate the temperature of the kit, etc., which may be necessary to keep the kit in good condition during transportation and storage.

[0152] In some embodiments, the kit includes a gelation chamber for forming the sample-gel complex as described above. The chamber is configured to include a cavity, or aperture, for containing a sample prior to gelation, such as in the form of a dish, like a MatTek dish. The gelation chamber may also include a cover (e.g., a coverslip) covering the cavity, or aperture.

[0153] In some embodiments, the kit also includes an expansion chamber configured to contain the sample after gelation. The kit may include multiple containers packaged together for ease of use.

[0154] In addition to the ingredients and components described above, the kit typically includes instructions for carrying out the methods of the invention using the kit's ingredients. These instructions for carrying out the methods of the invention are usually recorded on a suitable recording medium. For example, the instructions may be printed on a material such as paper or plastic. Thus, the instructions may exist as a packaging insert in the kit, or on the label of a container of the kit or its components. In other embodiments, the instructions are stored as electronic storage data files on a suitable computer-readable storage medium; for example, in other embodiments, there are no actual instructions in the kit, but rather the instructions are obtained from a remote source (e.g., an optical disc). An example of this embodiment is a kit that includes a website address at which the instructions can be viewed and / or downloaded. Like the instructions, the means for obtaining the instructions are recorded on a suitable material.

[0155] The ProteomEx 2.0 method provided by this invention offers several advantages, including:

[0156] 1. The method of this invention incorporates a secondary gel embedding step. By using secondary embedding, the volume of the gel particles increases to a visible size, approximately 5 mm in diameter, which facilitates the positioning of the gel particles during sample processing. This secondary gel embedding technique enables successful processing of tiny samples, is economical, and allows for proteomics processing of micron-sized samples without requiring high-precision instruments.

[0157] 2. The method of this invention can apply reductive and alkylation to the pretreatment of whole hydrogel samples. By performing protein reductive and alkylation treatment on the whole hydrogel (containing the sample) before staining, the overall operation process is simplified and made more efficient. Previous expansion proteomics treatments involved performing individual reductive alkylation treatment on each sample after cutting and sampling. This invention performs reductive alkylation treatment on the whole hydrogel, which greatly improves the efficiency of sample processing and shortens the time.

[0158] 3. The method of this invention can combine the entire technical process with fluorescence staining imaging. The method of this invention can be easily combined with fluorescence staining imaging, providing clearer, higher-resolution fluorescence imaging without affecting proteomics identification.

[0159] 4. The method of this invention combines technical processes with laser capture microdissection (LCM). For the precise cutting and sampling of micro-samples smaller than 1 mm, laser microdissection technology can be used, enabling effective identification of micron-sized samples.

[0160] 5. The entire technical process has been optimized compared to first-generation expanded proteomics, including processing time, operational steps, proteome retention, and staining effects. Denaturation conditions have been optimized, and the workflow is applicable to hard samples such as hearts and kidneys.

[0161] 6. Immobilization Method. The sample immobilization method has been improved, and the optimized technique is also applicable to tissue samples with high rigidity, such as those from the heart and kidneys. Paraformaldehyde and protein-anchoring molecules (N-succinimide acrylate) are added during tissue sample immobilization. While maintaining the immobilization effect, protein retention is greatly improved, achieving higher and better proteomics detection depth and coverage.

[0162] The following examples are provided to better illustrate the claimed invention and are not to be construed as limiting the scope of the invention. All the specific compositions, materials, and methods described below fall in whole or in part within the scope of the invention. These specific compositions, materials, and methods are not intended to limit the invention, but merely illustrative of specific embodiments falling within the scope of the invention.

[0163] Preparation of reagents and materials for the example

[0164] Table 1: Solution preparation and reagent preparation

[0165]

[0166]

[0167] Example 1: Sample-gel swelling sampling and mass spectrometry analysis

[0168] 1. Irrigation fixation

[0169] Mice were first perfused with 1xPBS, then perfused with 1% paraformaldehyde (PFA) + 0.1 mg / ml N-succinimide acrylate (NSA) solution. Brain, liver, kidney, and heart samples were collected separately. The specific organs were then fixed by immersion in 1% paraformaldehyde (PFA) + 0.1 mg / ml N-succinimide acrylate (NSA) solution for 1-5 hours. The samples were then prepared into tissue sections.

[0170] 2. Dewaxing (only for paraffin-embedded sections)

[0171] Soak paraffin sections in xylene or pentane for 5 minutes, repeating once. Elute sequentially with a gradient of ethanol, repeating each gradient twice (100%, 95%, 75%, 50%). Finally, wash twice with ultrapure water.

[0172] 3. Protein anchoring

[0173] Use a small brush to transfer the sample into a 24-well plate, with 500 μL of PBS solution added to each well beforehand. Remove the PBS solution and wash the sample three times with 100 mM MES buffer (pH 6.0).

[0174] Incubate with 200 μL of protein anchoring solution at room temperature for 6 hours.

[0175] 4. Gel-forming reaction

[0176] The anchoring reaction was terminated by rinsing three times with 200 μL of 100 mmol MOPS (3-morpholinopropanesulfonic acid) buffer, pH 7.0. The sample was incubated at 4°C for 6 hours or more with 200 μL of activated monomer solution. The tissue section was then laid flat in the center of a custom-made, well-deep glass slide using a soft brush and allowed to air dry, ensuring the tissue section adhered tightly to the bottom of the well. Approximately 100 μL of activated monomer solution was added to the well of the slide, and the tissue section in the well was covered with a custom-made coverslip, leaving a small gap to prevent air bubbles from forming.

[0177] Place the glass slide into the humidified chamber, then place the humidified chamber into a vacuum drying oven preheated to 37°C. Use a vacuum pump to evacuate the vacuum drying oven to a semi-vacuum, then purge with nitrogen to normal atmospheric pressure. Repeat this process four times to increase the nitrogen ratio. Allow the free radical polymerization reaction to proceed for 3.5 hours to fully polymerize the activated monomer solution into a hydrogel, and encapsulate the sample inside the hydrogel.

[0178] 5. Protein denaturation:

[0179] Remove the humidified chamber from the vacuum drying oven and take out the glass slide. Add approximately 100 μL of protein denaturing buffer 1 around the sample, let stand for 3 minutes, and gently pry open the coverslip with a blade. Add approximately 200 μL of protein denaturing buffer 1, let stand for 10 minutes to allow the hydrogel to fully absorb water and detach. Trim off any excess hydrogel from the slide with a knife. Immerse the hydrogel containing the sample in protein denaturing buffer 2 and place it in a 95°C incubator (12 hours for brain samples, 16 hours for liver samples, 36 hours for kidney samples, and 48 hours for heart samples) or a 105°C autoclave (1 hour for brain samples, 1.5 hours for liver samples, and 2 hours for kidney samples).

[0180] 6. Reductive alkylation

[0181] Remove the denaturing hydrogel and immerse it in 100 mmol Tris (pH 8.0) buffer for 10 minutes, repeating this process three times to remove SDS. Place the hydrogel in 20 mmol DTT solution, shake well, and react for half an hour to perform whole-gel reduction treatment. Remove the DTT solution and add 55 mmol iodoacetamide (IAA) solution to submerge the hydrogel block. React in the dark for half an hour, then remove the IAA solution and immerse the hydrogel block in 100 mmol Tris (pH 8.0) buffer for 10 minutes, repeating this process three times.

[0182] 7. Staining

[0183] A. Coomassie Brilliant Blue Staining: Immerse the hydrogel in a 100 mmol Tris (pH 8.0) Coomassie Brilliant Blue solution and incubate at 95°C for 2 hours.

[0184] B. Fluorescent staining: Immerse the hydrogel in SYPRO series dye diluted 1:1000 and allow it to react for 1 hour in a shaker under dark conditions.

[0185] 8. Expansion

[0186] The stained hydrogel was immersed in 100 mmol Tris (pH 7.4) buffer and rinsed three times. The hydrogel was then immersed in a series of Tris (pH 7.4) buffer concentrations for approximately 15 minutes each (50 mmol, 10 mmol, 5 mmol, 1 mmol, 0.5 mmol). The staining of the hydrogel after swelling was photographed and recorded.

[0187] 9. Sampling and cutting

[0188] A. Sample taking (for sample sizes of 1 mm and above): Select the target area under bright field (for Coomassie blue staining) or fluorescence microscopy (for fluorescence staining), and use a sampler of a specific size to punch holes in the gel to collect the sample. Collect the cut gel pieces in 200 μL PCR tubes.

[0189] B. LCM Sampling (for samples smaller than 1 mm): At room temperature, apply the hydrogel onto a specially designed LCM slide and allow it to dry overnight. Place the LCM slide containing the fully dried hydrogel on the LCM machine and select the target area for cutting under a microscope. Attach the cut sample to a specially designed LCM collection tube.

[0190] 10. Secondary embedding

[0191] For samples cut by the sampler, drop freshly prepared secondary gel solution onto the bottom of the PCR tube to coat the gel block. For samples cut by the LCM, drop freshly prepared secondary gel solution onto the center of the collection tube to coat the cut sample. Place the reaction tube at 37°C and react for 15 minutes to form a secondary gel network.

[0192] 11. Enzymatic extraction of protein

[0193] A. Washing the gel block: Add 200 μL of 100 mmol ammonium bicarbonate (ABB) buffer solution, react in a constant temperature shaker at 600 rpm and 20°C for 15 minutes, discard the solution, and repeat this step twice.

[0194] B. Coomassie / fluorescent staining elution: Add 200 μL of 50% / 50% (v / v) acetonitrile / 100 mM ammonium bicarbonate buffer to elute Coomassie stain. In a constant-temperature shaker, incubate at 60°C and 600 rpm for 15 minutes, discard the solution, and repeat once.

[0195] C. Acetonitrile dehydration: Add 200 μL of acetonitrile and mix well in a constant temperature shaker at 600 rpm and 37°C for 15 minutes each time. Repeat until the gel turns into white granules.

[0196] D. Enzymatic digestion: Gently transfer the white particles to a 1.5 mL test tube. Ensure the gel is sufficiently dry. React on ice, adding 10 μL of working concentration trypsin solution to immerse the gel particles in the trypsin solution. Incubate on ice for at least 2 hours to allow the gel to fully soak in the trypsin solution. Add 5 μL of 25 mmol ammonium bicarbonate solution to keep the gel moist. Invert the EP tube containing the gel particles and place it in a 37°C incubator, then place the gel particles in digestion buffer for 12 hours.

[0197] E. Peptide Extraction: Transfer the remaining digestion buffer from the collection tube to a new EP collection tube. Add 100 μL of 100 mM ammonium bicarbonate buffer to the tube containing the gel, and shake at 600 rpm for 20 minutes at 37°C. Transfer the supernatant to the peptide collection tube. Add 100 μL of 10% / 90% (v / v) acetonitrile / 100 mM ammonium bicarbonate buffer, and shake at 600 rpm for 20 minutes at 37°C. Transfer the supernatant to the peptide collection tube. Add 100 μL of 50% / 50% (v / v) acetonitrile / 5% formic acid buffer, and shake at 600 rpm for 20 minutes at 37°C. Transfer the supernatant to the peptide collection tube. Add 100 μL of 70% / 30% (v / v) acetonitrile / 5% formic acid buffer and shake at 600 rpm for 20 minutes at 37°C. Transfer the supernatant to a peptide collection tube. Add 150 μL of acetonitrile and shake at 600 rpm for 20 minutes at 37°C. Transfer the supernatant to a peptide collection tube. Vacuum-dry all peptide collection solutions and store at -20°C. Resuspend in MS buffer and desalt.

[0198] 12. Desalination

[0199] Place the Micro Spin column in a new long EP tube, wash the Micro Spin column with 20 μl MeOH by centrifugation, then place it in a new long EP tube, wash with 20 μl 80% ACN and 0.1% TFA by centrifugation, equilibrate with 20 μl 2% ACN, load with 30 μl peptide solvent, and centrifuge. Wash with 20 μl 2% ACN and centrifuge.

[0200] Elution: Replace the cup (with final label) and elute twice with 30 μl of 40% ACN and 0.1% TFA, centrifuging at 1200 rpm for 5 minutes. Dry the sample in a SpeedVac. Centrifuge at 45°C and 6–7 mbar for 4 hours.

[0201] 13. Redissolve

[0202] The peptide was redissolved in 10 μL MS buffer (2% ACN, 0.1% FA) and then centrifuged. The supernatant was then transferred to a tube for mass spectrometry analysis.

[0203] 14. Data Acquisition

[0204] 8 μL (8 / 10) of peptide was injected into HPLC-MS / MS (timsTOF) over 90 minutes.

[0205] HPLC settings

[0206] Buffer A: 100% H2O, 0.1% FA

[0207] Buffer B: 100% ACN, 0.1% FA

[0208] Trap column: Thermo, 3um

[0209] Mass spectrometry column: BPRC, 1.9 μm, 15 cm

[0210] Flow rate: 0.3 μL / min

[0211] Gradient: 5%-27% Buffer B (0-80 min), 27%-40% Buffer B (80-90 min), 40%-80% Buffer B (90-92 min), 80%-80% Buffer B (92-95 min).

[0212] 15. Data Query

[0213] The obtained peptide samples were acquired via data-dependent acquisition (DDA) using HPLC-MS / MS. Finally, the proteomics data were analyzed using MSFragger software to determine the number of proteins identified.

[0214] result

[0215] like Figure 1 As shown, based on the method of ProteomEx 1.0, the method in this application further optimizes the entire experimental workflow of expanded proteomics, shortening the working time; the entire workflow takes approximately 41 hours. Figure 3 The results showed that, when using the ProteomEx2.0 method and the ProteomEx1.0 method respectively to determine and analyze peptides and proteins in 5.9 μL of mouse liver tissue under the same conditions (same sampling area size, tissue thickness, data acquisition mode (DDA), tissue type, and expansion coefficient), the number of proteins identified by the ProteomEx2.0 method was significantly higher than that of the ProteomEx1.0 method.

[0216] Secondary encapsulation

[0217] The method of this invention employs a secondary embedding step (step 10) after sampling and cutting. Traditional sample processing methods involve in-gel digestion and peptide extraction of the gel cut from the sampler. However, this method has several drawbacks. First, after adding acetonitrile, the gel particles dehydrate and shrink to a size difficult to observe with the naked eye, which may cause sample loss during experimental processing. Second, the mechanical nature of the primary gel often leads to easy breakage during sample processing. Broken gel fragments can cause blockage of the NanoLC column when loaded into the LC-MS instrument. Finally, for high-resolution (less than 1 mm) micro-samples, it is difficult to visually process these tiny samples. To address these problems, we have developed a new sample processing method called secondary gel embedding technology. Secondary gel embedding overcomes the limitations of previous techniques and brings several improvements. Secondary gel embedding involves embedding a first gel (containing the protein sample) into a second gel (polyacrylamide gel). The second gel has better mechanical properties, preventing gel fragment breakage during sample processing. By using secondary embedding, the volume of the gel particles is increased to a visible size, approximately 5 millimeters in diameter, which facilitates the positioning of the gel particles during sample processing. This secondary gel embedding technique enables successful processing of tiny samples, is cost-effective, and allows for proteomics processing of micron-sized samples without requiring high-precision instruments.

[0218] Furthermore, this invention tested the compatibility of the micro-space imaging expanded proteomics technique with laser microdissection. For sampling of samples smaller than 1 mm, laser microdissection can be used for precise measurement. Combining secondary embedding gel technology and laser microdissection, micro-scale visualized expanded proteomics can be achieved at a size of 57*57*30 μm. 3 909 specific proteins were identified in mouse liver tissue. Figure 5 The protein identification results demonstrate the applicability and effectiveness of this method for trace samples.

[0219] The effects of using or not using secondary embedding on the number of proteins identified were compared, and the results are as follows: Figure 4 As shown, the gel particles without secondary embedding are smaller than those with secondary embedding.

[0220] Figure 5 The analysis of 28*28*30μm in PulseDDA mode is shown. 3 57*57*30μm 3 Results of the number of identified proteins, the number of identified peptides, and the missed cleavage rate in mouse liver tissue of this size. The missed cleavage rate was less than 10%, compared to approximately 20% using the ProteomEx 1.0 method.

[0221] Irrigation fixation

[0222] As shown in this embodiment, the tissue fixation conditions were optimized by adding paraformaldehyde (PFA) and protein anchoring molecules (NSA) while fixing the tissue sample. This significantly improved protein retention while maintaining the fixation effect, resulting in higher and better proteomics detection depth and coverage.

[0223] The effects of using different fixation solutions in step 1 on the number of proteins identified were compared. For example... Figure 6 As shown, the results were compared with those of other methods that were the same except for the methods that used 1% PFA, 1% PFA + 0.1 mg / ml NSA, and 4% PFA for sample perfusion fixation. The number of proteins and peptides obtained by perfusion fixation with the mixed solution of 1% PFA + 0.1 mg / ml NSA was significantly higher than that of the other two fixation solutions.

[0224] protein anchoring

[0225] The effect of different protein anchoring times in step 3 on the number of proteins identified was compared. For example... Figure 7 As shown in b, the results were compared between methods with the same steps but different protein anchoring times of 4 hours and 6 hours. The number of proteins and peptides identified with a protein anchoring time of 6 hours was significantly higher than that with a protein anchoring time of 4 hours, indicating that appropriately increasing the anchoring time is beneficial for obtaining a more comprehensive proteome.

[0226] protein denaturation

[0227] The effect of different protein denaturation temperatures in step 5 on the number of proteins identified was compared. Figure 7 As shown in c, the results of methods with the same steps but different protein denaturation temperatures of 95℃ and 105℃ were compared. There was no significant difference in the number of proteins and peptides identified by the two denaturation temperatures.

[0228] Furthermore, this embodiment used two different protein denaturation buffers to optimize the protein denaturation conditions, achieving high-quality preservation of expanded proteins in samples with high mechanical stress, such as the heart and kidneys. By first using an acidic denaturation buffer to inhibit hydrogel swelling, and then replacing it with a neutral buffer to denature the tissue samples, expansion with a smaller deformation coefficient was successfully achieved in tissues such as the heart, kidneys, and intestines.

[0229] Reductive alkylation

[0230] This invention innovatively applies reductive and alkylation to the pretreatment of the entire hydrogel sample. As described in step 6 above, the entire hydrogel (containing the sample) is subjected to protein reductive and alkylation treatment before staining, simplifying and improving the overall operation process. Previous ProteomEx 1.0 expansion proteomics processing involved performing individual reductive alkylation on each sample after cutting and sampling. This invention performs reductive alkylation on the entire hydrogel, significantly improving sample processing efficiency and reducing time.

[0231] dyeing

[0232] In addition to Coomassie blue staining, this invention can also be applied to combining expanded proteomics with fluorescent dyes. In step 7, fluorescent protein dyes are applied to the expanded samples, and the staining efficiency and specificity are verified. Figure 9-11 This fluorescent staining method enables higher-resolution imaging of the target sample, thereby improving the precision of microdissection using a fluorescence image-guided microdissection system. The selected fluorescent dye does not affect mass spectrometry analysis and allows for super-resolution images of 70 nm under diffraction-limited microscopy. By optimizing the dissection process settings and combining them with fluorescence image guidance, this invention successfully achieves micro-space imaging proteomics, which allows for precise and targeted micro-space proteomics analysis in expanded tissue samples.

[0233] Destaining

[0234] The effects of using different solutions to elute Coomassie blue staining in step 11 on the number of proteins identified were compared. Figure 7 As shown in a, the results showed that the number of proteins and peptides identified by the two solutions after elution were comparable, with no significant difference. The other steps were the same, except that the staining of the gel block was eluted with either 50% / 50% volume ratio acetonitrile / 100 mmol ammonium bicarbonate buffer or 50% / 50% volume ratio methanol / 100 mmol ammonium bicarbonate buffer.

[0235] In summary, this application employed a novel secondary gel embedding technique and explored various conditions, including whole-sheet reduction alkylation, fixation of different samples, protein denaturation, staining, and destaining. This new method (ProteomEx2.0) not only reduces the time required and identifies a significantly higher number of proteins than ProteomEx1.0, but also significantly reduces the rate of missed cleavage.

Claims

1. A method for processing biological samples for protein identification, the method comprising: (a) The biological sample is treated with an anchoring agent; (b) Add hydrogel monomer solution to form sample-hydrogel composite through polymerization of hydrogel monomer solution; (c) Denature the protein in the sample-hydrogel complex; (d) Perform protein reduction alkylation treatment on the sample-hydrogel composite. (e) Staining the sample-hydrogel complex. (f) Swell the sample-hydrogel composite; (g) Cutting samples from the expanded sample-hydrogel composite, embedding the cut samples with a secondary gel solution to form a secondary gel-embedded sample; and (h) Peptides are extracted from the secondary gel-embedded samples for protein identification.

2. The method of claim 1, wherein the biological sample is a specimen, tissue section, biopsy sample, organ or part thereof, or whole organism.

3. The method of claim 2, wherein the biological sample is a liver tissue section, a heart tissue section, a kidney tissue section, a brain section, or a cancer tissue section.

4. The method of claim 1, wherein adding the hydrogel monomer solution in step (b) comprises perfusing the biological sample with the hydrogel monomer solution or embedding the biological sample in the hydrogel monomer solution.

5. The method according to any one of claims 1-4, wherein steps (a) and (b) are performed simultaneously.

6. The method of claim 5, wherein an anchoring agent is added to the hydrogel monomer solution to simultaneously perform anchoring and polymerization.

7. The method according to any one of claims 1-4, wherein the hydrogel monomer solution comprises one or more monomers capable of polymerizing to form a hydrogel.

8. The method of claim 7, wherein the monomer comprises sodium methacrylate (SMA), N,N-dimethylacrylamide (DMAA), and pentaerythritol allyl ether (PAE).

9. The method of claim 8, wherein the molar ratio of DMAA to SMA is in the range of 30:1 to 3:1, and the molar ratio of SMA:PAE is in the range of 10:0.004 to 10:

3.

10. The method according to claim 8 or 9, wherein the molar ratio of DMAA:SMA:PAE is (30-50):(5-15):(0.004-2).

11. The method of claim 10, wherein the molar ratio of DMAA:SMA:PAE is from 40:10:0.02 to 40:10:0.

2.

12. The method according to any one of claims 1-4, wherein a polymerization activator and / or accelerator are added thereto before using the hydrogel monomer solution.

13. The method of claim 12, wherein the polymerization activator and / or accelerator is selected from VA-044, V50, ammonium persulfate, potassium persulfate, and TEMED.

14. The method according to any one of claims 1-4, wherein the secondary adhesive solution is a solution of 30% acrylamide / methylenebisacrylamide solution diluted in water.

15. The method of claim 14, wherein the secondary adhesive solution is a solution of mixing 30% acrylamide / methylenebisacrylamide solution with water in a volume ratio ranging from 10:5.5 to 12:4.

5.

16. The method of claim 15, wherein the 30% acrylamide / methylenebisacrylamide solution is mixed with water at a volume ratio of 11:

5.

17. The method of claim 14, wherein a polymerization activator and / or accelerator are added thereto before using the secondary adhesive solution.

18. The method of claim 17, wherein the polymerization activator and / or accelerator is selected from ammonium persulfate, potassium persulfate, and TEMED.

19. The method according to any one of claims 1-4, wherein the anchoring agent comprises biomolecular reactive chemical groups and hydrogel reactive chemical groups.

20. The method according to any one of claims 1-4, wherein the anchoring time is 4-12 hours.

21. The method of claim 19, wherein the anchoring agent is selected from N-succinimide acrylate (NSA), N-(allyloxycarbonyloxy)-succinimide (NAS), and combinations thereof.

22. The method according to any one of claims 1-4, further comprising perfusion fixation of the biological sample prior to step (a).

23. The method of claim 22, wherein the fixative solution used for perfusion fixation comprises 1% paraformaldehyde and 0.1 mg / ml of N-succinimide acrylate (NSA).

24. The method according to any one of claims 1-4, wherein in step (c), protein denaturation of the sample-hydrogel complex comprises treatment with two protein denaturation buffers, wherein the first protein denaturation buffer is an acidic denaturation buffer and the second protein denaturation buffer is a neutral denaturation buffer.

25. The method of claim 24, wherein both the first protein denaturing buffer and the second protein denaturing buffer contain a detergent, wherein the first protein denaturing buffer is acid-treated to be acidic.

26. The method of claim 25, wherein the detergent is SDS or sodium dodecylbenzenesulfonate.

27. The method according to any one of claims 1-4, wherein the reductive alkylation in step (d) is carried out by treatment with a disulfide reducing agent selected from TCEP, DTT and BME.

28. The method according to any one of claims 1-4, wherein the reductive alkylation further comprises treatment with an alkylating agent selected from iodoacetamide (IAA), chloroacetamide, iodoacetic acid, and N-ethylmaleimide.

29. The method according to any one of claims 1-4, wherein the staining is selected from Coomassie blue staining, fluorescent staining, Coomassie brilliant blue staining, immunostaining, silver staining, and isotope staining.

30. The method of any one of claims 1-4, wherein swelling of the sample-gel complex is performed by incubating it with water or an aqueous buffer.

31. The method of claim 30, wherein swelling of the sample-gel complex is performed by incubating it with Tris.

32. The method according to any one of claims 1-4, wherein the sampling is a local sampling.

33. The method of claim 32, wherein the local sampling is microsampling, microsampling performed by a punch, or laser capture microdissection (LCM) sampling.

34. The method of claim 32, wherein the diameter of the sample obtained by local sampling is less than 1 mm.

35. The method according to any one of claims 1-4, wherein step (h) further comprises destaining the secondary gel-embedded sample.

36. The method of claim 35, wherein the destaining is performed by eluting the gel block with a 50% / 50% volume ratio of acetonitrile / 100mM ammonium bicarbonate buffer or a 50% / 50% volume ratio of methanol / 100mM ammonium bicarbonate buffer.

37. The method according to any one of claims 1-4, wherein protein identification is performed by mass spectrometry analysis.

38. The method of claim 37, wherein the mass spectrometry analysis is HPLC-MS.

39. The method of claim 37, wherein the mass spectrometry analysis comprises enzymatic digestion of the sample-gel complex, peptide extraction, and mass spectrometry detection.

40. The method of claim 39, wherein desalting is performed after peptide extraction.

41. A kit comprising DMAA, SMA, and PAE in one or more containers, and a secondary gel solution for secondary gel embedding of a cut sample, said secondary gel solution being an acrylamide / methylenebisacrylamide solution.

42. The kit of claim 41, wherein DMAA, SMA and PAE are each in separate containers.

43. The kit according to claim 41 or 42, wherein DMAA and SMA are in solid form and PAE is in liquid form soluble in an organic solvent.

44. The kit according to claim 41 or 42, wherein DMAA, SMA and PAE are mixed together in a molar ratio in the range of (20-60):(5-15):(0.005-2).

45. The kit according to claim 44, wherein DMAA, SMA and PAE are mixed together in a molar ratio within the range of (40±10):(5-15):(0.005-2), (40±15):(10±3.5):(0.4±0.2), (40±10):(10±2.5):(0.4±0.2), (40±5):(10±2):(0.4±0.2), (40±5):(10±1.2):(0.4±0.2), or (40±5):(10±1):(0.005±0.1).

46. ​​The kit according to claim 44, wherein DMAA, SMA and PAE are dissolved in water to form a solution.

47. The kit according to any one of claims 41-42, wherein the kit is suitable for storage at a temperature of 0°C-4°C or at room temperature.

48. The kit according to any one of claims 41-42, wherein the kit further comprises one or more of the following: A container containing the anchoring agent; Containers containing disulfide bond reducing agents; Containers containing alkylating agents; A container containing one or more polymerization activators or accelerators selected from VA-044, V50, TEMED, ammonium persulfate and potassium persulfate; Containers containing fixatives; A container containing protein denaturation buffer; Containers containing protein staining solutions; and A container containing enzymes for digesting proteins in a sample.

49. The kit of claim 48, wherein the anchoring agent is NAS and / or NSA.

50. The kit of claim 48, wherein the fixative is paraformaldehyde and NSA.

51. The kit of claim 48, wherein the protein denaturation buffer is an acidic first protein denaturation buffer and a neutral second protein denaturation buffer.

52. The kit of claim 48, wherein the protein denaturation buffer is a protein denaturation buffer containing SDS or sodium dodecyl sulfate.

53. The kit according to any one of claims 41-42, wherein the kit further comprises one or more of the following: A gel chamber, configured to hold the sample prior to gelation; The staining chamber is configured for staining proteins in samples; Gel handling tools; Hole punch; Cooling device.

54. The kit of claim 53, wherein the gel chamber is a dish having a cavity or groove.

55. A system for spatial proteomics analysis of biological samples, comprising: Anchoring module, which is used to anchor proteins in biological samples; A gel reaction module in which polymerization occurs to form a sample-gel complex; Protein denaturation module, in which proteins are denatured; The reduction alkylation module performs reduction and alkylation treatment on the sample-gel complex. The staining module is used to stain proteins. as well as The secondary gelation module performs secondary gel embedding on the acquired biological samples.

56. The system of claim 55 further comprises a mass spectrometry analysis module, wherein the locally sampled sample undergoes mass spectrometry pretreatment and is connected in series with a mass spectrometry detection device.

57. The system of claim 55 or 56, wherein local sampling is performed in the staining module.

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