Phosphorylated peptide fragment preparation and space phosphorylated proteomics detection method

Abstract

The invention relates to the field of protein detection, in particular to a phosphorylated peptide fragment preparation and space phosphorylated proteomics detection method. By means of the preparation method, enough phosphorylated peptide fragments can be efficiently extracted from a trace sample, the method can be compatible with LCM for use, operation is easy and convenient, the recovery rate is high, and the method is suitable for achieving high-depth space phosphorylated proteomics analysis. Meanwhile, the analysis method shows high sensitivity on the trace level, and compared with a traditional method, the identification number of phosphorylation sites is remarkably increased. Compared with the homogenization treatment of the whole section, the analysis method provided by the invention can more accurately reflect the specificity of the cells in the target area and better reflect the intra-tumor heterogeneity.

Description

Technical Field

[0001] This invention relates to the field of protein detection, specifically to a method for preparing phosphorylated peptides and detecting spatially phosphorylated proteomics. Background Technology

[0002] Proteins, as the core executors of life activities, play a crucial role in tumorigenesis and development through post-translational modifications. Among various modification forms, phosphorylation is the most common and widespread, affecting approximately one-third of all proteins. This modification, catalyzed by protein kinases, directly regulates protein activity, conformation, stability, and signaling pathway networks. Abnormal protein phosphorylation can drive oncogene expression and promote tumor progression by activating pro-cancer signaling pathways and regulating transcription factor function. Therefore, phosphorylated proteins and related kinases have become important potential therapeutic targets.

[0003] While whole-tissue lysis-based phosphorylated proteomics has been widely used for drug target screening, its homogenization process mixes phosphorylation signals from different cell types within a tumor, leading to the loss of spatial distribution information. This "averaging" of signals inevitably masks the specific biological behaviors of particular cell populations. Studies have shown that in highly heterogeneous tumors such as GBM, whole-tissue lysis-based phosphorylated proteomics analysis may overlook phosphorylation changes in key signaling proteins within spatially heterogeneous tumor cells, limiting its application in elucidating tumor malignancy and predicting treatment outcomes. Therefore, developing phosphorylated proteomics that can preserve spatial resolution or achieve cell type-specific analysis is crucial.

[0004] Laser Capture Microdissection (LCM) can enrich target cells while preserving spatial coordinates, but current sample preparation still follows the traditional process: multiple rounds of xylene dewaxing, decrosslinking, SP3 / S-Trap magnetic bead purification, repeated washing and desalting, enzymatic digestion and enrichment are performed on slices larger than 15 mm² (area) × 10 μm (thickness). This process has three major bottlenecks: (1) Dewaxing-hydration requires multiple rounds of xylene dewaxing, centrifugation and liquid exchange, which is time-consuming, has a high risk of wax residue, and each transfer step is accompanied by protein / peptide loss; (2) The single-pot, solid phase-enhanced sample preparation (SP3) or suspension trapping (S-Trap) based on magnetic beads has many washing steps, and the recovery rate of phosphorylated peptides is particularly low; (3) The operation is cumbersome, the organic reagent exposure is large, and it is difficult to match the small sample size (<10,000 cells) obtained by LCM. Although reverse phase protein arrays (RPPA) bind to LCM, they rely on antibodies, cannot discover new sites, and also suffer from sample loss due to multiple washes, resulting in a detection depth far inferior to LC-MS.

[0005] In summary, existing technologies suffer from the following main drawbacks: 1) lack of spatial information; 2) cumbersome sample processing steps with significant losses; 3) low recovery rate of phosphorylated peptides, making it difficult to achieve deep coverage; and 4) antibody dependence, limiting throughput and discovery capabilities. Therefore, there is an urgent need to establish a spatially resolved phosphorylated peptide preparation method that is compatible with LCM, simple to operate, has high recovery rates, and is suitable for micro-samples (such as FFPE samples) to achieve high-depth spatial phosphorylated proteomics analysis. Summary of the Invention

[0006] The first objective of this invention is to provide a method for preparing phosphorylated peptides for analysis, comprising the following steps: S1, extracting, reducing, and alkylating a sample containing phosphorylated protein to obtain a denatured protein solution; wherein the sample is selected from tissue samples or cell samples; S2, purifying the denatured protein solution obtained in step S1; S3, enzymatically digesting the purified protein using a mixture of trypsin and Lys-C protease to obtain a peptide mixture; S4, eluting the peptide mixture, collecting the peptides, and drying them; S5, enriching phosphorylated peptides from the dried peptides; wherein, in step S3, no auxiliary enzymatic digestion reagents, including urea, are used during the enzymatic digestion.

[0007] The second objective of this invention is to provide a spatial phosphorylated proteomics detection method, comprising: (1) preparing a slide based on a sample to be tested and staining the slide; (2) selecting a target region using LCM technology and cutting to obtain a sample of the target region; (3) obtaining phosphorylated peptides from the sample of the target region using the method for preparing phosphorylated peptides for analysis; and (4) analyzing the phosphorylated peptides.

[0008] The preparation method of this invention can efficiently extract a sufficient number of phosphorylated peptides from trace samples. It is compatible with LCM, and is simple to operate with high recovery rate, making it suitable for high-depth spatial phosphorylated proteomics analysis.

[0009] The spatial phosphorylation proteomics detection method of this invention can screen thousands of proteins and phosphorylated proteins without pre-defined criteria and can discover novel biomarkers. Simultaneously, the analytical method of this invention exhibits high sensitivity at the micro-level, significantly increasing the number of phosphorylation sites identified compared to traditional methods. Compared to whole-slice homogenization, the analytical method of this invention can more accurately reflect the specificity of cells in the target region and better reflect intratumoral heterogeneity. Furthermore, this method is applicable not only to paraffin-embedded tissues but also to frozen tissues and cell samples, showing broad application prospects. Attached Figure Description

[0010] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0011] Figure 1 This is a schematic diagram of the spatial phosphorylation proteomics process based on laser capture microdissection technology in an embodiment of the present invention.

[0012] Figure 2 This is a schematic diagram of a glioblastoma FFPE section covered with a liquid coverslip after hematoxylin and eosin (HE) staining, as described in an embodiment of the present invention. In this diagram, the red dashed line outlines the sarcomatous portion of the glioblastoma, and the gray dashed line outlines the glial portion of the glioblastoma.

[0013] Figure 3This figure shows a comparison of the phosphorylated protein profiles of FFPE tissue treated with protease digestion buffer and conventional protease digestion buffer containing 1 M urea in this embodiment of the invention. In this figure, a) the results of the number of protein phosphorylation sites identified are compared; b) the results of the number of protein phosphorylated peptides identified are compared; and c) the results of the number of phosphorylated proteins identified are compared.

[0014] Figure 4 This represents the number of quantitative protein phosphorylation sites identified by the phosphorylated proteomics detection method in various sample types in this embodiment of the invention.

[0015] Figure 5 The figures show the results of phosphorylated protein spectra obtained by laser capture microdissection of different areas in this embodiment of the invention; in the figure, a) results of the number of protein phosphorylation sites identified; b) results of the number of protein phosphorylated peptides identified; c) results of the number of phosphorylated proteins identified. Detailed Implementation

[0016] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the invention. Those skilled in the art can make various modifications and variations to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment can be used in another embodiment to produce further embodiments.

[0017] Unless otherwise stated, all terms used to disclose this invention (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Further guidance is provided below for a better understanding of the teachings of this invention. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0018] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B.

[0019] The terms “containing,” “comprising,” and “including” as used in this invention are synonyms and are inclusive or open-ended, not excluding additional, uncited members, elements, or method steps.

[0020] In this invention, the numerical range represented by endpoints includes all numerical values ​​and fractions contained within that range, as well as the endpoints mentioned.

[0021] The numerical values ​​involved in this invention include fluctuations within a certain range. For example, fluctuations are allowed within a corresponding precision range. For instance, 1% can fluctuate within ±0.05%. For larger values ​​or values ​​that do not require overly precise control, even greater fluctuations are permitted. For example, 75% can fluctuate within ranges of ±1%, ±2%, ±5%, etc.

[0022] In this invention, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity of 2 or more.

[0023] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0024] In this invention, terms such as "preferred," "better," "more suitable," and "ideal" are merely descriptions of more effective implementation methods or embodiments, and should be understood not to limit the scope of protection of this invention.

[0025] In this invention, "optionally," "optionally," "optionally," "optionally," "optionally," and "optional" mean that they are optional, that is, they are selected from either "with" or "without." If multiple "optional" or "optional" terms appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, then each "optional" or "optional" term is independent.

[0026] This invention first provides a method for preparing phosphorylated peptides for analysis, comprising the following steps: S1, extracting, reducing, and alkylating a sample containing phosphorylated protein to obtain a denatured protein solution; the sample is selected from tissue samples or cell samples; S2, purifying the denatured protein solution obtained in step S1; S3, enzymatically digesting the purified protein using a mixture of trypsin and Lys-C protease to obtain a peptide mixture; S4, eluting the peptide mixture, collecting the peptides, and drying them; S5, enriching phosphorylated peptides from the dried peptides; wherein, in step S3, no auxiliary enzymatic digestion reagents, including urea, are used during the enzymatic digestion.

[0027] This invention discovers that by avoiding the use of auxiliary enzymatic digestion reagents, including urea, during enzymatic digestion, the enrichment of phosphorylated peptides can be significantly increased.

[0028] In some embodiments, in step S1, a lysis buffer is used to extract and reduce the phosphorylated protein from the sample; the lysis buffer contains 8-12 mM tris(2-carboxyethyl)phosphine, 70-90 mM 4-hydroxyethylpiperazine ethanesulfonic acid, and 3-5% w / v sodium dodecyl sulfate, with a pH of 7.5 ± 0.5. Using the aforementioned lysis buffer further improves the extraction efficiency of phosphorylated proteins.

[0029] In some embodiments, the lysis buffer is mixed with a sample containing phosphorylated proteins and incubated at 97-99°C and 900-1100 rpm for 25-35 minutes, followed by sonication in a water bath at 35-45 kHz and 140-160 W for 8-12 minutes; then the incubation and sonication process is repeated once.

[0030] In some embodiments, in step S1, the protein is alkylated using methyl S-methylthiosulfonate at a final concentration of 15-25 mM, under conditions of incubation at room temperature in the dark for 8-12 minutes.

[0031] In some embodiments, step S2 specifically includes: S21, mixing equal volumes of hydrophilic and hydrophobic magnetic beads equilibrated at room temperature, rinsing successively with pure water and 45-55% v / v acetonitrile aqueous solution, and resuspending in 45-55% v / v acetonitrile aqueous solution to obtain pretreated magnetic beads; S22, mixing the pretreated magnetic beads with the denatured protein solution obtained in step S1, and then adding acetonitrile two or more times sequentially to gradually increase the final acetonitrile concentration for stepwise protein adsorption; S23, discarding the supernatant, washing the magnetic beads 1-2 times with 65-75% v / v ethanol aqueous solution, and then washing the magnetic beads 1-3 times with 65-75% v / v acetonitrile aqueous solution to remove liquid residue. This method further improves the adsorption and purification effect of phosphorylated proteins.

[0032] In some embodiments, in step S3, the pH of the enzymatic digest solution is controlled to be 8.0 ± 0.5.

[0033] In some embodiments, in step S3, triethylammonium bicarbonate is used to adjust the pH of the enzymatic digestion solution. Using this pH adjuster will further improve the quality of the obtained peptide mixture.

[0034] In some embodiments, in step S3, the enzymatic digestion solution contains a mixture of trypsin and Lys-C protease at a mass ratio of 0.01 to 0.03 mg / mL of 1:1 to 10:1 and 80 to 120 mM triethylammonium bicarbonate, with a pH of 7.5 to 8.5.

[0035] In practice, those skilled in the art can obtain a mixture of trypsin and Lys-C protease through commercial channels. For example, in some embodiments, the mixture of trypsin and Lys-C protease can be purchased from Thermo Scientific. TM The product number is A40007.

[0036] In some embodiments, step S3 specifically includes: mixing the enzymatic digestion solution with magnetic beads adsorbed with purified protein, sonicating in a water bath for 8 to 12 minutes at room temperature, 35 to 45 kHz, and 140 to 160 W, and then incubating overnight at 37±1℃ and 250 to 350 rpm to obtain magnetic beads adsorbed with a mixture of peptides.

[0037] In some embodiments, in step S4, peptides are first eluted from the peptide mixture using 40-60% v / v acetonitrile and 1-5% v / v dimethyl sulfoxide aqueous solutions, respectively, and then eluted again from the peptide mixture using 0.1-1% v / v formic acid aqueous solution. The peptide eluent is then collected and dried. The present invention further finds that the elution effect obtained by the above elution sequence is better.

[0038] In some specific embodiments, those skilled in the art can adapt the order of elution using 40-60% V / V acetonitrile and 1-5% V / V dimethyl sulfoxide aqueous solution in step S4, which has no particular impact on the elution effect of the present invention.

[0039] In some embodiments, an aqueous solution of formic acid at a concentration of 0.1–1% V / V (e.g., 0.1% V / V, 0.3% V / V, 0.4% V / V, 0.5% V / V, 0.6% V / V, 0.7% V / V, or 1.0% V / V, etc.) is used to elute the peptides. Using this eluent yields better elution results.

[0040] In some embodiments, step S4 specifically includes: S41, separating and collecting the initial supernatant from the magnetic beads adsorbed with the peptide mixture; S42, sequentially using four eluents—40-60% V / V acetonitrile, 1-5% V / V dimethyl sulfoxide aqueous solution, 0.1-1% V / V formic acid aqueous solution, and water—to perform room temperature ultrasonic-vortex combined extraction on the magnetic beads, separating and collecting the supernatant after each extraction; S43, combining all supernatants and drying to obtain peptide powder.

[0041] In some embodiments, in room temperature ultrasound-vortex combined extraction, the ultrasound conditions are 35-45 kHz and 140-160 W, and the vortex conditions are 1800-2200 rpm.

[0042] In some implementations, the sample is a target region sample obtained based on LCM technology.

[0043] In some implementations, the sample contains 1,000-3,000 cells.

[0044] Those skilled in the art can confirm, based on common sense, that any steps or specific operations not specifically described in this invention can achieve the technical effects described in this invention. For example, in some specific embodiments, those skilled in the art can confirm the specific operation of enriching phosphorylated peptides from dried peptides in step S5 by referring to commercially available reagent kits and their instructions.

[0045] Those skilled in the art can combine the above-mentioned embodiments with common sense to obtain more embodiments of the method for preparing phosphorylated peptides for analysis according to the present invention.

[0046] The present invention also provides a spatial phosphorylated proteomics detection method, which includes: (1) preparing a slice based on the sample to be tested and staining the slice; (2) selecting a target region by LCM technology and cutting to obtain a target region sample; (3) obtaining phosphorylated peptides from the target region sample using the method for preparing phosphorylated peptides for analysis; and (4) analyzing the phosphorylated peptides.

[0047] In some embodiments, the sample to be tested is a paraffin tissue sample, a frozen tissue sample, or a cell sample.

[0048] In some specific embodiments, the sample to be tested is a formalin-fixed paraffin-embedded (FFPE) tissue sample. For example, the sample to be tested can be an FFPE tissue sample of a glioma.

[0049] In some specific embodiments, the sample to be tested can be paraffin-embedded tissue (including clinical archive blocks, paraffin rolls of puncture tissue, or TMA), frozen tissue (OCT-embedded sections or freshly excised specimens), in vitro / primary cells (which can be made into cell clusters or cell slides and then fixed and embedded), fine needle biopsy and endoscopic biopsy FFPE, paraffin-embedded circulating tumor cell blocks or pleural and peritoneal fluid cell sediments, FFPE blocks and frozen sections of mouse / rat / PDX model tumors, etc. After the above samples are laser-captured microdissection to obtain a target area of ​​2.1~10 mm² × 5~10 μm thickness, they can all be used to extract phosphorylated peptides and perform mass spectrometry analysis to achieve spatially resolved, trace-level, or even high-throughput phosphorylated proteomics detection.

[0050] In some embodiments, when the sample to be tested is a paraffin tissue sample (such as an FFPE tissue sample), step (1) before staining the slides further includes: dewaxing the slides; the dewaxing process specifically includes: (11) placing the slides baked at 50~70℃ in fresh xylene 2~4 times (e.g., 2 times, 3 times or 4 times), each time reacting for 2~4 minutes; (12) placing the slides in fresh anhydrous ethanol 2~4 times (e.g., 2 times, 3 times or 4 times), each time reacting for 20~40 seconds; (13) placing the slides in 95±2% ethanol, 85±2% ethanol, and 75±2% ethanol in sequence, reacting for 20~40 seconds respectively. Through the above method, the dewaxing process of paraffin tissue samples can be completed quickly while taking into account the extraction effect of phosphoprotein.

[0051] In some specific embodiments, the slides are stained with hematoxylin staining solution and water-soluble eosin staining solution in step (1).

[0052] In some specific embodiments, the staining treatment in step (1) specifically includes: placing the slide in hematoxylin staining solution and reacting for 5-10 minutes, rinsing the slide for 15-30 minutes until blue reflection is observed under a microscope, then rinsing the slide for 0.2-2 minutes and placing it in water-soluble eosin staining solution and reacting for 20-40 seconds, rinsing the slide for 20-40 seconds, and then placing the slide in 85±2% ethanol, 95±2% ethanol, and 98-100% ethanol in sequence, reacting for 20-40 seconds each time before taking it out.

[0053] In some embodiments, step (2) specifically includes: constructing a temporary chamber liquid coverslip with anhydrous ethanol on the front side of a polyethylene naphthalate (PEN) slide carrying stained sections; removing the anhydrous ethanol and drying the slide after outlining the target area; and then using a laser to cut the sample from the target area into a collection tube. This invention has found that the ethanol coverslip has a high refractive index matching degree with the tissue, which can improve transparency and resolution, and avoid the inaccurate identification of the target area due to tissue drying. At the same time, ethanol is volatile; after the target area is selected, removing the liquid coverslip allows the section to dry quickly, without affecting the sample obtained by laser cutting.

[0054] In some embodiments, step (4) involves analyzing the phosphorylated peptides using liquid chromatography-mass spectrometry (LC-MS), specifically including: reconstituted the phosphorylated peptides with 0.05-0.2% V / V formic acid aqueous solution, followed by separation using C18 nano-level liquid chromatography, data acquisition in data-dependent acquisition (DDA) mode using a trapping ion mobility spectrometer, and real-time matching analysis between peptide spectra and protein databases, adding peptide information with a matching confidence threshold ≥99% to the library.

[0055] In some specific embodiments, the reconstituted phosphorylated peptide is injected into a liquid chromatograph (Bruker, nanoElute 2 UHPLC) and separated by liquid phase using a liquid mass spectrometry column (Bruker PepSep C18, 25 cm).

[0056] In some specific embodiments, the trapping ion mobility spectrometer-mass spectrometer is a Bruker timsTOFpro2.

[0057] In some specific implementations, the Bruker ProteoScape software is used, and the dda_PASEF_TIMSrescore_Human_phospho_RT method is selected for the real-time matching analysis.

[0058] In practice, those skilled in the art may use other instruments and software, and select data acquisition methods that provide a deeper peptide identification than conventional DDA for the real-time matching analysis.

[0059] Using the analytical method of this invention, approximately 1000 phosphorylation sites, over 600 phosphorylated peptides, and over 300 phosphorylated proteins can be detected using only 2.1~10 mm² × 5~10 μm thick paraffin tissue. Furthermore, the entire process from section preparation to mass spectrometry detection of phosphorylated proteins can be completed within two days.

[0060] Those skilled in the art can combine the above-described embodiments with common sense to obtain more embodiments of the spatial phosphorylation proteomics detection method of the present invention.

[0061] The embodiments of the present invention will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this invention, or follow experimental manuals or conventional conditions in the art, or other experimental methods known in the art, or follow the conditions recommended by the manufacturer.

[0062] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0063] Example 1 This embodiment provides a method for detecting spatial phosphorylation proteomics, the flowchart of which is shown below. Figure 1 As shown, the specific steps are as follows: I. Slice Preparation 1. Cover the front side of the PEN slide (ZEISS, 415190-9041-001) with a 0.01% (w / v) poly-L-lysine solution; 2. Place the PEN glass slide covered with poly-L-lysine in an oven and bake at 60°C for 1 hour; 3. Pre-cool the paraffin-embedded glioblastoma tissue block to -20℃; 4. Fill the water tank with pure water and heat it to 40℃; 5. Set the section thickness to 10µm and use a paraffin microtome to perform the sectioning; 6. Transfer the sections to 40℃ pure water for spreading; 7. Use the processed PEN slide to retrieve the section from the water bath; 8. Once all slices are finished, bake them in an oven at 60°C for 1 hour.

[0064] II. Rapid dewaxing and staining of sections 1. Place the baked slices into fresh xylene three times while they are still hot, reacting for 3 minutes each time; 2. Place the slices in fresh anhydrous ethanol three times, reacting for 30 seconds each time; 3. Place the slices in 95% ethanol, 85% ethanol, and 75% ethanol in sequence, and react for 30 seconds each time; 4. Rinse the slices under running tap water for 1 minute. 5. Place the sections into the hematoxylin staining solution and react for 7 minutes; 6. Rinse the slides under running tap water for 15-30 minutes until they turn blue under a microscope; 7. After rinsing the sections with pure water under slow flow for 1 minute, place them in water-soluble eosin staining solution and react for 30 seconds; 8. Rinse the sections with pure water under a slow flow for 30 seconds; 9. Place the slices in 85% ethanol, 95% ethanol, and 100% ethanol in sequence, and remove them after reacting for 30 seconds each.

[0065] III. Microdissection for tissue collection 1. Prepare a chambered liquid coverslip: Construct a chamber on the front side of the stained tissue section, add 1 mL of anhydrous ethanol to the chamber to fully cover the tissue section; 2. For example Figure 2 As shown, the target area in the tissue section was delineated and selected using a micro-laser cutting system (ZESIS, PALM-icroBeam system). In this figure, the red dashed line delineates the selected area as the glioblastoma sarcoma part, while the gray dashed line delineates the selected area as the glioblastoma glial part. 3. Remove the anhydrous ethanol from the chamber and allow the remaining ethanol to air dry naturally; 4. Use a laser to cut the target area sample from the tissue section and eject it into the cap of the corresponding collection tube (ZEISS, 415190-9201-000).

[0066] IV. Protein Extraction and Reductive Alkylation 1. Preparation of lysis buffer: The lysis buffer contains 10 mM tris(2-carboxyethyl)phosphine, 80 mM 4-hydroxyethylpiperazine ethanesulfonic acid and 4% (w / v) sodium dodecyl sulfate, with a pH of 7.5; 2. Invert the collection tube onto a table, open the cap, add 100µL of lysis buffer to the cap, resuspend the harvested tissue, and transfer to a 1.5mL low-protein-binding microcentrifuge tube (Thermo Scientific). TM Pierce TM (90410) 3. Place the low protein binding microcentrifuge tubes in a metal bath (Eppendorf, Thermo Mixer C), incubate at 99°C and 1000 rpm for 30 minutes, then sonicate in a water bath (40 kHz, 150 W) for 10 minutes; repeat once. 4. Add methyl S-methylthiosulfonate to a concentration of 20 mM, mix well, and incubate at room temperature in the dark for 10 min.

[0067] V. Protein Purification 1. Take hydrophobic (Cytiva, 65152105050250) and hydrophilic (Cytiva, 45152105050250) magnetic beads and equilibrate at room temperature for 30 minutes, then invert the beads until the microbead suspension is homogeneous; 2. Take 20µL each of hydrophilic and hydrophobic magnetic beads, mix them, add 160µL of pure water, mix well, place on a magnetic rack, and remove the supernatant; 3. Add 200µl of 50% (V / V) acetonitrile aqueous solution to the magnetic beads, mix well, place on a magnetic rack, remove the supernatant, repeat twice, and then resuspend the magnetic beads in 100µl of 50% (V / V) acetonitrile aqueous solution; 4. Add 20 µL of resuspended magnetic beads to the reduced alkylated sample, mix well, and react for 2 minutes; 5. Add 60µL of acetonitrile to the test tube containing the magnetic beads and the sample, mix well, and react for 3 minutes; 6. Add 180µL of acetonitrile, mix well, and react for 3 minutes; 7. Incubate the sample on a magnetic rack for 2 minutes, then remove the supernatant; 8. Clean the magnetic beads once with 500µL of 70% (V / V) ethanol aqueous solution, and then clean the magnetic beads twice with 500µL of 70% (V / V) acetonitrile aqueous solution; 9. Air-dried liquid residue.

[0068] VI. One-step pancreatic enzyme digestion 1. Preparation of enzymatic digestion solution: Prepare a mixture containing 0.02 mg / mL trypsin / Lys-C protease using mass spectrometry-grade pure water (Thermo Scientific). TM Enzymatic digestion solution with pH 8, containing 100mM triethylammonium bicarbonate (Sigma, T7408) and A40007; 2. Add 100µL of enzymatic digestion solution to the magnetic beads; 3. Ultrasonic treatment in a water bath at room temperature (40kHz, 150W) for 10 minutes; 4. Incubate overnight in a metal bath at 37℃ and 300 rpm.

[0069] VII. Peptide Elution and Drying 1. Place the digested sample on a magnetic rack and collect the supernatant into a low-protein-binding microcentrifuge tube; 2. Add 120µL of 50% (V / V) acetonitrile aqueous solution to the magnetic beads, sonicate in a water bath at room temperature (40kHz, 150W) for 3 minutes, vortex at 2000 rpm for 3 minutes, place the magnetic beads on a magnetic rack, and collect the supernatant into a low protein binding microcentrifuge tube. 3. Add 120µL of 2% (V / V) dimethyl sulfoxide aqueous solution to the magnetic beads, sonicate in a water bath at room temperature (40kHz, 150W) for 3 minutes, vortex at 2000 rpm for 3 minutes, place the magnetic beads on a magnetic rack, and collect the supernatant into a low protein binding microcentrifuge tube. 4. Add 120µL of 0.5% (V / V) formic acid aqueous solution to the magnetic beads, sonicate in a water bath at room temperature (40kHz, 150W) for 3 minutes, vortex at 2000 rpm for 3 minutes, place the magnetic beads on a magnetic rack, and collect the supernatant into a low protein binding microcentrifuge tube. 5. Add 120µL of mass spectrometry grade pure water to the magnetic beads, sonicate in a water bath at room temperature (40kHz, 150W) for 3 minutes, vortex at 2000 rpm for 3 minutes, place the magnetic beads on a magnetic rack, and collect the supernatant into a low protein binding microcentrifuge tube. 6. Place the collected supernatant in a vacuum dryer and dry for 6 hours to obtain dried peptide powder.

[0070] 8. Enrichment of phosphorylated peptides 1. The main lyophilized peptides were mixed with 150 μL Binding / Equilibration Buffer (ThermoScientific TM (A32993) Fully resuspended; 2. Insert the centrifuge column adapter into the centrifuge tube, and then insert the enrichment pipette tip filled with TiO2; 3. Add 20 μL of Wash Buffer to the enrichment pipette tip and centrifuge at 3000×g for 2 minutes; then add 20 μL of Binding / Equilibration Buffer and centrifuge at 3000×g for 2 minutes. 4. Reload the centrifuge column adapter and enrichment tip into a new low-protein binding microcentrifuge tube; 5. Add 150 μL of dissolved peptide sample to the enrichment tip, centrifuge at 1000×g for 5 minutes, add the liquid from the bottom of the centrifuge tube back to the same enrichment tip, and centrifuge at 1000×g for 5 minutes. 6. Reinstall the centrifuge column adapter and enrichment tip into the new centrifuge tube; 7. Add 20 μL of Binding / Equilibration Buffer to the enrichment pipette tip, centrifuge at 3000×g for 2 minutes, then add 20 μL of Wash Buffer and centrifuge at 3000×g for 2 minutes; repeat once. 8. Add 20 μL of mass spectrometry grade pure water to the enrichment pipette tip and centrifuge at 3000×g for 2 minutes; 9. Reload the centrifuge column adapter and enrichment tip into a new low-protein binding microcentrifuge tube; 10. Add 50 μL of Phosphhopeptide Elution Buffer to the enrichment pipette tip, centrifuge at 1000×g for 5 minutes; repeat once, collect a total of 100 μL of eluent, and immediately place it in a vacuum dryer to dry for 2 hours to obtain dried phosphorylated peptide powder.

[0071] IX. Mass Spectrometry Detection 1. Add 10µL of 0.1% (V / V) formic acid aqueous solution to the collection tube of phosphorylated peptide powder, mix well, and then transfer to the corresponding mass spectrometer loading liner tube; 2. The sample peptides were injected into a liquid chromatograph (Bruker, nanoElute 2 UHPLC) and then injected into a mass spectrometer (Bruker, timsTOF pro2) through a liquid chromatography-mass spectrometry column (Bruker PepSep C18, 25cm). 3. For phosphorylated peptide powder samples, mass spectrometry data were collected using data-dependent acquisition (DDA) mode, and protein library search and acquisition were performed in real time using Bruker ProteoScape software (method selected as dda_PASEF_TIMSrescore_Human_phospho_RT). Peptides with a range greater than 99% were added to the library.

[0072] Example 2 This embodiment uses 50mm. 2 Phosphorylated peptides were prepared and phosphorylated proteomics were detected using FFPE tissue sections of human gliomas measuring ×10µm. The effects of different enzymatic digestion solutions on the final identification of phosphorylation sites, phosphorylated peptides, and the number of phosphorylated proteins in tissue samples of the same volume were compared.

[0073] Add 200 µL of the enzymatic digestion solution used in Example 1 of this invention and a conventional enzymatic digestion solution containing 1 M urea (containing a 0.02 mg / mL trypsin / Lys-C protease mixture (Thermo Scientific)) to the purified protein beads. TMThe enzyme digestion solution contained 1M urea (A40007), 100mM triethylammonium bicarbonate (Sigma, T7408), and 1M urea at pH 8 was prepared. The sample was ultrasonicated in a water bath at 40kHz, 150W for 10 minutes at room temperature; then incubated overnight in a metal bath at 37°C, 300 rpm. The elution steps for the enzyme digestion solution group using this invention were the same as described in Example 1. The elution steps for the conventional enzyme digestion solution group containing 1M urea were as follows: 500µL of acetonitrile was added to the enzyme digestion solution group containing 1M urea and incubated at room temperature for 8 minutes. The sample was then placed on a magnetic rack and incubated for 2 minutes. After removing the supernatant, 1000µL of 70% acetonitrile was added to wash the magnetic beads. The supernatant was discarded, and the washing was repeated once with 1000µL of 70% acetonitrile. Subsequent peptide elution, phosphorylated peptide enrichment, and mass spectrometry detection were performed using the same methods as described in Example 1.

[0074] The results are as follows Figure 3 As shown, compared with the enzymatic digestion solution group containing 1M urea, the number of protein phosphorylation sites identified in the enzymatic digestion solution group used in this invention is ( Figure 3 The number of phosphorylated peptides identified in protein a)) Figure 3 (b) and the number of phosphorylated proteins identified ( Figure 3 c)) increased significantly.

[0075] Example 3 This embodiment uses 100mm. 2 Phosphorylated peptides were prepared and phosphorylated proteomics were performed on 10µm × 10µm human glioma FFPE tissue sections, 1000µg of frozen human glioma tissue proteins, and 1000µg of U87 protein from human glioma cells. 1000µg of frozen tissue and cells were collected from each group using conventional methods. The paraffin in the FFPE tissue was removed using the dewaxing method described in Example 1 of this invention. The same protein extraction, reductive alkylation, and subsequent processing methods were applied to all three groups, with the corresponding steps as described in Example 1.

[0076] The results are as follows Figure 4 As shown in the results, the method of the present invention is applicable to a variety of common clinical samples.

[0077] Example 4 In this embodiment, human glioma FFPE groups with different areas and a thickness of 10µm were dissected. Sample pretreatment and liquid chromatography-mass spectrometry (LC-MS) detection were performed according to the method in Example 1 of this invention. The obtained protein phosphorylation sites, phosphorylated peptides, the number of phosphorylated proteins, and the corresponding tissue areas are as follows: Figure 5 As shown in the figure. The results show that the detection method of the present invention can still identify more than 1,000 phosphorylation sites, more than 600 phosphorylated peptides, and more than 300 phosphorylated proteins even when the sample area is as small as 5 mm², and can effectively perform spatial phosphorylated proteomics analysis.

[0078] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of protection of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A method for preparing phosphorylated peptides for analysis, comprising the following steps: S1. The sample containing phosphorylated protein is subjected to protein extraction, reduction and alkylation to obtain a denatured protein solution; the sample is selected from tissue samples or cell samples; S2. Purify the denatured protein solution obtained in step S1; S3. The purified protein was enzymatically digested using a mixture of trypsin and Lys-C protease to obtain a mixture of peptide fragments. S4. Elute the peptide mixture, collect the peptides and dry them; S5. Phosphorylated peptides are enriched from the dried peptides. in, When performing enzymatic digestion in step S3, no auxiliary enzymatic digestion reagents, including urea, are used.

2. The method for preparing phosphorylated peptides for analysis according to claim 1, wherein, In step S1, lysis buffer is used to extract and reduce proteins from samples containing phosphorylated proteins. The lysis buffer contains 8-12 mM tris(2-carboxyethyl)phosphine, 70-90 mM 4-hydroxyethylpiperazine ethanesulfonic acid and 3-5% w / v sodium dodecyl sulfate, with a pH of 7.5 ± 0.5; Preferably, the lysis buffer is mixed with the sample containing phosphorylated protein and incubated at 97-99°C and 900-1100 rpm for 25-35 minutes, followed by sonication in a water bath at 35-45 kHz and 140-160 W for 8-12 minutes; then the incubation and sonication process is repeated once.

3. The method for preparing phosphorylated peptides for analysis according to claim 1 or 2, wherein, In step S1, the protein is alkylated using methyl S-methylthiosulfonate at a final concentration of 15-25 mM under the condition of incubation at room temperature in the dark for 8-12 minutes.

4. The method for preparing the phosphorylated peptide for analysis according to any one of claims 1 to 3, wherein, Step S2 specifically includes: S21. Mix equal volumes of hydrophilic and hydrophobic magnetic beads that have been equilibrated at room temperature, rinse successively with pure water and 45-55% V / V acetonitrile aqueous solution, and then resuspend in 45-55% V / V acetonitrile aqueous solution to obtain pretreated magnetic beads. S22. The pretreated magnetic beads are mixed with the denatured protein solution obtained in step S1 and reacted. Acetonitrile is then added twice more in sequence to gradually increase the final acetonitrile concentration so as to adsorb the protein in a stepwise reaction. S23. After discarding the supernatant, wash the magnetic beads 1-2 times with a 65-75% V / V ethanol aqueous solution, and then wash the magnetic beads 1-3 times with a 65-75% V / V acetonitrile aqueous solution to remove any liquid residue.

5. The method for preparing the phosphorylated peptide for analysis according to any one of claims 1 to 4, wherein, In step S3, the pH of the enzymatic digestion solution is controlled to be 8.0 ± 0.5; preferably, triethylammonium bicarbonate is used to adjust the pH of the enzymatic digestion solution.

6. The method for preparing phosphorylated peptides for analysis according to claim 1, wherein, Step S3 specifically includes: The enzymatic digestion solution was mixed with magnetic beads adsorbed with purified protein, and the mixture was sonicated in a water bath for 8-12 minutes at room temperature, 35-45 kHz, and 140-160 W. Then, it was incubated overnight at 37±1℃ and 250-350 rpm to obtain magnetic beads adsorbed with a mixture of peptides. The enzymatic digestion solution contains a mixture of trypsin and Lys-C protease at a mass ratio of 1:1 to 10:1 (0.01–0.03 mg / mL) and 80–120 mM triethylammonium bicarbonate, with a pH of 7.5–8.

5.

7. A method for preparing the phosphorylated peptide for analysis according to any one of claims 1 to 3, wherein, In step S4, peptides are first eluted from the peptide mixture using 40-60% V / V acetonitrile and 1-5% V / V dimethyl sulfoxide aqueous solutions, respectively. Then, peptides are eluted again from the peptide mixture using 0.1-1% V / V formic acid aqueous solution. The peptide eluent is collected and dried.

8. The method for preparing phosphorylated peptides for analysis according to claim 7, wherein, Step S4 specifically includes: S41. Separate and collect the primary supernatant from the magnetic beads containing the peptide mixture; S42. The magnetic beads were subjected to room temperature ultrasonic-vortex extraction using four eluents in sequence: 40-60% V / V acetonitrile, 1-5% V / V dimethyl sulfoxide aqueous solution, 0.1-1% V / V formic acid aqueous solution, and water. The supernatant was separated and collected after each extraction. S43. Combine all supernatants and dry to obtain peptide powder; Preferably, in the room temperature ultrasonic-vortex combined extraction, the ultrasonic conditions are 35~45 kHz and 140~160 W, and the vortex conditions are 1800~2200 rpm.

9. The method for preparing the phosphorylated peptide for analysis according to any one of claims 1 to 8, wherein, The sample is a target region sample obtained based on LCM technology; preferably, the sample contains 1000 to 3000 cells.

10. A method for detecting spatial phosphorylation proteomics, comprising: (1) Prepare slides based on the sample to be tested, and stain the slides; (2) Select the target region using LCM technology and cut it to obtain the target region sample; (3) Obtaining phosphorylated peptides from the target region sample using the method for preparing phosphorylated peptides for analysis according to any one of claims 1 to 9; (4) Analyze the phosphorylated peptides.

11. The method for detecting spatial phosphorylation proteomics according to claim 10, wherein, The sample to be tested is a paraffin tissue sample, a frozen tissue sample, or a cell sample.

12. The spatial phosphorylation proteomics detection method according to claim 10, wherein, When the sample to be tested is a paraffin tissue sample, step (1) further includes, before staining the section, dewaxing the section; the dewaxing process specifically includes: (11) Place the slices baked at 50~70℃ into fresh xylene 2~4 times, each time for 2~4 minutes; (12) Place the slices in fresh anhydrous ethanol 2 to 4 times, each time for 20 to 40 seconds; (13) Place the slices in 95±2% ethanol, 85±2% ethanol and 75±2% ethanol in sequence, and react for 20~40 seconds respectively.

13. The method for detecting spatial phosphorylation proteomics according to any one of claims 10-12, wherein, Step (2) specifically includes: On the front side of a polyethylene naphthalate slide containing stained sections, a temporary chamber liquid cover slide is constructed with anhydrous ethanol. After the target area is delineated, the anhydrous ethanol is removed and the slide is dried. Subsequently, the sample from the target area is cut and transferred to a collection tube using a laser.

14. The method for detecting spatial phosphorylation proteomics according to any one of claims 10-13, wherein, In step (4), the phosphorylated peptide is analyzed by liquid chromatography-mass spectrometry, specifically including: Phosphorylated peptides were reconstituted in 0.05–0.2% V / V formic acid aqueous solution, separated by C18 nano-level liquid chromatography, and data were acquired using trap ion mobility spectrometry-mass spectrometry in data-dependent mode. Real-time matching analysis of peptide spectra with protein databases was performed, and peptide information with a matching confidence threshold ≥99% was added to the library.

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