A rapid and low-cost sample pretreatment kit for phosphorylated proteomics

Abstract

This invention discloses a rapid and low-consumption phosphorylated proteomics sample pretreatment kit, belonging to the field of sample pretreatment kits. Specifically, it relates to a sample pretreatment kit comprising a protein extraction reagent A and a phosphopeptide enrichment reagent C. Protein extraction reagent A includes at least one of ethylphenyl polyethylene glycol, sodium deoxycholate, and sodium dodecyl sulfate. Phosphopeptide enrichment reagent C includes titanium dioxide. The content of ethylphenyl polyethylene glycol is 0.1-2 wt%; or, the content of sodium deoxycholate is 0.1-1 wt%; or, the content of sodium dodecyl sulfate is 0.05-0.2 wt%. This invention discloses a rapid, low-consumption, high-extraction, and high-detection phosphorylated proteomics sample pretreatment kit.

Description

Technical Field

[0001] This invention belongs to the field of sample pretreatment kits, specifically relating to a rapid and low-consumption sample pretreatment kit for phosphorylated proteomics. Background Technology

[0002] Protein phosphorylation plays a crucial role in cell signal transduction, metabolic regulation, and disease development, making it a core topic in life sciences. Phosphoryproteomics, as a powerful tool for studying this phenomenon, is profoundly changing our understanding of biology and medicine. Phosphoryproteomics involves the systematic identification and quantitative analysis of all phosphorylated protein peptides and their modification sites in biological samples at a global level. This process typically requires first enzymatically digesting the protein sample into peptides, and then specifically isolating and enriching phosphorylated peptides. However, the abundance of phosphorylated peptides in complex biological samples such as cells, tissues, and blood is usually low, and their mass spectrometry detection is easily interfered with by other peptides in the sample. Therefore, in phosphoryproteomics analysis, specific enrichment of phosphorylated peptides is generally required before mass spectrometry detection.

[0003] Currently, commonly used methods for enriching phosphorylated peptides mainly include: (1) Immobilized Metal Affinity Chromatography (IMAC): selective capture is achieved by binding fixed metal ions (such as Fe³⁺, Ga³⁺) to phosphate groups. IMAC has the advantage of strong binding ability, but it is often accompanied by high non-specific binding, such as interference from acidic peptides, thus reducing the specificity of the results. (2) Metal Oxide Affinity Chromatography (MOAC), such as TiO2 and ZrO2: this method uses the affinity of metal oxides for phosphate groups for enrichment, and TiO2 is one of the most widely used methods. Although TiO2 enrichment has high selectivity, it is prone to non-specific binding problems, and usually requires optimization of sample loading and elution conditions, which is complicated to operate.

[0004] To improve the coverage of phosphorylated peptides, existing technologies often combine the above methods with fractionation strategies, such as strong cation exchange chromatography (SCX) or high pH reversed-phase chromatography fractionation. These multi-stage enrichment processes can increase the number of detected phosphorylation sites, but they typically have the following drawbacks: (1) Large sample requirements, often requiring milligram-level protein samples, making them unsuitable for trace samples. (2) Long experimental cycle, as multi-stage separation and enrichment operations greatly increase experimental time and are cumbersome. (3) High loss rate, as samples are easily lost during multi-step separation and enrichment, which can reduce peptide enrichment yield and the accuracy of quantitative analysis.

[0005] Therefore, existing phosphorylated proteomics sample pretreatment methods still have significant shortcomings in terms of small sample size and rapid detection. Summary of the Invention

[0006] The purpose of this invention is to provide a rapid, low-consumption, high-efficiency phosphorylated proteomics sample pretreatment kit with good extraction and detection effects.

[0007] The technical solution adopted by the present invention to achieve the above objectives is as follows: A sample pretreatment kit includes a protein extraction reagent A and a phosphopeptide enrichment reagent C. Protein extraction reagent A comprises at least one of ethylphenyl polyethylene glycol, sodium deoxycholate, and sodium dodecyl sulfate. Phosphopeptide enrichment reagent C comprises titanium dioxide. The method of this invention is based on metal oxide affinity chromatography. Through optimized binding and elution conditions, it achieves rapid and efficient enrichment of phosphorylated peptides with a total protein sample volume of less than 100 micrograms, suitable for subsequent mass spectrometry-based phosphorylated proteomics detection. In this invention, phosphopeptide enrichment reagent C comprises titanium dioxide, which is anatase TiO2 microbeads with a pore size of approximately 100 Å, a specific surface area of ​​approximately 100 m² / g, and a pH operating range of 2–12. In this invention, protein extraction reagent A is mixed with biological samples, followed by enzymatic hydrolysis, and finally enrichment is performed to obtain a detectable phosphorylated peptide extract. The kit of this invention is suitable for solid trace biological samples such as cells, bacteria, and tissues, avoiding the use of multi-stage fractionation steps, such as strong cation exchange chromatography (SCX) or high pH reversed-phase chromatography fractionation, so as to reduce operation time and sample loss.

[0008] Preferably, in protein extraction reagent A, the content of ethyl phenyl polyethylene glycol is 0.1-2 wt%; or, the content of sodium deoxycholate is 0.1-1 wt%; or, the content of sodium dodecyl sulfate is 0.05-0.2 wt%.

[0009] Preferably, the sample pretreatment kit further includes at least one of 100× protein digestion buffer B, enrichment buffer D, and elution buffer E.

[0010] More preferably, the enzyme in 100× protein digestion buffer B is trypsin.

[0011] More preferably, the enrichment buffer D includes at least one of potassium dihydrogen phosphate and trifluoroacetic acid, wherein the content of potassium dihydrogen phosphate is 0.2-1 wt% and the content of trifluoroacetic acid is 0.05-0.2 wt%.

[0012] Preferably, the elution buffer E is an ammonia solution with a concentration of 1-5 mg / mL.

[0013] Preferably, protein extraction reagent A further includes N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid. In this invention, during enzymatic extraction of biological samples, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid can be added to protein extraction reagent A. Then, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, ethylphenyl polyethylene glycol, sodium deoxycholate, and sodium dodecyl sulfate are used together, and extraction is carried out under trypsin enzymatic hydrolysis. With the appropriate use of N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid and sodium deoxycholate, the enzymatic extraction effect on biological samples can be greatly improved, more phosphorylated peptides can be enriched, and the required amount of biological sample is low, with a fast processing speed.

[0014] More preferably, the content of N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid is 0.05-0.3 wt%.

[0015] Preferably, the sample includes at least one of cells, bacteria, punctured tissue, and protein.

[0016] Preferably, the sample pretreatment kit also includes isopropanol.

[0017] Preferably, the pretreatment kit includes protein extraction reagent A, 100× protein digestion buffer B, phosphopeptide enrichment reagent C, enrichment buffer D, and elution buffer E, as well as trypsin, isopropanol, and trifluoroacetic acid.

[0018] More preferably, in the pretreatment kit, protein extraction reagent A includes ethylphenyl polyethylene glycol, sodium deoxycholate, sodium dodecyl sulfate and water, wherein the content of ethylphenyl polyethylene glycol in protein extraction reagent A is 0.1-2 wt%, the content of sodium deoxycholate is 0.1-1 wt%, and the content of sodium dodecyl sulfate is 0.05-0.2 wt%.

[0019] More preferably, in the pretreatment kit, protein extraction reagent A contains N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, and the content of N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid is 0.05-0.3 wt%.

[0020] More preferably, in the pretreatment kit, protein extraction reagent A contains succinamide, with a succinamide content of 0.01-0.1 wt%. In the enzymatic hydrolysis of biological samples, after using N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, ethylphenyl polyethylene glycol, sodium deoxycholate, and sodium dodecyl sulfate, succinamide can be added for combined use. In the presence of N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, ethylphenyl polyethylene glycol, sodium deoxycholate, and sodium dodecyl sulfate, the use of succinamide further improves the enzymatic extraction effect on biological samples, enriching more phosphorylated peptides, and requiring less biological sample while offering faster processing speed.

[0021] Preferably, in the preparation of the protein extract, the solid biological sample is mixed with protein extraction reagent A and allowed to stand at 20-40°C for 10-60 minutes to obtain the protein extract.

[0022] More preferably, in the preparation of the protein extraction solution, the mass-volume relationship between the amount of solid biological sample and protein extraction reagent A used is 1-10 mg: 1-10 mL. The solid biological sample is A549 cell protein.

[0023] Preferably, in the preparation of the peptide mixture, trypsin solution is added to the protein extraction solution, enzymatically hydrolyzed at 30-40℃, the pH is adjusted to 2-3, the enzymatic hydrolysis is terminated, and the precipitate is removed by centrifugation to obtain the peptide mixture.

[0024] More preferably, in the preparation of the peptide mixture, in the preparation of the trypsin solution, 100× protein digestion buffer B is diluted 100 times with deionized water, and then used as a diluent to prepare the trypsin solution. The trypsin content in the trypsin solution is 0.5-2wt%, and the volume of the trypsin solution used is 1000-3000% of the volume of the protein extract. The pH adjustment reagent is trifluoroacetic acid.

[0025] Preferably, in the preparation of the phosphopeptide enrichment product, phosphopeptide enrichment reagent C and enrichment buffer D are added to the peptide mixture, incubated at 20-40℃ for 10-30 min, the supernatant is discarded by centrifugation, and the phosphopeptide enrichment product is obtained by centrifugation with washing buffer.

[0026] More preferably, in the preparation of the phosphopeptide enrichment product, the phosphopeptide enrichment reagent C is TiO2, and the mass-volume relationship between the amount of phosphopeptide enrichment reagent C and the peptide mixture is 1-2 mg: 1-10 mL.

[0027] More preferably, in the preparation of the phosphopeptide enrichment product, the enrichment buffer D includes potassium dihydrogen phosphate, trifluoroacetic acid and water. In the enrichment buffer D, the content of potassium dihydrogen phosphate is 0.2-1 wt%, the content of trifluoroacetic acid is 0.05-0.2 wt%, and the volume of enrichment buffer D used is 100-300% of the volume of peptide mixture used.

[0028] More preferably, in the preparation of the phosphopeptide enrichment product, the washing solution is composed of isopropanol, trifluoroacetic acid and deionized water, wherein the content of isopropanol in the washing solution is 10-70 vol%, and the content of trifluoroacetic acid is 0.1-5 vol%.

[0029] More preferably, in the preparation of the phosphopeptide enrichment product, washing is performed using washing buffers A, B, and C. Washing buffer A contains 60 vol% isopropanol and 5 vol% trifluoroacetic acid; washing buffer B contains 60 vol% isopropanol and 1 vol% trifluoroacetic acid; washing buffer C contains 10 vol% isopropanol and 0.2 vol% trifluoroacetic acid. Sequential washing with three buffers of different compositions efficiently removes non-specifically bound non-phosphorylated peptides, thereby improving the purity of the phosphorylated peptides.

[0030] Preferably, in the preparation of the phosphorylated peptide extract, the phosphopeptide enriched product is added to elution buffer E and incubated for 10-30 min, centrifuged to obtain the supernatant, formic acid aqueous solution is added, centrifuged again to obtain the supernatant, and thus the phosphorylated peptide extract is obtained.

[0031] More preferably, in the preparation of the phosphorylated peptide extract, the elution buffer E is an ammonia solution with a concentration of 1-5 mg / mL; the volume-mass relationship between the elution buffer E and the amount of phosphopeptide enriched product used is 1-10 mL: 1-10 mg.

[0032] More preferably, in the preparation of the phosphorylated peptide extract, the formic acid content in the formic acid aqueous solution is 0.1-0.5 wt%, and the volume of the formic acid aqueous solution used is 400-600% of the volume of the elution buffer E used.

[0033] After obtaining the phosphorylated peptide extract, LC-MS / MS can be used for phosphorylated proteomics analysis. After enrichment, the background peptides are reduced, and the ion competition of phosphorylated peptides is significantly reduced during MS analysis, so that low-abundance phosphorylated peptides can be detected more sensitively and in greater quantities.

[0034] This invention involves mixing protein extraction reagent A with a solid biological sample, then adding trypsin solution diluted with 100× protein digestion buffer B for enzymatic digestion. After digestion, phosphopeptide enrichment reagent C and enrichment buffer D are added to separate the phosphopeptide-enriched product. The product is then incubated with elution buffer E, centrifuged, and the supernatant is added to formic acid aqueous solution to prepare a phosphorylated peptide extract. Finally, tandem mass spectrometry is used for phosphorylated proteomics analysis. The phosphorylated peptide extract prepared by this method can effectively enrich phosphorylation sites. Protein extraction reagent A can be selected from ethylphenyl polyethylene glycol, sodium deoxycholate, or sodium dodecyl sulfate. Under trypsin digestion, more digestion products can be prepared, resulting in a greater number of phosphorylated peptides after enrichment. N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid can also be added to protein extraction reagent A, thus providing the following advantages: rapid sample pretreatment, low sample usage, good extraction effect on phosphorylated peptides, and good detection effect. Therefore, this invention is a rapid, low-consumption, high-efficiency phosphorylated proteomics sample pretreatment kit with good extraction and detection effects. Attached Figure Description

[0035] Figure 1 Flowchart for reagent kit usage.

[0036] Figure 2 This is a diagram showing the number of phosphorylation sites in the DDA mode.

[0037] Figure 3 This is a diagram showing the number of phosphorylation sites in the DIA mode. Detailed Implementation

[0038] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] The concepts involved in this application will first be described with reference to the accompanying drawings. It should be noted that the following descriptions of various concepts are only for the purpose of making the content of this application easier to understand and do not constitute a limitation on the scope of protection of this application; furthermore, the embodiments and features in the embodiments of this application can be combined with each other unless otherwise specified. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0040] Example 1: A method for extracting phosphorylated peptides Pretreatment kit: Protein extraction reagent A, 100× protein digestion buffer B, phosphopeptide enrichment reagent C, enrichment buffer D, and elution buffer E, as well as trypsin, isopropanol, and trifluoroacetic acid. Protein extraction reagent A consists of ethylphenyl polyethylene glycol, sodium deoxycholate, sodium dodecyl sulfate, and water. In protein extraction reagent A, the content of ethylphenyl polyethylene glycol is 1 wt%, the content of sodium deoxycholate is 0.5 wt%, and the content of sodium dodecyl sulfate is 0.1 wt%.

[0041] Preparation of protein extraction solution: The solid biological sample was mixed with protein extraction reagent A and allowed to stand at 25°C for 30 min to obtain the protein extraction solution. The mass-volume relationship between the solid biological sample and protein extraction reagent A was 1 mg: 1 mL. The solid biological sample was A549 cells.

[0042] Preparation of peptide mixture: Trypsin solution was added to protein extraction solution, and enzymatic hydrolysis was performed at 37℃. The pH was adjusted to 2 to terminate the hydrolysis. The precipitate was removed by centrifugation to obtain peptide mixture. In the preparation of trypsin solution, 100× protein hydrolysis buffer B was diluted 100 times with deionized water to prepare the trypsin solution. The trypsin content in the trypsin solution was 1 wt%, and the volume of trypsin solution used was 2000% of the volume of protein extraction solution used. Trifluoroacetic acid was used to adjust the pH.

[0043] Preparation of phosphopeptide enriched products: Phosphopeptide enrichment reagent C and enrichment buffer D were added to the peptide mixture and incubated at 25°C for 20 min. The supernatant was discarded by centrifugation, and the product was washed with washing buffer to obtain the phosphopeptide enriched products. Phosphopeptide enrichment reagent C was TiO2, and the mass-volume relationship between phosphopeptide enrichment reagent C and peptide mixture was 1 mg:1 mL. Enrichment buffer D consisted of potassium dihydrogen phosphate, trifluoroacetic acid, and water. The content of potassium dihydrogen phosphate in enrichment buffer D was 0.6 wt%, and the content of trifluoroacetic acid was 0.1 wt%. The volume of enrichment buffer D used was 200% of the volume of the peptide mixture. The washing solution consisted of isopropanol, trifluoroacetic acid, and deionized water. Washing was performed using washing solutions A, B, and C. Washing solution A contained 60 vol% isopropanol and 5 vol% trifluoroacetic acid; washing solution B contained 60 vol% isopropanol and 1 vol% trifluoroacetic acid; washing solution C contained 10 vol% isopropanol and 0.2 vol% trifluoroacetic acid. Phosphopeptide enrichment products. Preparation of phosphorylated peptide extract: The phosphopeptide enriched product was added to elution buffer E and incubated for 15 min. The supernatant was collected by centrifugation, and formic acid aqueous solution was added. The supernatant was collected by centrifugation again to obtain the phosphorylated peptide extract. Elution buffer E was an ammonia solution with a concentration of 3 mg / mL. The volume-to-mass ratio of elution buffer E to the phosphopeptide enriched product was 2 mL:1 mg. The formic acid aqueous solution contained 0.15 wt% formic acid, and its volume was 500% of the volume of elution buffer E.

[0044] Example 2: A method for extracting phosphorylated peptides The difference between this embodiment and Example 1 is in protein extraction reagent A.

[0045] Pretreatment kit: Protein extraction reagent A, 100× protein digestion buffer B, phosphopeptide enrichment reagent C, enrichment buffer D, and elution buffer E, as well as trypsin, isopropanol, and trifluoroacetic acid. Protein extraction reagent A includes ethylphenyl polyethylene glycol, sodium deoxycholate, sodium dodecyl sulfate, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, and water. In protein extraction reagent A, the content of ethylphenyl polyethylene glycol is 1 wt%, the content of sodium deoxycholate is 0.5 wt%, the content of sodium dodecyl sulfate is 0.1 wt%, and the content of N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid is 0.25 wt%.

[0046] Example 3: A method for extracting phosphorylated peptides The difference between this embodiment and Example 2 is in protein extraction reagent A.

[0047] Pretreatment kit: Protein extraction reagent A, 100× protein digestion buffer B, phosphopeptide enrichment reagent C, enrichment buffer D, and elution buffer E, as well as trypsin, isopropanol, and trifluoroacetic acid. Protein extraction reagent A includes ethylphenyl polyethylene glycol, sodium deoxycholate, sodium dodecyl sulfate, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, and water. In protein extraction reagent A, the content of ethylphenyl polyethylene glycol is 1 wt%, the content of sodium deoxycholate is 0.5 wt%, the content of sodium dodecyl sulfate is 0.1 wt%, and the content of N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid is 0.1 wt%.

[0048] Example 4: A method for extracting phosphorylated peptides The difference between this embodiment and Example 2 is in protein extraction reagent A.

[0049] Pretreatment kit: Protein extraction reagent A, 100× protein digestion buffer B, phosphopeptide enrichment reagent C, enrichment buffer D, and elution buffer E, as well as trypsin, isopropanol, and trifluoroacetic acid. Protein extraction reagent A includes ethylphenyl polyethylene glycol, sodium deoxycholate, sodium dodecyl sulfate, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, succinamide, and water. In protein extraction reagent A, the content of ethylphenyl polyethylene glycol is 1 wt%, sodium deoxycholate is 0.5 wt%, sodium dodecyl sulfate is 0.1 wt%, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid is 0.25 wt%, and succinamide is 0.08 wt%.

[0050] Example 5: A method for extracting phosphorylated peptides The difference between this embodiment and Example 4 is in protein extraction reagent A.

[0051] Pretreatment kit: Protein extraction reagent A, 100× protein digestion buffer B, phosphopeptide enrichment reagent C, enrichment buffer D, and elution buffer E, as well as trypsin, isopropanol, and trifluoroacetic acid. Protein extraction reagent A includes ethylphenyl polyethylene glycol, sodium deoxycholate, sodium dodecyl sulfate, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, succinamide, and water. In protein extraction reagent A, the content of ethylphenyl polyethylene glycol is 1 wt%, sodium deoxycholate is 0.5 wt%, sodium dodecyl sulfate is 0.1 wt%, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid is 0.25 wt%, and succinamide is 0.03 wt%.

[0052] Comparative Example 1: A method for extracting phosphorylated peptides The difference between this comparative example and Example 2 is the protein extraction reagent A.

[0053] Pretreatment kit: Protein extraction reagent A, 100× protein digestion buffer B, phosphopeptide enrichment reagent C, enrichment buffer D, and elution buffer E, as well as trypsin, isopropanol, and trifluoroacetic acid. Protein extraction reagent A consists of ethylphenyl polyethylene glycol, sodium dodecyl sulfate, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, and water. In protein extraction reagent A, the content of ethylphenyl polyethylene glycol is 1 wt%, the content of sodium dodecyl sulfate is 0.1 wt%, and the content of N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid is 0.25 wt%.

[0054] Comparative Example 2: A method for extracting phosphorylated peptides The difference between this comparative example and Example 2 is the protein extraction reagent A.

[0055] Pretreatment kit: Protein extraction reagent A, 100× protein digestion buffer B, phosphopeptide enrichment reagent C, enrichment buffer D, and elution buffer E, as well as trypsin, isopropanol, and trifluoroacetic acid. Protein extraction reagent A includes ethylphenyl polyethylene glycol, sodium deoxycholate, sodium dodecyl sulfate, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, and water. In protein extraction reagent A, the content of ethylphenyl polyethylene glycol is 1 wt%, the content of sodium deoxycholate is 0.5 wt%, the content of sodium dodecyl sulfate is 0.1 wt%, and the content of N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid is 0.02 wt%.

[0056] Experimental example: In this invention, after preparing the phosphorylated peptide extract using the method described in the examples, an appropriate volume of sample was taken for phosphorylated proteomics analysis using LC-MS / MS. The detection procedure is as follows: Figure 1 As shown.

[0057] In this invention, a single sample requires 1-1.5 mg of TiO2 microbeads, priced at 10 yuan / mg, thus costing approximately 10-15 yuan per sample. A single 100-microgram protein sample requires 2 micrograms of trypsin, priced at 7.64 yuan / microgram, thus costing approximately 15.28 yuan per sample. Protein extraction takes 1 hour; protein digestion takes 2 hours; and phosphopeptide enrichment takes 2 hours. The required sample size is less than 100 micrograms. In contrast, this invention, through a two-stage TiO2 enrichment combined with a multi-buffer washing strategy, achieves highly efficient enrichment of phosphorylated peptides in protein samples smaller than 100 micrograms without additional fractionation, completing the entire process in just a few hours. Therefore, this invention significantly shortens experimental time and improves the detection coverage of phosphorylation sites while reducing sample requirements and experimental costs.

[0058] This invention employs both data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes. DDA is a commonly used mass spectrometry acquisition method. It first detects precursor ions in the full-scan mass spectrum, then selects several ions with the strongest signals for fragmentation and analysis in MS / MS. Its advantage is high accuracy in identifying individual peptides, but it is prone to missing low-abundance peptides. DIA is a mass spectrometry acquisition method that performs parallel fragmentation analysis of all ions in the full-scan mass spectrum according to a specific m / z range, thereby obtaining more comprehensive peptide information in a single experiment. Its advantages are broad coverage and high reproducibility, but data interpretation relies on complex algorithms.

[0059] In this invention, the phosphorylated peptide extracts prepared using the methods of the examples and comparative examples were detected using a Bruker Tims-TOF mass spectrometry platform. 100 micrograms of A549 cell protein were used as the solid biological sample. Phosphorylation sites were identified in DDA mode. A control group of A549 cell protein was not enriched and was directly analyzed for overall proteomics. The results are as follows: Figure 2 As shown, S1 is Example 1, S2 is Example 2, S3 is Example 3, S4 is Example 4, S5 is Example 5, D1 is Comparative Example 1, D2 is Comparative Example 2, and B is the control group. In this invention, protein extraction reagent A is mixed with a solid biological sample, and then trypsin solution diluted with 100× protein digestion buffer B is added for enzymatic digestion. After digestion, phosphopeptide enrichment reagent C and enrichment buffer D are added to separate the phosphopeptide-enriched product. Then, elution buffer E is added for incubation, and after centrifugation, the supernatant is added to formic acid aqueous solution to prepare a phosphorylated peptide extract. Finally, tandem mass spectrometry is used for phosphorylated proteomics analysis. The phosphorylated peptide extract prepared by this method can effectively enrich phosphorylation sites. Protein extraction reagent A in this invention can be selected from ethylphenyl polyethylene glycol, sodium deoxycholate, or sodium dodecyl sulfate. Under trypsin digestion, more enzymes can be prepared. The digestion products, after final enrichment treatment, yield a greater number of phosphorylated peptides. In this invention, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid can also be added to protein extraction reagent A. Treatment with ethylphenyl polyethylene glycol, sodium deoxycholate, sodium dodecyl sulfate, and N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, along with trypsin hydrolysis, can further increase the number of phosphorylated peptides. This invention has found that the combined use of N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid and sodium deoxycholate has a better effect. Without N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid or sodium deoxycholate, the number of phosphorylated peptides after enzymatic hydrolysis will decrease, while the combined use of N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid and sodium deoxycholate yields a better effect. Furthermore, succinamide can be added to protein extraction reagent A to increase the number of phosphorylated peptides after enzymatic hydrolysis and enrichment.

[0060] In this invention, the phosphorylated peptide extracts prepared using the methods of the examples and comparative examples were detected using a Bruker Tims-TOF mass spectrometry platform. 100 micrograms of A549 cell protein were used as the solid biological sample. Phosphorylation sites were identified in DIA mode. A control group of A549 cell protein was not enriched and was directly analyzed for overall proteomics. The results are as follows: Figure 3As shown, S1 is Example 1, S2 is Example 2, S3 is Example 3, S4 is Example 4, S5 is Example 5, D1 is Comparative Example 1, D2 is Comparative Example 2, and B is the control group. In this invention, protein extraction reagent A is mixed with a solid biological sample, and then trypsin solution diluted with 100× protein digestion buffer B is added for enzymatic digestion. After digestion, phosphopeptide enrichment reagent C and enrichment buffer D are added to separate the phosphopeptide-enriched product. Then, elution buffer E is added for incubation, and after centrifugation, the supernatant is added to formic acid aqueous solution to prepare a phosphorylated peptide extract. Finally, tandem mass spectrometry is used for phosphorylated proteomics analysis. The phosphorylated peptide extract prepared by this method can effectively enrich phosphorylation sites. Protein extraction reagent A in this invention can be selected from ethylphenyl polyethylene glycol, sodium deoxycholate, or sodium dodecyl sulfate. Under trypsin digestion, more enzymes can be prepared. The digestion products, after final enrichment treatment, yield a greater number of phosphorylated peptides. In this invention, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid can also be added to protein extraction reagent A. Treatment with ethylphenyl polyethylene glycol, sodium deoxycholate, sodium dodecyl sulfate, and N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, along with trypsin hydrolysis, can further increase the number of phosphorylated peptides. This invention has found that the combined use of N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid and sodium deoxycholate has a better effect. Without N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid or sodium deoxycholate, the number of phosphorylated peptides after enzymatic hydrolysis will decrease, while the combined use of N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid and sodium deoxycholate yields a better effect. Furthermore, succinamide can be added to protein extraction reagent A to increase the number of phosphorylated peptides after enzymatic hydrolysis and enrichment.

[0061] The embodiments and / or implementation methods described above are merely preferred embodiments and / or implementation methods for implementing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any way. Any person skilled in the art can make some modifications or alterations to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as the technology or embodiments that are substantially the same as the present invention.

[0062] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. The above descriptions are only preferred embodiments of this application. It should be noted that due to the limitations of written expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner. These improvements, modifications, changes, or combinations, or the direct application of the inventive concept and technical solution to other situations without modification, should all be considered within the scope of protection of this application.

Claims

1. A sample pretreatment kit, comprising: Protein extraction reagent A and phosphopeptide enrichment reagent C. Protein extraction reagent A includes N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, ethylphenyl polyethylene glycol, sodium deoxycholate, sodium dodecyl sulfate, and succinamide. Phosphopeptide enrichment reagent C includes titanium dioxide. In the protein extraction reagent A, the content of ethyl phenyl polyethylene glycol is 0.1-2 wt%; the content of sodium deoxycholate is 0.1-1 wt%; the content of sodium dodecyl sulfate is 0.05-0.2 wt%; the content of N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid is 0.05-0.3 wt%; and the content of succinamide is 0.01-0.1 wt%. The sample pretreatment kit is used for the extraction of phosphorylated peptides; The sample includes at least one of cells, bacteria, and punctured tissue.

2. The sample pretreatment kit according to claim 1, characterized in that, The sample pretreatment kit also includes at least one of 100× protein digestion buffer B, enrichment buffer D, and elution buffer E.

3. The sample pretreatment kit according to claim 2, characterized in that, The enzyme in the 100× protein digestion buffer B is trypsin.

4. The sample pretreatment kit according to claim 2, characterized in that, The enrichment buffer D includes at least one of potassium dihydrogen phosphate and trifluoroacetic acid, with the content of potassium dihydrogen phosphate being 0.2-1 wt% and the content of trifluoroacetic acid being 0.05-0.2 wt%.

5. The sample pretreatment kit according to claim 2, characterized in that, The elution buffer E is an ammonia solution with a concentration of 1-5 mg / mL.

6. The sample pretreatment kit according to claim 1, characterized in that, The sample pretreatment kit also includes isopropanol.

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