A method for efficient extraction of protein polypeptides and deep identification of glycopeptides from paraffin-embedded section samples

Abstract

The application discloses a kind of paraffin-embedded section sample protein polypeptide high-efficiency extraction and glycopeptide deep identification method, including to paraffin-embedded section carries out dewaxing and rehydration treatment;To the section after rehydration carries out multiple cycles of antigen repair, reduction alkylation and enzymolysis, and enzyme hydrolysate is collected;Enzymolysis peptide is desalted;To the peptide after desalination is isotopically labeled and glycopeptide enrichment.The core of the present application is "multiple cycle protein repair and enzymolysis" step, which is repeated by multiple antigen repair and enzymolysis to the same section, iteratively uncrosslinked and released peptide segment, significantly improve the total amount and coverage of recovered peptide segment from FFPE section.On this basis, combined with TMT labeling and glycopeptide enrichment strategy, the deep identification of low-abundance glycopeptide is realized.

Description

Technical Field

[0001] This invention belongs to the field of glycoproteomics technology, specifically relating to a method for efficient extraction of proteins and peptides and in-depth identification of glycopeptides from paraffin-embedded sectioned samples. Background Technology

[0002] Formalin-fixed paraffin-embedded (FFPE) sections are the most widely used form of tissue preservation in clinical pathology diagnosis and biomedical research. Through formaldehyde fixation and paraffin embedding, the morphological structure and molecular information of tissue cells can be stably preserved at room temperature for a long period. Therefore, FFPE samples are widely used in disease pathological classification, clinical diagnosis, prognostic assessment, and basic research fields such as oncology, immunology, and molecular biology. With the rapid development of precision medicine and space omics technologies, the vast amount of clinical FFPE samples has become a core resource for retrospective cohort studies and disease biomarker mining. Genomic, transcriptomic, and proteomic analysis technologies based on FFPE sections are constantly maturing, providing important technical support for disease mechanism analysis and clinical translational research.

[0003] Protein glycosylation is one of the most prevalent and functionally critical post-translational modifications in eukaryotes, participating in various physiological and pathological processes such as cell recognition, signal transduction, immune response, and tumor invasion and metastasis. Abnormal protein glycosylation modifications are highly correlated with the occurrence and development of various diseases, including tumors and autoimmune diseases, and serve as important biomarkers and targets for early disease diagnosis, prognosis, and targeted therapy. In-depth glycoproteomic analysis based on clinical tissue samples has significant clinical value and scientific implications for discovering novel disease molecular biomarkers and developing precise targeted therapies.

[0004] However, during formaldehyde fixation, FFPE samples form numerous covalent cross-links between and within protein molecules, hindering the effective release and complete enzymatic digestion of proteins within the tissue. Compared to fresh tissue samples, FFPE samples exhibit low peptide extraction efficiency and poor sequence coverage, which remains a core bottleneck restricting FFPE proteomics research. Current conventional FFPE protein extraction methods mostly employ a standard strategy of "single heat-induced antigen retrieval + single enzymatic digestion," which only releases partially uncross-linked proteins from the tissue surface. A large number of deeply cross-linked proteins remain inside the tissue sections, resulting in limited peptide yield and identification depth, making it difficult to meet the initial sample volume requirements for subsequent modified proteomics analysis.

[0005] Meanwhile, glycopeptides account for only 1% to 10% of total peptides naturally, belonging to low-abundance modified peptides. Their enrichment and in-depth identification usually require a high amount of starting peptides. Limited by the peptide extraction efficiency of FFPE samples, most existing technologies require multiple FFPE slices to be stacked for extraction to obtain sufficient peptides for glycopeptide enrichment. This not only significantly increases the consumption of precious clinical samples but also makes it difficult to meet the research needs of micro-puncture samples and analysis of specific spatial regions. In addition, repeated high-temperature antigen retrieval can cause degradation and loss of glycosylation modifications, further limiting the development of glycoproteomics analysis technology for FFPE samples. Existing FFPE glycopeptide identification methods still have significant shortcomings in terms of the number of glycoproteins identified, glycosylation site coverage, and the depth of intact glycopeptide identification. The identification level of most methods is far lower than that of fresh tissue samples, failing to fully realize the research value of clinical FFPE samples.

[0006] Therefore, developing a method that can efficiently extract proteins and peptides from a single FFPE slice and achieve in-depth identification of glycopeptides, thereby improving the peptide yield and glycopeptide identification coverage of FFPE samples, is of great practical significance for making full use of existing clinical FFPE sample resources and conducting high-quality retrospective glycoproteomics research. Summary of the Invention

[0007] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0008] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0009] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a method for efficient extraction of proteins and peptides and in-depth identification of glycopeptides from paraffin-embedded section samples.

[0010] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for efficient extraction of proteins and peptides and in-depth identification of glycopeptides from paraffin-embedded section samples, characterized in that it includes: Dewaxing and rehydration of sections: The sections are heated and then dewaxed and rehydrated; Antigen repair: Perform antigen repair to break the cross-links between proteins; Reductive alkylation: Reduction and alkylation treatment is performed on the slices; Multi-cycle protein repair and enzymatic digestion: Add trypsin solution for enzymatic digestion, collect the digestion solution, repeat antigen repair, perform multiple enzymatic digestions on the slide, collect the digestion solutions separately and then combine them, and then continue antigen repair and complete protein digestion in the solution tube. Finally, adjust the pH with acidic solution and collect the samples. Desalting of enzymatically hydrolyzed peptides: The sample was washed, activated, and equilibrated using a reversed-phase chromatography column. The sample was repeatedly loaded and eluted through a high organic phase to obtain the eluent. After evaporation and drying, the eluent was reconstituted and the peptide concentration was determined. Tandem Mass Tag labeling of peptides and enrichment of glycopeptides: The reconstituted peptides were mixed with isotope labeling reagents, the labeling reaction was carried out and the reaction was terminated, followed by sample mixing and pH adjustment. Then, the samples were washed and eluted using a reversed-phase chromatography column. After adjusting the organic phase concentration of the eluent, the glycopeptide samples were collected through a hydrophilic interaction chromatography column, dried, and prepared for mass spectrometry analysis.

[0011] As a preferred embodiment of the method described in this invention, the slices are heated and dewaxed and rehydrated, wherein the heating temperature is 50~70℃ and the heating time is [not specified]; dewaxing is performed using xylene or an environmentally friendly dewaxing solution at 37℃; and rehydration is performed using a gradient of ethanol solutions with concentrations decreasing gradually.

[0012] In a preferred embodiment of the method described in this invention, the antigen retrieval is performed using thermally induced epitope retrieval.

[0013] As a preferred embodiment of the method described in this invention, the alkylation treatment uses dithiothreitol (DTT), iodoacetic acid (IAA), or iodoacetamide (IAM), with the DTT solution concentration being 10-20 mM and the IAA or IAM concentration being 20-40 mM.

[0014] In a preferred embodiment of the method described in this invention, the pH is adjusted using an acidic solution, wherein the acidic solution is selected from formic acid or trifluoroacetic acid, and the pH is adjusted to 2-3.

[0015] In a preferred embodiment of the method described in this invention, the desalting of the enzymatically hydrolyzed peptides includes: using a C18 solid-phase extraction column for peptide enrichment, with a capacity of 50-100 mg; using a high organic phase solution, selected from acetonitrile or methanol, with a concentration of 80-100%; using an acidic low organic phase solution, selected from acetonitrile or methanol, with a concentration of 0-10%; using a high organic phase solution, selected from acetonitrile or methanol, with a concentration of 50-80%; and using an alkaline 2-hydroxyethylpiperazine-2'-ethanesulfonic acid buffer (HEPES) or triethylammonium bicarbonate buffer (TEAB), with a concentration of 50-100 mM, to reconstitute and evaporate the sample.

[0016] In a preferred embodiment of the method described in this invention, the reconstituted peptide is mixed with an isotope labeling reagent, a labeling reaction is performed, and the reaction is terminated. Specifically, 20-50 μg of the reconstituted peptide is taken from each sample for labeling; the labeling reagent is selected from TMT10plex. TM Use TMTpro 11-plex, TMTpro 16-plex, or TMTpro 18-plex for labeling. The reaction time is 60-90 min, the reaction temperature is room temperature, and the mixture is shaken at 800-1200 rpm. The quenching reagent is 5% hydroxylamine reagent. The quenching reaction time is 15-30 min, the reaction temperature is room temperature, and the mixture is shaken at 800-1200 rpm.

[0017] In a preferred embodiment of the method described in this invention, the mixing and pH adjustment of the sample are carried out, wherein the pH adjustment is performed by using an acidic reagent to adjust the pH of the solution to 2-3.

[0018] As a preferred embodiment of the method described in this invention, the washing and elution are performed using a reversed-phase chromatography column, wherein the acetonitrile concentration of the eluent is adjusted to above 93% using a high-acetonitrile solution.

[0019] As a preferred embodiment of the method described in this invention, the hydrophilic interaction chromatographic column is selected as a HILIC or MAX solid phase extraction column. The column is activated using a high acetonitrile phase solution with a concentration of 80-100%. After activation, it is blocked using 100mM TEAB reagent. After washing with pure water, it is equilibrated using an acetonitrile solution with a concentration of 93% or higher. The elution solution is a high acetonitrile solution with a concentration of 40-50%.

[0020] Beneficial effects of this invention: (1) Efficient extraction of peptides from paraffin-embedded sections was achieved: Through a multi-cycle protein repair and enzymatic digestion strategy, the recovery rate and number of peptides identified were significantly improved. A total of 54,699 unique peptides were identified in three cycles. The number of peptides identified by multiple cycles increased by about 15% compared with single cycles. The newly identified peptides showed shorter sequence lengths and lower missed digestion rates.

[0021] (2) High-yield peptide extraction from single sections was achieved: This method can extract sufficient peptides from a single paraffin-embedded section for in-depth analysis. The peptide extraction yield exceeded 50 μg in about 90% of the sections, and the highest extraction yield in a single section reached 210 μg. Moreover, the extraction yield was significantly positively correlated with the tissue area of ​​the section (R=0.57, p=2.76e-08).

[0022] (3) Achieved in-depth identification of glycopeptides in paraffin-embedded sections: This method successfully performed glycoproteomics analysis on FFPE sections, identifying 906 glycoproteins, 1323 glycosylation sites, 8306 intact glycopeptides, and 241 N-linked glycans. Furthermore, the identification depth reached or surpassed that of fresh tissue. Regarding the number of intact glycopeptides identified, the results obtained by this method on FFPE sections significantly exceeded those of published literature on fresh tissue, and the number of glycoproteins identified was comparable to or even superior to the results of fresh tissue studies.

[0023] (4) Comprehensive analysis of glycosylation modification characteristics: This method can analyze the modification type of glycopeptides in detail. Among the identified glycopeptides, 49% are modified with fucose, 26% are modified with sialic acid, and 21% are modified with high mannose. Among the identified N-linked glycans, 61% are modified with fucose, 43% are modified with sialic acid, and 6% are modified with high mannose. The high abundance is mainly high mannose type and double-antenna glycan type.

[0024] (5) The similarities and differences between FFPE and fresh tissue glycosylation profiles were revealed: Through comparison, it was found that paraffin-embedded sections and fresh tissues from different studies share some glycoproteins, glycosylation sites and glycans, while also having their own unique parts, providing a reliable basis for retrospective glycomics studies using FFPE samples. Attached Figure Description

[0025] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein: Figure 1 This diagram illustrates the strategy for efficient extraction of peptides and glycopeptide analysis from paraffin-embedded sections according to the present invention.

[0026] Figure 2 This is the analysis result of the effect of the multi-cycle protein repair and enzymatic hydrolysis strategy of the present invention on the extraction of peptides from paraffin-embedded sections.

[0027] Figure 3 This is an analytical diagram showing the results of peptide extraction and glycopeptide identification from paraffin-embedded sections in this invention.

[0028] Figure 4 This is a compositional analysis diagram of the types of glycopeptides and N-linked glycans identified in paraffin-embedded sections according to the present invention.

[0029] Figure 5 The relative abundance of N-linked glycans on paraffin-embedded sections identified in this invention shows the relative abundance of major N-linked glycans and the glycan structure type.

[0030] Figure 6 The results show the identification of glycopeptides in fresh mouse kidney tissue and formalin-embedded mouse kidney tissue in Example 2 of this invention. Detailed Implementation

[0031] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0032] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0033] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0034] The raw materials used in this invention are as follows: the dewaxing solution is Savill environmentally friendly dewaxing solution; the antigen retrieval solution is Beyotime sodium citrate antigen retrieval solution; acetonitrile and trifluoroacetic acid were purchased from Sigma; the desalting column was purchased from Waters Corporation; and the labeling reagent was purchased from Thermo Fisher Scientific.

[0035] The instrument used in this embodiment of the invention is a Thermo Fisher Orbitrap Fusion Lumos mass spectrometer.

[0036] Example 1 This embodiment provides a method for efficient extraction of proteins and peptides and in-depth identification of glycopeptides from paraffin-embedded section samples, specifically including the following steps: (1) Dewaxing and rehydration of FFPE tissue sections from liver cancer: The sections were heated at 60°C for 1 hour, placed in an environmentally friendly dewaxing solution at 37°C for 20 minutes, and then placed in ethanol for 10 minutes. This process was repeated once.

[0037] Then immerse them in 95%, 90%, 80%, 70%, and 50% ethanol solutions for 5 minutes each, and finally place them in pure water to complete the rehydration.

[0038] (2) Antigen retrieval on FFPE tissue sections of liver cancer: Check the dewaxing effect and dry the product. Place it in the antigen retrieval solution at 95°C for 30 minutes, then replace the solution with pure water to complete the antigen retrieval.

[0039] (3) Reduction alkylation on FFPE tissue sections of liver cancer: The slices were coated with 10 mM dithiothreitol (DTT) and reduced at 37 °C for 1 h. Then, they were alkylated with 20 mM iodoacetamide at room temperature in the dark for 45 min.

[0040] (4) Multi-cycle antigen retrieval and trypsin digestion on FFPE tissue sections of liver cancer: After drying the liquid, add 20 mmol of trypsin solution (Promega, Madison, WI) to cover the tissue, and enzymatically hydrolyze for 4 h. Collect the solution into a peptide solution centrifuge tube.

[0041] Antigen retrieval was performed on the slides again. The slides were placed in the antigen retrieval solution and incubated at 95°C for 30 min. Then, the solution was replaced with pure water, and 20 mmol of trypsin solution (Promega, Madison, WI) was added to cover the tissue. Enzymatic digestion was carried out for 4 h, and the solution was collected into peptide solution centrifuge tubes.

[0042] After combining the peptide digestion solutions, add an appropriate amount of trypsin solution to the collected peptide solution centrifuge tube and digest for 8 hours. Then acidify the solution with 50% TFA to 2000 ppm. <pH<3。

[0043] (5) Desalting of enzymatically hydrolyzed peptides: The column was activated three times with pure ACN using 50 mg C18, then activated three times with 80% ACN (v / v) and 0.1% TFA (v / v), and equilibrated five times with 0.1% TFA (v / v).

[0044] The above sample was loaded twice.

[0045] The C18 column was washed four times with 0.1% TFA, and then eluted with 500 μl of 50% ACN (v / v) and 0.1% TFA (v / v) to obtain the eluent.

[0046] After freeze-drying, the peptide sample concentration was measured by reconstitution with 100 mm triethylammonium bicarbonate buffer (TEAB, Sigma-Aldrich).

[0047] (6) Tandem Mass Tag labeling of peptides and enrichment of glycopeptides: Take 50 μg of each reconstituted peptide, and group 9 samples together. Add 126, 127N, 127C, 128N, 128C, 129N, 129C, 130N, and 130C labeling reagents from Tandem Mass Tag10Plex. Use the mixture of peptides from each slice sample as a reference calibration channel. Add 131 labeling reagent and incubate at 1200 rpm for 90 min.

[0048] Add 5% hydroxylamine and quench the reaction at 1200 rpm for 15 min. Mix each sample separately and add 50% TFA to adjust the pH to between 2 and 3.

[0049] The column was activated three times with pure ACN using 100 mg C18, then activated three times with 80% ACN (v / v), 0.1% TFA (v / v), and equilibrated five times with 0.1% TFA (v / v).

[0050] The above sample was loaded twice.

[0051] The C18 column was washed four times with 0.1% TFA, and then eluted with 400 μl of 50% ACN (v / v) and 0.1% TFA (v / v) and 400 μl of 80% ACN (v / v) and 0.1% TFA (v / v) to obtain the eluent.

[0052] Add 10 ml of 1% TFA / 95% ACN (v / v) and 14.4 μl of 50% TFA to the above C18 eluent.

[0053] The MAX column was washed three times with pure ACN, blocked three times with 200 mM TEAB, washed five times with pure water, and washed five times with 1% TFA / 95% ACN (v / v). The sample was then added, washed three times with 1% TFA / 95% ACN (v / v), and finally eluted with 400 μl of 0.1% TFA / 50% ACN (v / v). The resulting glycopeptide sample was dried in a speed-vac and awaited mass spectrometry analysis.

[0054] (7) NanoLC-MS / MS analysis and data analysis: The samples were resuspended in 0.1% FA, with each sample weighing 2 μg. Data were acquired using an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific), with mass spectrometry parameters referencing published work (Comprehensive Analysis of the Glycome and Glycoproteome of Bovine Milk-Derived Exosomes. J Agric Food Chem. 2020 Nov 11;68(45):12692-12701). Data were searched and analyzed using the glycopeptide analysis software GPquest.

[0055] (8) Efficient extraction of peptides from slices and in-depth identification of glycopeptides: use Figure 1The flowchart shown first involves enzymatic digestion and peptide extraction of proteins from 10 slices, and then separating the peptide solutions extracted in each cycle, and identifying each extracted peptide separately.

[0056] Experimental results are as follows Figure 2 As shown, where, Figure 2 (a) The amount of peptides extracted and the number of peptides identified in different cycles; Figure 2 (b) shows the overlap relationship of peptide segments in different cycles; Figure 2 (c) represents the peptide length in different cycles; Figure 2 (d~f) represent the missed cuts in different loops.

[0057] A total of 54,699 unique peptides were identified across three cycles. Multiple cycles can increase the amount of peptides extracted and the number of peptides identified (e.g., ...). Figure 2 As shown in a), multiple cycles can increase the number of peptides identified by 15% compared to single cycles (e.g., Figure 2 (as shown in b).

[0058] Newly identified peptides in multiple cycles exhibited shorter peptide sequences and lower peptide cleavage miss rates (e.g., ...). Figure 2 As shown in cf), this method demonstrates the high efficiency of peptide extraction.

[0059] Next, peptide extraction and glycopeptide enrichment were performed on 89 slices. The amount of peptide extracted and the area on the slices showed a linear relationship.

[0060] Figure 3 This is an analytical diagram showing the results of peptide extraction and glycopeptide identification from paraffin-embedded sections in this invention. Figure 3 (a) Shows the correlation between peptide extraction amount and tissue area of ​​the slice; Figure 3 (b) Statistical analysis of the number of identified intact glycopeptides, glycans, glycosylation sites, and glycoproteins; Figure 3 (c~f) Comparison of the results of intact glycopeptides (c), glycans (d), glycosylation sites (e), and glycoproteins (f) between FFPE sections and fresh tissue.

[0061] like Figure 3 As shown in a, approximately 90% of the slices yielded peptides exceeding 50 μg, with the highest extraction yield on a single slice being 210 μg and the lowest being 36 μg.

[0062] In mass spectrometry analysis, a total of 241 N-linked glycans were identified, covering 1323 glycosylation sites, 8306 intact glycopeptides and 906 glycoproteins. The identification results of glycopeptides on FFPE sections exceeded the identification level of glycopeptides in fresh tissue samples (N-glycosylation Modification of CTSD Affects Liver Metastases in Colorectal Cancer. Adv Sci (Weinh). 2025 Feb;12(7):e2411740.; GlycoPCT: Pressure Cycling Technology-Based Quantitative Glycoproteomics Reveals Distinctive N-Glycosylation in Human Liver Biopsy Samples of Nonalcoholic Fatty Liver Disease. J Proteome Res. 2025 Jan 3;24(1):202-209; Deciphering a profiling based on multiple post-translational modifications functionally associated regulatory patterns and therapeutic opportunities in human hepatocellularcarcinoma. Mol Cancer. 2024 Dec 28;23(1):283) (eg Figure 3 (as shown in b).

[0063] Figure 4 This is a compositional analysis diagram of the types of glycopeptides and N-linked glycans identified in paraffin-embedded sections according to the present invention. Figure 4 (a) Shows the compositional proportions of glycopeptides with different modification types; Figure 4 (a) Shows the composition ratio of N-linked sugar chains with different modification types.

[0064] Among the modification characteristics of glycopeptides, 49% were modified with fucose, 26% with sialic acid, and 21% with high-mannose modification (e.g., Figure 4 (As shown in a). Of the N-linked glycans identified, 61% were fucose-modified, 43% were sialic acid-modified, and 6% were high-mannose-modified (e.g., ...). Figure 4 (as shown in b).

[0065] Abundance analysis of the identified N-linked glycans revealed that highly abundant N-linked glycans were mainly of the high-mannose type and the biantennary type (e.g., Figure 5 (As shown).

[0066] Example 2 This embodiment provides the identification results of glycopeptides in fresh mouse kidney tissue and formalin-embedded mouse kidney tissue.

[0067] Peptide extraction and glycopeptide identification from FFPE tissue sections of mouse kidney: (1) Dewaxing and rehydration of mouse kidney FFPE tissue sections: Nine mouse kidney FFPE tissue sections were heated at 60°C for 1 hour, placed in an environmentally friendly dewaxing solution at 37°C for 20 minutes, and then placed in ethanol for 10 minutes. This process was repeated once.

[0068] Then immerse them in 95%, 90%, 80%, 70%, and 50% ethanol solutions for 5 minutes each, and finally place them in pure water to complete the rehydration.

[0069] (2) Antigen retrieval on mouse kidney FFPE tissue sections: Check the dewaxing effect and dry the product. Place it in the antigen retrieval solution at 95°C for 30 minutes, then replace the solution with pure water to complete the antigen retrieval.

[0070] (3) Reduction alkylation on mouse kidney FFPE tissue sections: The slices were coated with 10 mM dithiothreitol (DTT) and reduced at 37 °C for 1 h. Then, they were alkylated with 20 mM iodoacetamide at room temperature in the dark for 45 min.

[0071] (4) Multi-cycle antigen retrieval and trypsin digestion in mouse kidney FFPE tissue sections: After drying the liquid, add 20 mmol of trypsin solution (Promega, Madison, WI) to cover the tissue, and enzymatically hydrolyze for 4 h. Collect the solution into a peptide solution centrifuge tube.

[0072] Antigen retrieval was performed on the slides again. The slides were placed in the antigen retrieval solution and incubated at 95°C for 30 min. Then, the solution was replaced with pure water, and 20 mmol of trypsin solution (Promega, Madison, WI) was added to cover the tissue. Enzymatic digestion was carried out for 4 h, and the solution was collected into peptide solution centrifuge tubes.

[0073] After combining the peptide digestion solutions, add an appropriate amount of trypsin solution to the collected peptide solution centrifuge tube and digest for 8 hours. Then acidify the solution with 50% TFA to 2000 ppm. <pH<3。

[0074] (5) Reverse-phase desalting of enzymatically digested peptides on mouse kidney FFPE tissue sections: The column was activated three times with pure ACN using 50 mg C18, then activated three times with 80% ACN (v / v) and 0.1% TFA (v / v), and equilibrated five times with 0.1% TFA (v / v).

[0075] The above sample was loaded twice.

[0076] The C18 column was washed four times with 0.1% TFA, and then eluted with 500 μl of 50% ACN (v / v) and 0.1% TFA (v / v) to obtain the eluent.

[0077] After freeze-drying, the peptide sample concentration was measured by reconstitution with 100 mm triethylammonium bicarbonate buffer (TEAB, Sigma-Aldrich).

[0078] (6) Tandem Mass Tag labeling of peptides and enrichment of glycopeptides: Take 45 μg of each of the reconstituted peptides, and add 126, 127N, 127C, 128N, 128C, 129N, 129C, 130N, and 130C labeling reagents from Tandem Mass Tag10Plex ​​to the nine samples respectively. The mixture of peptides from the nine sliced ​​samples is used as a reference calibration channel. Add 131 labeling reagent and incubate at 1200 rpm for 90 min.

[0079] Add 5% hydroxylamine and quench the reaction at 1200 rpm for 15 min. Mix each sample separately and add 50% TFA to adjust the pH to between 2 and 3.

[0080] The column was activated three times with pure ACN using 100 mg C18, then activated three times with 80% ACN (v / v), 0.1% TFA (v / v), and equilibrated five times with 0.1% TFA (v / v).

[0081] The above sample was loaded twice.

[0082] The C18 column was washed four times with 0.1% TFA, and then eluted with 400 μl of 50% ACN (v / v) and 0.1% TFA (v / v) and 400 μl of 80% ACN (v / v) and 0.1% TFA (v / v) to obtain the eluent.

[0083] Add 10 ml of 1% TFA / 95% ACN (v / v) and 14.4 μl of 50% TFA to the above C18 eluent.

[0084] The MAX column was washed three times with pure ACN, blocked three times with 200 mM TEAB, washed five times with pure water, and washed five times with 1% TFA / 95% ACN (v / v). The sample was then added, washed three times with 1% TFA / 95% ACN (v / v), and finally eluted with 400 μl of 0.1% TFA / 50% ACN (v / v). The resulting glycopeptide sample was dried in a speed-vac and awaited mass spectrometry analysis.

[0085] (7) NanoLC-MS / MS analysis and data analysis: The samples were resuspended in 0.1% FA, with each sample weighing 2 μg. Data were acquired using an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific), with mass spectrometry parameters referencing published work (Comprehensive Analysis of the Glycome and Glycoproteome of Bovine Milk-Derived Exosomes. J Agric Food Chem. 2020 Nov 11;68(45):12692-12701). Data were searched and analyzed using the glycopeptide analysis software GPquest.

[0086] Peptide extraction and glycopeptide identification from fresh mouse kidney tissue: (1) Extraction of protein from fresh mouse kidney tissue by grinding with liquid nitrogen Mouse kidney tissue preserved in liquid nitrogen was placed in a pre-chilled mortar, liquid nitrogen was added, and the tissue was ground into powder. The powder was then poured into 8M Urea lysis buffer. The mixture was sonicated for 10 min using a probe sonicator, centrifuged at 12000 rpm for 15 min, and the supernatant was collected. Protein concentration was determined using a Bradford assay kit.

[0087] (2) Extraction of peptides from fresh mouse kidney tissue Take 500 μg of protein and add dithiothreitol (DTT) to a final concentration of 10 mg / mL. Reduce at 37°C for 1 h. Then add iodoacetamide to a final concentration of 20 mg / mL and react at room temperature in the dark for 45 min. Add 40 mg / mL ammonium bicarbonate solution to reduce the urea concentration to 1 M. Add trypsin at a protein:enzyme ratio of 40:1 and hydrolyze for 16 h. Acidify the solution to 20 mg / mL with 50% TFA. <pH<3。

[0088] (3) Reverse-phase desalting and glycopeptide enrichment of peptides in fresh mouse kidney tissue: The column was activated three times with pure ACN using 100 mg C18, then activated three times with 80% ACN (v / v), 0.1% TFA (v / v), and equilibrated five times with 0.1% TFA (v / v).

[0089] The above sample was loaded twice.

[0090] The C18 column was washed four times with 0.1% TFA, and then eluted with 400 μl of 50% ACN (v / v) and 0.1% TFA (v / v) and 400 μl of 80% ACN (v / v) and 0.1% TFA (v / v) to obtain the eluent.

[0091] Add 10 ml of 1% TFA / 95% ACN (v / v) and 14.4 μl of 50% TFA to the above C18 eluent.

[0092] The MAX column was washed three times with pure ACN, blocked three times with 200 mM TEAB, washed five times with pure water, and washed five times with 1% TFA / 95% ACN (v / v). The sample was then added, washed three times with 1% TFA / 95% ACN (v / v), and finally eluted with 400 μl of 0.1% TFA / 50% ACN (v / v). The resulting glycopeptide sample was dried in a speed-vac and awaited mass spectrometry analysis.

[0093] Figure 6 This image compares the glycopeptide identification results of fresh mouse tissue and formalin-fixed paraffin-embedded tissue. Figure 6 (a) Shows the number of intact glycopeptides, glycoproteins, glycosylation sites and glycans identified in the two types of samples; Figure 6 (b) Shows the overlap of intact glycopeptides, glycoproteins, glycosylation sites and glycans in the two types of samples.

[0094] like Figure 6 As shown, a comparison of glycopeptide identification in fresh mouse liver tissue and formalin-fixed paraffin-embedded mouse liver tissue revealed similar numbers of glycopeptides, glycoproteins, glycosylation sites, and glycan types in both samples. Figure 6 a) Through multi-cycle protein repair, more than 75% of intact glycopeptides, more than 80% of glycoproteins, nearly 75% of glycosylation sites, and more than 80% of glycans can be restored. Figure 6 b).

[0095] This invention discloses a method for efficient extraction of proteins and peptides and in-depth identification of glycopeptides from paraffin-embedded tissue sections, belonging to the fields of glycoproteomics and clinical sample pretreatment technology. Addressing the technical problems of severe protein cross-linking, low peptide extraction efficiency, and difficulty in in-depth identification of low-abundance glycopeptides in formalin-fixed paraffin-embedded sections, this invention employs a multi-cycle protein repair and enzymatic digestion core strategy: through multiple rounds of iterative operations of "heat-induced antigen repair - trypsin digestion - digestion solution collection," deeply cross-linked protein peptides within the tissue are gradually released; after merging multiple rounds of digestion solutions, antigen repair and complete enzymatic digestion are further supplemented in the solution system to improve peptide recovery rate. The obtained peptides are then subjected to reverse-phase desalting and isotope labeling, followed by reverse-phase purification and hydrophilic interaction chromatography to enrich glycopeptides for mass spectrometry detection. This invention can significantly improve the peptide yield and identification coverage of a single paraffin-embedded section, with glycopeptide identification depth reaching or even exceeding that of fresh tissue samples, making it suitable for retrospective glycoproteomics studies and disease biomarker mining of clinical paraffin-embedded samples.

[0096] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A method for efficient extraction of proteins and peptides and deep identification of glycopeptides from paraffin-embedded sectioned samples, characterized in that: The process includes steps such as dewaxing and rehydration of tissue sections, antigen retrieval, reductive alkylation, multi-cycle protein retrieval and trypsin digestion, as well as reverse-phase desalting of enzymatically digested peptides, isotope labeling, and glycopeptide enrichment. The trypsin hydrolysis employs a multi-cycle protein repair and hydrolysis process, which includes, after completing the initial antigen repair and reductive alkylation treatment, adding trypsin solution to the tissue slices for the first round of hydrolysis, and collecting the first round of hydrolysis solution. The remaining tissue sections after the first round of enzymatic digestion were subjected to antigen retrieval and enzymatic digestion again, and the corresponding enzymatic digestion solutions were collected. The above antigen retrieval-enzymatic digestion-collection operation was repeated for a total of 2 to 3 rounds. After combining the enzymatic digestion solutions from each round, in-solution antigen retrieval was performed on the combined enzymatic digestion system, and trypsin was added to complete enzymatic digestion. The pH of the system was adjusted to 2 to 3 using an acidic solution to obtain the total peptide sample. After reverse-phase desalting and resolution, the peptide fragments were sequentially subjected to isotope labeling reaction, sample mixing and pH adjustment, reverse-phase purification, organic phase concentration adjustment, and hydrophilic interaction chromatography for glycopeptide enrichment. Finally, a dried glycopeptide sample was obtained for mass spectrometry detection.

2. The method as described in claim 1, characterized in that: The dewaxing and rehydration process specifically involves heating the paraffin-embedded sections at 50-70°C for 30-100 minutes; performing the dewaxing reaction using xylene or an environmentally friendly dewaxing solution at 37°C; and gradually rehydrating the sections using a gradient of ethanol solutions with varying concentrations.

3. The method as described in claim 1, characterized in that: The antigen retrieval is a heat-induced epitope retrieval, with a retrieval temperature of 90~98℃ and a retrieval time of 20~60 min.

4. The method as described in claim 1, characterized in that: The reduction alkylation process uses dithiothreitol (DTT) for reduction and iodoacetic acid (IAA) or iodoacetamide (IAM) for alkylation. The final concentration of DTT is 10-20 mM, and the final concentration of IAA or IAM is 20-40 mM. The reduction alkylation process is performed only once after the first round of antigen retrieval.

5. The method as described in claim 1, characterized in that: The acidic solution is a formic acid solution or a trifluoroacetic acid solution.

6. The method as described in claim 1, characterized in that: The reverse-phase desalting process uses a C18 solid-phase extraction column with a packing material size of 50-100 mg. The C18 solid-phase extraction column is activated with 80-100% acetonitrile or methanol solution, washed with 0-10% acidic acetonitrile or methanol solution, and eluted with 50-80% acetonitrile or methanol solution. The desalted peptides are reconstituted with 50-100 mM 2-hydroxyethylpiperazine-2'-ethanesulfonic acid buffer or triethylammonium bicarbonate buffer.

7. The method as described in claim 1, characterized in that: The isotope labeling was performed using TMT series labeling reagents, with 20-50 μg of peptide fragments from each sample labeled. The labeling reaction conditions were room temperature, shaking speed of 800-1200 rpm, and reaction time of 60-90 min. A 5% hydroxylamine solution was used as a quenching reagent to terminate the reaction, with the quenching reaction conditions being room temperature, shaking speed of 800-1200 rpm, and reaction time of 15-30 min.

8. The method as described in claim 1, characterized in that: After the samples were mixed, the pH of the solution was adjusted to 2-3 using an acidic reagent. After elution by reversed-phase chromatography, the volume fraction of acetonitrile in the eluent was adjusted to above 93% using a high-concentration acetonitrile solution.

9. The method as described in claim 1, characterized in that: The hydrophilic interaction chromatographic enrichment of glycopeptides was performed using a HILIC solid-phase extraction column or a MAX solid-phase extraction column. The column was activated with 80-100% acetonitrile solution, treated with 100mM triethylammonium bicarbonate solution, washed with pure water, and then equilibrated with acetonitrile solution of 93% or higher. Glycopeptides were eluted with 40-50% acetonitrile solution.

10. The method as described in claim 1, characterized in that: In the multi-cycle protein repair and enzymatic hydrolysis strategy, the enzymatic hydrolysis time on each slice is 3-6 h, and the time for supplemental enzymatic hydrolysis in the solution is 6-16 h; the concentration of trypsin in each round of enzymatic hydrolysis is 10 mmol-40 mmol.

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