A liP-ms sample preparation method for reducing non-specific peptide fragment interference

Abstract

The application discloses a LiP-MS sample preparation method for reducing non-specific peptide segment interference. After limited hydrolysis of proteins by using a non-specific protease, purification treatment is performed to remove non-specific peptide segments. The purification treatment uses an organic solvent to adjust the polarity of a first enzyme solution, so that target proteins are precipitated, and non-specific peptide segments are not precipitated. Non-specific peptide segments are removed by centrifugal separation, and potential target proteins after purification are obtained. The organic solvent includes a mixed reagent of acetone, trichloroacetic acid, methanol and trichloromethane. The purified proteins are prepared into LiP-MS samples, which are used for mass spectrometry detection and analysis. Compared with a traditional method for preparing LiP-MS samples, the method effectively removes interference substances such as non-specific peptide segments and drug molecules, reduces the interference of non-target ions, makes the signal of target peptide segments clearer, improves the confidence of spectrum matching, and reduces false positive results.

Description

Technical Field

[0001] This invention belongs to the field of functional proteomics detection and analysis technology, and more specifically, relates to a LiP-MS sample preparation method that reduces interference from non-specific peptides. Background Technology

[0002] LiP-MS (Limited Proteolysis-Mass Spectrometry) is a proteomics technique that has emerged in recent years. It first involves limited hydrolysis of proteins using non-specific proteases (such as proteinase K) under non-denaturing conditions, followed by enzymatic cleavage with specific proteases (such as trypsin) to produce fully cleaved peptides, semi-specifically cleaved peptides, and non-specifically cleaved peptides. Limited hydrolysis with proteinase K primarily produces non-specifically cleaved peptides. Finally, combined with mass spectrometry detection, it can detect conformational changes of proteins under specific conditions (such as binding to small molecule drugs). This technique has broad application prospects in drug target identification, protein function research, and the elucidation of interaction mechanisms.

[0003] For example, patent CN119395303A uses LiP-MS technology to screen for targets of shikonin. However, non-specific peptides generated during the limited hydrolysis with proteinase K in sample preparation are not removed. This is mainly because existing technologies do not recognize the need to remove these non-specific peptides before trypsin digestion. These non-specific peptides can severely interfere with mass spectrometry detection and data analysis later, and may even ultimately affect the detection results of LiP-MS. Patent CN120294192A uses LiP-MS and phosphorylated proteomics to analyze the interaction between small molecules and target proteins. While this can be used to elucidate the mechanism of action of small molecule compounds such as pomolic acid (PA) on Ki67, it also fails to remove the non-specific peptides generated during the limited hydrolysis with proteinase K in sample preparation, and cannot reduce or avoid the interference of non-specific enzymatically digested peptides on mass spectrometry detection and data analysis. Therefore, it is unclear whether this technology can be directly applied to the analysis of the mechanism of action of other target proteins.

[0004] To develop a LiP-MS technique widely applicable to the analysis of target protein mechanisms of action, it is necessary to remove non-specific enzymatic peptides generated by limited hydrolysis using proteinase K to reduce their interference with subsequent mass spectrometry analysis. One study utilized acetonitrile (ACN) precipitation to enrich low molecular weight proteins in serum (DOI:10.1002 / rcm.3729), a method designed to selectively remove high molecular weight and abundant proteins from serum. However, direct application to LiP-MS may result in the ineffective removal of non-specific enzymatic peptides or the loss of some target proteins during removal, thus affecting the analytical results.

[0005] Therefore, how to remove as many non-specific enzymatically cleaved peptides as possible while ensuring no loss of the target protein, so as to reduce the interference they cause, is a pressing challenge that needs to be addressed. Summary of the Invention

[0006] To address the aforementioned technical deficiencies or improvement needs, this invention provides a LiP-MS sample preparation method that reduces interference from non-specific peptides. The aim is to perform purification after limited protein hydrolysis for the first time. By using an organic solvent to adjust the polarity of the first enzymatic digest, the target protein precipitates while non-specific peptides do not. Centrifugation removes the non-specific peptides, avoiding interference from these non-target ions. This solves the technical problem of non-specific peptide interference in samples prepared by existing LiP-MS technology, which affects the mass spectrometry detection and data analysis of the target peptides and easily leads to false positives.

[0007] To achieve the above objectives, according to a first aspect of the present invention, a method for preparing LiP-MS samples to reduce nonspecific peptide interference is provided, comprising the following steps: (1) Limited hydrolysis of protein: The total protein extracted from the sample is dissolved in the lysis buffer to obtain the protein lysis buffer. Non-specific protease is added and limited hydrolysis is carried out under non-denaturing conditions to obtain the first enzymatic hydrolysate containing non-specific peptides and target proteins.

[0008] (2) Sample purification: terminate the limited hydrolysis and purify the first enzymatic hydrolysate containing non-specific peptides and target protein obtained in step (1) to remove non-specific peptides; the purification process uses an organic solvent to adjust the polarity of the first enzymatic hydrolysate, so that the target protein precipitates and the non-specific peptides do not precipitate, and centrifuges to remove the non-specific peptides to obtain the purified potential target protein; the organic solvent includes acetone, trichloroacetic acid, and a mixture of methanol and trichloroacetic acid.

[0009] (3) Specific enzymatic digestion: The purified protein in step (2) is subjected to a reduction alkylation reaction, and a specific protease is added to perform specific enzymatic digestion of the target protein under denaturing conditions to obtain a second enzymatic digestion solution including fully digested peptides and semi-specifically digested peptides. After terminating the enzymatic digestion, desalting is performed to obtain a LiP-MS sample for subsequent mass spectrometry detection and analysis.

[0010] Preferably, in the method, the non-specific peptide is <5kD.

[0011] Preferably, in the method, step (2) of the purification process involves adding 4 times the volume of icy acetone, 1 / 4 volume of trichloroacetic acid, or an equal volume of methanol and 1 / 4 volume of trichloromethane as a purification reagent based on the volume of the first enzymatic hydrolysate to separate and remove non-specific peptides.

[0012] Preferably, in the method, the purification process in step (2) involves first adjusting the pH of the first enzymatic hydrolysate to 0.5-3 using an organic acid or its solution, then adding an organic solvent to induce the precipitation of the target protein and prevent the precipitation of nonspecific peptides, thereby separating and removing the nonspecific peptides.

[0013] Preferably, in the method, the purification process in step (2) involves first adjusting the pH of the first enzymatic hydrolysate to 0.8-1 using an organic acid or its solution, then adding an organic solvent to induce the precipitation of the target protein and prevent the precipitation of nonspecific peptides, thereby separating and removing the nonspecific peptides.

[0014] Preferably, in the method, the organic acid includes trifluoroacetic acid.

[0015] Preferably, the method involves purifying the peptides by combining a 10% TFA solution with icy acetone to remove nonspecific peptides.

[0016] Preferably, in the purification process of the method, the pH of the first enzymatic hydrolysate is first adjusted to 0.5-3 using a 10% trifluoroacetic acid solution, and then four times the volume of ice-cold acetone is added to induce precipitation of the target protein and prevent precipitation of non-specific peptides.

[0017] Preferably, the method is used to analyze the interaction between small molecules and target proteins. In step (1), the protein lysis buffer is first incubated with the small molecule at room temperature, and then a non-specific protease is added for limited hydrolysis under non-denaturing conditions. The non-specific protease includes proteinase K.

[0018] Preferably, in the case where the target of the small molecule and the target protein is unknown, the method collects the protein and the supernatant containing non-specific peptides during the purification process in step (2) to obtain two LiP-MS samples, and detects them by mass spectrometry and integrates the two sets of data to analyze the potential target of the small molecule.

[0019] Preferably, in the method, the non-specific protease includes proteinase K, and the first enzymatic hydrolysate is obtained by digestion for 3-5 minutes at a substrate to non-specific protease ratio of 100:1.

[0020] Preferably, in the method, the specific protease includes trypsin, and the second enzymatic hydrolysate is obtained by enzymatic hydrolysis for more than 10 hours at a substrate to non-specific protease ratio of 50:1.

[0021] In summary, compared with the prior art, the above-described technical solutions conceived by this invention can achieve the following beneficial effects: The preparation method provided by this invention, through purification treatment with the addition of organic solvents, can effectively remove interfering substances such as non-specific peptides and drug molecules, reduce interference from non-target ions, make the signal of the target peptide clearer, improve the confidence of spectral matching, and reduce false positive results. At the same time, the higher signal-to-noise ratio allows for the identification of more real, small molecule-induced protein conformational change sites in a single experiment, improving data output quality and screening throughput, and facilitating the rapid and effective screening of small molecule targets for protein interaction. Attached Figure Description

[0022] Figure 1 This is the experimental flowchart.

[0023] Figure 2 This is the BSA verification result.

[0024] Figure 3 It is a BSA structural analysis.

[0025] Figure 4 This is a comparison of the protein precipitation efficiency under different organic solvents and acidification conditions.

[0026] Figure 5 It is a comparison of the number of proteins identified.

[0027] Figure 6 It refers to the distribution of the physicochemical properties of proteins.

[0028] Figure 7 It represents the percentage of semi-specific enzyme-digested peptides.

[0029] Figure 8 It is a comparison of experimental results. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0031] Conventional proteomics sample preparation often involves precipitating proteins with organic solvents (typically 4 volumes of acetone or 1 / 4 volume of trichloroacetic acid). This step removes lipids, nucleic acids, polysaccharides, and small metabolic molecules from the sample, allowing for protein separation. The obtained proteins are then completely digested with trypsin under denaturing conditions, followed by mass spectrometry analysis to maximize the identification of protein types and abundance. LiP-MS, however, utilizes a two-step differential enzymatic digestion process to generate peptides for mass spectrometry detection while preserving protein structural information.

[0032] This invention, based on LiP-MS technology, investigated the removal effects of different treatment methods on non-specific peptides generated by limited hydrolysis of proteinase K using a bovine serum albumin model. The results showed that compared to traditional LiP-MS technology, treating the proteinase K hydrolysate with organic solvents (such as icy acetone, trichloroacetic acid, or a mixture of methanol and chloroform) effectively removed non-specific peptides. In particular, the combined treatment of the proteinase K hydrolysate with acidification and organic solvents improved sample detection accuracy while removing non-specific peptides (<5kD).

[0033] Based on this, the present invention provides a LiP-MS sample preparation method for reducing non-specific peptide interference, which includes the following steps: (1) Limited hydrolysis of protein: The total protein extracted from the sample is dissolved in the lysis buffer to obtain the protein lysis buffer. Non-specific protease is added and limited hydrolysis is carried out under non-denaturing conditions to obtain the first enzymatic hydrolysate containing non-specific peptides and target proteins.

[0034] (2) Sample purification: terminate the limited hydrolysis and purify the first enzymatic hydrolysate containing non-specific peptides and target protein obtained in step (1) to remove non-specific peptides; the purification process uses an organic solvent to adjust the polarity of the first enzymatic hydrolysate, so that the target protein precipitates and the non-specific peptides do not precipitate, and centrifuges to remove the non-specific peptides to obtain the purified potential target protein; the organic solvent includes acetone, trichloroacetic acid, and a mixture of methanol and trichloroacetic acid.

[0035] (3) Specific enzymatic digestion: Dissolve the protein precipitate obtained in step (2) and carry out a reduction alkylation reaction; add trypsin to perform specific enzymatic digestion of the target protein under denaturing conditions to obtain a second enzymatic digestion solution including fully digested peptides and semi-specifically digested peptides; terminate the enzymatic digestion and desalt to obtain LiP-MS samples for subsequent mass spectrometry detection and analysis.

[0036] The non-specific peptide is <5kD; the non-specific protease includes proteinase K; the specific protease includes trypsin.

[0037] Step (2) Sample purification is preferably performed using a combination of acidification and organic solvents. First, the pH of the first enzymatic hydrolysate is adjusted to 0.5-3 using an organic acid or its solution. Then, an organic solvent is added to induce precipitation of the target protein while preventing precipitation of non-specific peptides. The non-specific peptides are then removed by centrifugation. More preferably, the pH of the first enzymatic hydrolysate is adjusted to 0.8-1 using an organic acid or its solution. Then, an organic solvent is added and centrifuged to remove the non-specific peptides. The organic acid includes trifluoroacetic acid (TFA). For example, a 10% TFA solution is used in combination with ice-cold acetone. The pH of the first enzymatic hydrolysate is adjusted to 0.5-3 using a 10% TFA solution. Then, four times the volume of ice-cold acetone is added and the mixture is treated overnight.

[0038] In some embodiments, the nonspecific peptides are <5kD. When purifying the sample, add 4 times the volume of icy acetone, 1 / 4 volume of trichloroacetic acid, or an equal volume of methanol and 1 / 4 volume of chloroform as a purification reagent based on the volume of the first enzymatic hydrolysate. Then, centrifuge to remove the nonspecific peptides.

[0039] In some embodiments, the protein undergoes limited hydrolysis by adding a non-specific protease at a substrate-to-non-specific protease ratio of 100:1, and performing limited hydrolysis on the protein surface under non-denaturing conditions; the non-specific protease includes proteinase K.

[0040] In some embodiments, the specific enzymatic digestion involves adding a specific protease at a substrate-to-specific protease ratio of 50:1 and performing specific enzymatic cleavage of the target protein under denaturing conditions; the specific protease includes trypsin.

[0041] To analyze the interaction between small molecules and target proteins, the method involves step (1) first incubating the protein lysate with the small molecule at room temperature, and then adding a non-specific protease for limited hydrolysis under non-denaturing conditions; the non-specific protease includes proteinase K.

[0042] For example, this method is used to screen the target of the small molecule drug asteroidin. In step (1), the protein lysis buffer is incubated with asteroidin at room temperature (30 min at 25°C), and then proteinase K is added for limited hydrolysis (5 min at 25°C) to obtain the first hydrolysate. The first hydrolysate is first purified by sample treatment to remove non-specific peptides, and then subjected to reduction alkylation reaction. Trypsin is added for enzymatic hydrolysis overnight (12 h). The enzymatic hydrolysis is terminated and desalted to obtain LiP-MS sample. The sample without drug is used as the control group. Liquid chromatography-mass spectrometry is used to analyze the hydrolyzed peptides. Quantitative proteomics and bioinformatics methods are combined to analyze the target of asteroidin. The target of asteroidin is analyzed by this method, which includes ATP-binding protein and protein kinase. It is said that asteroidin is a broad-spectrum protein kinase inhibitor, and its mechanism of action belongs to the typical ATP competitive inhibition type.

[0043] In cases where the target sites of small molecules and target proteins are unknown, during the purification process in step (2), the protein and the supernatant containing non-specific peptides are collected to obtain two LiP-MS samples. The two samples are then analyzed by mass spectrometry and the two sets of data are integrated to analyze the potential target sites of the small molecules.

[0044] For example, this method is used to screen potential targets for small molecule drugs. In cases where the potential drug target is unknown, the control group is the untreated group. In the drug group, in step (1), the protein lysate is incubated with the small molecule at room temperature, and then a non-specific protease is added for limited hydrolysis under non-denaturing conditions. In step (2), during purification, the protein and the supernatant containing the non-specific peptide are collected. The supernatant containing the non-specific peptide is used as the first LiP-MS sample. The protein undergoes a reductive alkylation reaction, and a specific protease is added for specific enzymatic digestion of the target protein under denaturing conditions to obtain the second enzymatic digest. After terminating the digestion, desalting is performed to obtain the second LiP-MS sample. The two LiP-MS samples are then analyzed by mass spectrometry, and the two sets of data are integrated to analyze the potential drug targets.

[0045] The following are examples. Example 1

[0046] Preparation of lysis buffer: PBS lysis buffer: 137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 1.8mM KH2PO4, pH 7.6, 1% Cocktail protease inhibitor.

[0047] Hepes lysis buffer: 50 mM HEPES, 150 mM NaCl, pH 7.4, 1% Cocktail protease inhibitor.

[0048] (1) Sample pretreatment Bovine serum albumin (BSA) was dissolved using PBS lysis buffer and HEPES lysis buffer, respectively, to achieve a BSA concentration of 1 μg / μL. Three groups of 50 μL BSA (PBS) solutions were taken and labeled as P1, P2, and P3, respectively; one group of 50 μL BSA (HEPES) solutions was taken and labeled as H3.

[0049] 0.5 μg of nonspecific protease (proteinase K) was added to each of the four sample groups to perform limited hydrolysis of bovine serum albumin under non-denaturing conditions. The digestion was carried out at 25°C for 5 min to obtain the first hydrolysate containing the target protein and nonspecific peptides (<5 kDa). The method for removing nonspecific peptides was as follows: Group P1: Heat at 95℃ for 3 min, add 5 μL of 10% SDC, and heat again for 3 min to terminate enzyme digestion.

[0050] P2 group: Heat at 95℃ for 10 min to terminate enzyme digestion; centrifuge at 12000g at 4℃ for 5 min to collect protein precipitate.

[0051] For P3 and H3 groups: Add 200 μL of ice-cold acetone to terminate the enzyme digestion, precipitate overnight at 4°C, centrifuge at 12000g for 5 min at 4°C (to remove non-specific peptides), and collect the protein precipitate.

[0052] (2) Reductive alkylation The P2 / P3 / H3 histone precipitates were dissolved in 14 μL of 8M urea solution, and the urea was diluted to 2M by adding 42 μL of 100 mM Tris-HCl solution. 6 μL of 100 mM MTCEP / 400 mM MCAA solution was added to the P1 / P2 / P3 / H3 samples, and the samples were incubated at 60°C with shaking for 30 min to carry out the reductive alkylation reaction.

[0053] (3) Specific enzymatic hydrolysis Add 1 μg of specific protease (trypsin) to each sample in step (2) for specific enzymatic hydrolysis, incubate at 37℃ and 1200 rpm overnight (12 h) with shaking to obtain a second enzymatic hydrolysate containing specific peptides.

[0054] (4) Desalination Add 1 / 9 volume of 10% TFA to each of the second enzymatic digest samples from step (3) to acidify and stop the enzymatic digestion. Centrifuge at 12000g for 5 min to obtain the supernatant. Desalt the supernatant using an SDB desalting column. Finally, evaporate the sample to dryness using a rotary evaporator to obtain a solid powder, and store at -20℃ for mass spectrometry analysis.

[0055] (5) Mass spectrometry detection and analysis The sample, after desalting and rotary evaporation in step (4), was reconstituted with 1% formic acid. Data-dependent acquisition (DDA) was performed on the sample using a Thermo UltiMate 3000RSLCnano nano-liquid chromatography-tandem QExactive HF mass spectrometer. The mass spectrometry analysis was conducted using a Thermo UltiMate 3000RSLCnano nano-liquid chromatography-tandem QExactive HF mass spectrometer. Peptide samples were injected via an autosampler and bound to a C18 trapping column (75µm*2cm, 3µm particle size, 100Å pore size, Thermo), followed by separation on a self-made analytical column (75µm*25cm, 1.9µm particle size, 100Å pore size). An analytical gradient was established using mobile phase A (0.1% formic acid / 3% DMSO / 97% H2O) and mobile phase B (0.1% formic acid / 3% DMSO / 97% ACN). The flow rate was set to 300 nL / min. Mass spectrometry was performed in DDA mode for data acquisition. MS1 full scan parameters were set as follows: resolution 60 K @ 200 m / z, scan range 350–1500 m / z, AGC target 3E6, and maximum injection time 30 ms. The precursor ion selection window was set to 1.4 Da, selecting the top 20 precursor ions for fragmentation, and the HCD collision energy was set to 28%. MS2 scan parameters were set as follows: resolution 15 K @ 200 m / z, AGC target 1E5, and maximum injection time 50 ms. The dynamic exclusion time was set to 30 seconds. Raw data were analyzed using Fragpipe (v23.0) software in LFQ-MBR mode.

[0056] The mass spectrometry data of each sample were analyzed, and the results are as follows: Figure 2 As shown. Figure 2 The right-middle figure shows the PSM / MS2 and BSASpectral / MS ratios for each sample, ranked as H3≥P3>P1>P2.

[0057] Figure 2 The H3 and P3 groups showed the fewest MS2 spectra, while the P1 and P2 groups showed the most. This indicates that the P1 and P2 groups detected not only the specific peptides from the digestion of the target protein but also non-specific peptides. This demonstrates that the organic solvent acetone can effectively precipitate the target protein without precipitating some non-specific peptides. Centrifugation can remove non-specific peptides, thus reducing their interference with mass spectrometry detection. This method is effective for buffer systems such as PBS / HEPES. However, heating to precipitate the protein does not work, indicating that heating treatment is not an effective method for precipitating the target protein without precipitating non-specific peptides.

[0058] The BSA structure and peptide identification of each sample were analyzed. The BSA structure and peptide identification of samples P1 (routine treatment) and P3 (organic solvent purification treatment) were also analyzed. Figure 3 As shown.

[0059] Figure 3 The orange markers indicate BSA peptides identified only in group P1, while the blue markers indicate BSA peptides identified only in group H3. The orange-marked peptides are all located on the protein surface and are theoretically non-specifically cleaved peptides; the blue-marked peptides are all located inside the protein (interacting with other regions), and one is a fully cleaved peptide, indicating a specifically cleaved peptide formed after specific enzyme digestion. This demonstrates that, compared to conventional methods, this method can remove protein surface sequences easily cleaved by non-specific proteases, retaining core / interaction-related characteristic peptides, effectively reducing the interference of non-specific peptides generated by limited hydrolysis on the detection of characteristic peptides, and enabling the identification of more characteristic peptides. Example 2

[0060] Hepes lysis buffer preparation: 50 mM MEPES, 150 mM NaCl, pH 7.4, 1% Cocktail protease inhibitor.

[0061] 1. Sample pretreatment (1) Compare the removal effects of different organic solvents on non-specific peptides. HeLa cells were lysed by sonication at 4°C using Hepes lysis buffer, followed by centrifugation at 12,000 rpm for 3 min at 4°C, and the supernatant was retained. Protein levels in the supernatant were quantified using the BCA quantification method, and the protein concentration was adjusted to 2 μg / μL using Hepes lysis buffer. 150 μL of sample was taken, and 2 μg of proteinase K was added for limiting hydrolysis at 25°C for 5 min to obtain an enzymatic digest containing the target protein and non-specific peptides. The non-specific peptides in the digest were removed using an organic solvent to purify the sample. The sample purification process is as follows: The enzyme digest sample containing the target protein and non-specific peptides was heated at 95°C for 10 min to terminate the enzyme digestion. The sample was then divided into three aliquots. Sample 1 (Acetone sample) was purified using acetone; Sample 2 (TCA sample) was purified using trichloroacetic acid (TCA); and Sample 3 (Methanol sample) was purified using a mixture of methanol and chloroform. Details are as follows: ① Add 4 times the volume of ice-cold acetone to the Acetone sample, let it stand at 4°C for 12 hours to precipitate, centrifuge to remove non-specific peptides, and collect the precipitate containing the target protein.

[0062] ②Add 1 / 4 volume of trichloroacetic acid to the TCA sample, let it stand at 4℃ for 12 hours to precipitate, centrifuge to remove non-specific peptides, and collect the precipitate containing the target protein.

[0063] ③ Add an equal volume of methanol and 1 / 4 volume of chloroform to the methanol sample, let it stand at 4℃ for 12 hours to precipitate, centrifuge to remove non-specific peptides, and collect the precipitate containing the target protein.

[0064] (2) Reductive alkylation The P2 / P3 / H3 histone precipitates were dissolved in 14 μL of 8M urea solution, and the urea was diluted to 2M by adding 42 μL of 100 mM Tris-HCl solution. 6 μL of 100 mM MTCEP / 400 mM MCAA solution was added to the P1 / P2 / P3 / H3 samples, and the samples were incubated at 60°C with shaking for 30 min to carry out the reductive alkylation reaction.

[0065] (3) Specific enzymatic hydrolysis Add 1 μg of specific protease (trypsin) to each sample in step (2) for specific enzymatic hydrolysis, incubate at 37℃ and 1200 rpm overnight (12 h) with shaking to obtain an enzymatic hydrolysate containing specific peptides.

[0066] (4) Desalination Add 1 / 9 volume of 10% TFA to each enzyme digest sample from step (3) to acidify and stop enzyme digestion, centrifuge at 12000g for 5 min, and obtain the supernatant. Desalt the supernatant using an SDB desalting column. Finally, evaporate the sample to dryness using a rotary evaporator and store at -20℃ for mass spectrometry analysis.

[0067] (5) Mass spectrometry detection and analysis The sample after desalting and evaporating in step (4) was reconstituted with 1% formic acid and detected by data-dependent acquisition (DDA) using a ThermoUltiMate3000RSLCnano nano-liquid chromatography-tandem QExactive HF mass spectrometer. The raw data were analyzed using the LFQ-MBR mode of Fragpipe (v23.0) software. The effects of different organic solvents on the removal of non-specific peptides were compared, and the results are shown in Table 1.

[0068] Table 1 Comparison of the effects of different organic solvents on the removal of nonspecific peptides Analysis of mass spectrometry data for each sample, as shown in Table 1, revealed that the number of protein / peptide identified under different organic solvent precipitation conditions was similar to the proportion of semi-specific enzyme digestion. This indicates that different organic solvents are equally effective at removing non-specific peptides. The number of protein / peptide identified in the acetone group was slightly higher than that in the trichloroacetic acid group. Acetone reduces the polarity of the solution, exposing and interacting the hydrophobic regions of proteins, leading to the aggregation and precipitation of large protein molecules (≥5kDa). Small non-specific peptides (<5kDa), however, have high solubility in organic solvents and are difficult to aggregate effectively. Even if they form small aggregates, their small size or loose structure prevents them from being separated by conventional centrifugation. Trichloroacetic acid (TCA) lowers the pH of the solution (usually to 2-4), denaturing the protein and neutralizing its surface charge, thereby disrupting its solvation layer and promoting protein precipitation. Small non-specific peptides (<5kDa) maintain high solubility at low pH, making precipitation difficult and thus achieving separation. Combining acetone and trichloroacetic acid may improve the separation effect. Example 3

[0069] (1) Sample pretreatment HeLa cells were lysed by sonication at 4°C using Hepes lysis buffer, followed by centrifugation at 12,000 rpm for 3 min at 4°C, and the supernatant was collected. Protein levels in the supernatant were quantified using the BCA quantification method, and the protein concentration was adjusted to 2 μg / μL using Hepes lysis buffer. Five 50 μL samples (R1-R5) were taken, and 1 μg of proteinase K was added to each sample, followed by digestion at 25°C for 5 min. The treatment to remove non-specific peptides was as follows: R1: Heat at 95℃ for 10 min to terminate enzyme digestion, add four times the volume (200 μL) of ice-cold acetone directly, precipitate overnight at 4℃, and collect the protein precipitate by centrifugation at 12000g for 5 min at 4℃.

[0070] R2: Heat at 95℃ for 10 min to terminate enzyme digestion. Add 2.5 μL of 10% TFA solution to adjust the pH (pH approximately 3), then add four times the volume (210 μL) of ice-cold acetone, precipitate overnight at 4℃, and collect the precipitate containing the target protein by centrifugation at 12000g for 5 min at 4℃.

[0071] R3: Heat at 95℃ for 10 min to terminate enzyme digestion. Add 5 μL of 10% TFA solution to adjust the pH (pH approximately 1), then add four times the volume (220 μL) of ice-cold acetone, precipitate overnight at 4℃, and collect the precipitate containing the target protein by centrifugation at 12000g for 5 min at 4℃.

[0072] R4: Heat at 95℃ for 10 min to terminate enzyme digestion. Add 10 μL of 10% TFA solution to adjust the pH (pH approximately 0.8), then add four times the volume (240 μL) of ice-cold acetone, precipitate overnight at 4℃, and collect the precipitate containing the target protein by centrifugation at 12000g for 5 min at 4℃.

[0073] R5: Heat at 95℃ for 10 min to terminate enzyme digestion. Add 20 μL of 10% TFA solution to adjust the pH (pH approximately 0.5), then add four times the volume (280 μL) of ice-cold acetone, precipitate overnight at 4℃, and collect the precipitate containing the target protein by centrifugation at 12000g for 5 min at 4℃.

[0074] (2) Reductive alkylation Dissolve the protein precipitates from each sample in step (1) using 28 μL of 8M urea solution, and dilute the urea to 2M by adding 84 μL of 100 mM Tris-HCl solution. Add 12 μL of 100 mM MTCEP / 400 mM MCAA solution to the sample and incubate at 60°C with shaking for 30 min to carry out the reductive alkylation reaction.

[0075] (3) Specific enzymatic hydrolysis Add 1 μg of specific protease (trypsin) to each sample in step (2) for specific enzymatic hydrolysis, incubate at 37℃ and 1200 rpm overnight (12 h) with shaking to obtain an enzymatic hydrolysate containing specific peptides.

[0076] (4) Desalination Add 1 / 9 volume of 10% TFA to each sample to acidify and stop enzyme digestion, then centrifuge at 12000g for 5 min. Desalt the supernatant using an SDB desalting column. Finally, evaporate the samples to dryness using a rotary evaporator and store at -20℃ for mass spectrometry analysis.

[0077] (5) Detection and analysis DIA detection of samples was performed using a Thermo UltiMate 3000 RSLC nano-Liquid Chromatography-Tandem Pro mass spectrometer. Raw data were analyzed using the DIASPECLIB_QUANT_DIAPASEF mode of Fragpipe (v23.0) software. The mass spectrometry data for each sample were analyzed, and the results are as follows: Figure 4 As shown.

[0078] Figure 4The results showed that group R1 had the fewest protein / peptide abbreviated identification numbers (5411 / 45665) and the highest semi-specific enzyme digestion rate (28.16%). In contrast, groups R2-R5 had significantly higher abbreviated protein / peptide abbreviated identification numbers (5613 / 49556, 5616 / 48304, 5622 / 48840, and 5613 / 47067) than group R1, and semi-specific enzyme digestion rates (20.08%, 17.77%, 18.64%, and 15.70%), all lower than group R1. This indicates that adjusting the solution pH to 0.5-3 with an organic acid solution before precipitation with an organic solvent can increase the number of protein / peptide identified (approximately 200 / 4000) and significantly reduce the semi-specific enzyme digestion rate (approximately 10%). This demonstrates that the combination of acidification and organic solvents can improve sample detection accuracy while removing non-specific enzyme digestion interference peptides.

[0079] Example 4 validates this method using the positive control drug astrocyte spores and HeLa cells. This embodiment uses LiP-MS technology to screen potential drug targets and compares the effectiveness of this method with the traditional LiP-MS method. Specific experiments are as follows: Hepes lysis buffer preparation: 50 mM MEPES, 150 mM NaCl, pH 7.4, 1% Cocktail protease inhibitor.

[0080] (1) Sample pretreatment HeLa cells were lysed by sonication at 4°C using Hepes lysis buffer, followed by centrifugation at 12,000 rpm for 3 min at 4°C, and the supernatant was collected. Protein levels in the supernatant were quantified using the BCA quantification method, and the protein concentration was adjusted to 2 μg / μL using Hepes lysis buffer. Twenty-four 50 μL samples were collected (Group G / N and their respective drug treatment and control groups, with six replicates per group). 0.5 μL of 2 mM asteroides solution was added to the experimental group samples, and 0.5 μL of DMSO solvent was added to the control group samples. The samples were incubated at 25°C for 30 min. 1 μg of proteinase K was added to each sample, and the samples were digested at 25°C for 5 min. The treatment to remove non-specific peptides was as follows: Group G follows the standard procedure: heat at 95°C for 3 min, add 5 μL of 10% SDC, and heat again for 3 min to terminate the digestion. Add 6 μL of 100 mMTCEP / 400 mMMCAA solution to the sample and incubate at 60°C with shaking for 30 min to perform the reductive alkylation reaction.

[0081] Group N's method: Deactivate enzyme digestion by heating at 95°C for 10 min. Add 2.5 μL of 10% TFA solution, followed by four volumes (210 μL) of ice-cold acetone, and incubate at 4°C overnight to precipitate. Collect the protein precipitate by centrifugation at 12000g for 5 min at 4°C. Dissolve the protein precipitate in 28 μL of 8M urea solution, and dilute the urea to 2M by adding 84 μL of 100 mM Tris-HCl solution. Add 12 μL of 100 mM MTCEP / 400 mM MCAA solution to the sample and incubate at 60°C with shaking for 30 min to perform the reductive alkylation reaction.

[0082] Both Group G and Group N had three replicates of the drug administration group and the control group.

[0083] (2) Specific enzymatic hydrolysis Add 2 μg of trypsin to each sample and incubate overnight (12 h) at 37°C and 1200 rpm with shaking.

[0084] (3) Desalination Add 1 / 9 volume of 10% TFA to each sample to acidify and stop enzyme digestion, then centrifuge at 12000g for 5 min. Desalt the supernatant using an SDB desalting column. Finally, evaporate the samples to dryness using a rotary evaporator and store at -20℃ for mass spectrometry analysis.

[0085] (4) Detection and analysis DIA detection of the samples was performed using a Thermo UltiMate 3000 RSLC nano-Liquid Chromatography-Tandem Pro mass spectrometer. Raw data were analyzed using the DIASPECLIB_QUANT_DIAPASEF mode in Fragpipe (v23.0) software. Results are as follows: Figures 5 to 8 As shown.

[0086] (DIA mass spectrometry method:) Mass spectrometry analysis of the samples was performed using an UltiMate 3000 RSLC nano-liquid chromatograph (Thermo) tandem timsTOFPro mass spectrometer (Bruker). Peptide samples were injected via autosampler and bound to a C18 trap column (75µm*2cm, 3µm particle size, 100Å pore size, Thermo), followed by separation in an analytical column (75µm*25cm, 1.6µm particle size, 100Å pore size, Ion Opticks). An analytical gradient was established using mobile phase A (0.1% formic acid) and mobile phase B (0.1% formic acid in ACN). The flow rate was set to 300 nL / min. Mass spectrometry data acquisition was performed in diaPASEF mode. The capillary voltage was set to 1400 V. The scan range for MS1 and MS2 spectra was set to 100–1700 m / z. The ion mobility range was set to 0.6–1.6 Vs / cm². Accumulation time and ramp time were set to 100 ms. Based on the mass-to-charge ratio-ion mobility distribution, the diaPASEF acquisition window was set using timsControl software. The collision energy was set to linearly increase from 59 eV (1 / K0 = 1.6 Vs / cm²) to 20 eV (1 / K0 = 0.6 Vs / cm²) based on ion mobility. The results of comparing the number of protein identifications are as follows: Figure 5 As shown, Figure 5 The results showed that our method (N) identified 174 more proteins and 5438 more peptides than the conventional method (G). The optimized method of this invention significantly improves the number of proteins / peptides identified compared to the conventional LiP-MS method.

[0087] The distribution results of protein physicochemical properties are as follows Figure 6 As shown, Figure 6 The results showed that the protein size distribution and subcellular localization distribution trends identified in both groups of experiments were the same. The molecular weight and subcellular localization distribution of the proteins identified by the optimized method of this invention did not differ statistically from those identified by conventional methods, indicating that this method does not exhibit selective bias towards the target protein.

[0088] The results of the proportion of semi-specific enzyme-digested peptides are as follows: Figure 7 As shown, Figure 7 The results showed that, compared with conventional methods, the proportion of semi-specific enzyme-digested peptides decreased from 41% to 21.1% using the method of the present invention. The method provided by the present invention significantly reduces the proportion of semi-specific enzyme digestion in the sample.

[0089] This method, compared with conventional LiP-MS experimental results, is as follows: Figure 8 As shown, Figure 8 The results showed that, using the same two-sample t-test with equal variances, this invention screened 591 differentially expressed proteins under the criteria of 2-fold difference and P-value ≤ 0.01, while the conventional method screened 362 differentially expressed proteins under the criteria of 1.5-fold difference and P-value ≤ 0.05. The difference between the two methods was significant. Furthermore, compared to the conventional method, this method increased the number of ATP-binding proteins and kinases among the differentially expressed proteins by 37% and 17%, respectively.

[0090] Asteroidesin is known to be a broad-spectrum kinase inhibitor, targeting almost all kinases, and ATP-binding proteins are also potential targets. Therefore, kinases and ATP-binding proteins can be used as evaluation criteria. The screening results of this method confirm that asteroidesin is a broad-spectrum, ATP-competitive protein kinase inhibitor. Moreover, compared with the traditional LiP-MS method for screening potential drug targets, this method can screen out more potential targets, indicating that this method is more accurate.

[0091] For cases where the potential targets of certain drugs are unknown, this method can separate proteins and peptides, collect and detect them separately, and integrate the two sets of data for analysis, thereby improving detection efficiency.

[0092] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing LiP-MS samples to reduce non-specific peptide interference, characterized in that, Includes the following steps: (1) Limited hydrolysis of protein: The total protein extracted from the sample is dissolved in the lysis buffer to obtain the protein lysis buffer. Non-specific protease is added and limited hydrolysis is carried out under non-denaturing conditions to obtain the first enzymatic hydrolysate containing non-specific peptides and target proteins. (2) Sample purification: Terminating the limiting hydrolysis, the first enzymatic hydrolysate containing non-specific peptides and target protein obtained in step (1) is purified to remove non-specific peptides; the purification process uses an organic solvent to adjust the polarity of the first enzymatic hydrolysate, causing the target protein to precipitate while the non-specific peptides do not precipitate, and centrifugation is used to remove the non-specific peptides to obtain the purified potential target protein; the organic solvent includes acetone, trichloroacetic acid, and a mixture of methanol and trichloroacetic acid. (3) Specific enzymatic digestion: The purified protein in step (2) is subjected to a reduction alkylation reaction, and a specific protease is added to perform specific enzymatic digestion of the target protein under denaturing conditions to obtain a second enzymatic digestion solution including fully digested peptides and semi-specifically digested peptides. After terminating the enzymatic digestion, desalting is performed to obtain a LiP-MS sample for subsequent mass spectrometry detection and analysis.

2. The method as described in claim 1, characterized in that, The non-specific peptide is <5kD.

3. The method as described in claim 2, characterized in that, The purification process described in step (2) involves adding 4 times the volume of icy acetone, 1 / 4 volume of trichloroacetic acid, or an equal volume of methanol and 1 / 4 volume of chloroform as a purification reagent based on the volume of the first enzymatic hydrolysate to separate and remove non-specific peptides.

4. The method as described in claim 3, characterized in that, The purification process described in step (2) involves first adjusting the pH of the first enzymatic hydrolysate to 0.5-3 using an organic acid or its solution, then adding an organic solvent to induce the precipitation of the target protein and prevent the precipitation of nonspecific peptides, thereby separating and removing the nonspecific peptides.

5. The method as described in claim 4, characterized in that, The purification process described in step (2) involves first adjusting the pH of the first enzymatic hydrolysate to 0.8-1 using an organic acid or its solution, then adding an organic solvent to induce the precipitation of the target protein and prevent the precipitation of nonspecific peptides, thereby separating and removing the nonspecific peptides.

6. The method as described in claim 4 or 5, characterized in that, The organic acid includes trifluoroacetic acid.

7. The method as described in claim 6, characterized in that, Purification was performed by combining 10% TFA solution with icy acetone to remove nonspecific peptides.

8. The method as described in claim 7, characterized in that, The purification process involves first adjusting the pH of the first enzymatic hydrolysate to 0.5-3 using a 10% trifluoroacetic acid solution, and then adding four times the volume of ice-cold acetone to induce precipitation of the target protein while preventing the precipitation of non-specific peptides.

9. The method according to any one of claims 1 to 8, characterized in that, To analyze the interaction between small molecules and target proteins, step (1) involves incubating the protein lysate with the small molecule at room temperature, and then adding a non-specific protease for limited hydrolysis under non-denaturing conditions; the non-specific protease includes proteinase K.

10. The method as described in claim 9, characterized in that, In response to the unknown target of small molecule substances and target proteins, during the purification process in step (2), the protein and the supernatant containing non-specific peptides were collected to obtain two LiP-MS samples. The two samples were then analyzed by mass spectrometry and the two sets of data were integrated to analyze the potential target of small molecule substances.

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