A method for monitoring the enrichment process of phosphorylated peptides in phosphoproteomics

Abstract

The application discloses a method for monitoring phosphorylation peptide enrichment process in phosphoproteomics, comprising the following steps: 1) setting at least two groups of exogenous peptide reference, each group of exogenous peptide reference comprising at least three peptides containing different phosphorylation modification sites, and for each peptide in each group, there is a corresponding peptide with the same sequence but different isotopic labels in other groups; 2) adding the different exogenous peptide references at different time points during sample processing, and the time points of adding any two groups of exogenous peptide references comprise the step of phosphorylation peptide enrichment; 3) sample injection and detection, data processing, exogenous peptide reference quantification, yield ratio calculation of different phosphorylation modification peptides, and evaluation of whether the phosphorylation enrichment process between the time points of adding two exogenous peptide references is qualified. The method can effectively evaluate the phosphorylation peptide enrichment efficiency.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and in particular, relates to a method for monitoring the enrichment process of phosphorylated peptides in phosphoproteomics. BACKGROUND

[0002] Protein phosphorylation is a process catalyzed by protein kinases, which transfers the phosphate group of ATP to the substrate amino acid residues (serine, threonine, tyrosine) and the like, and is a common and important regulation mode in vivo, which plays an important role in the process of cell signal transduction. Phosphorylation proteomics is a main research means, and mass spectrometry is a key tool for identifying protein phosphorylation sites and quantifying phosphorylation changes. The enrichment of phosphorylated peptides is crucial for successful phosphopeptide mass spectrometry analysis. Current enrichment methods include TiO2 enrichment method, antibody, immobilized metal affinity chromatography (IMAC), chemical modification and strong cation exchange chromatography (SCX) and the like.

[0003] However, protein phosphorylation is unstable. Firstly, protein phosphorylation is reversible, and the reverse process is to remove the corresponding phosphate group by protein phosphatase. Secondly, the phosphorylation group is easily degraded under high temperature or high pH conditions. Therefore, it is necessary to improve the enrichment efficiency of phosphorylated peptides and quality control the enrichment process of phosphorylated peptides. At present, most of the phosphorylated peptide enrichment processes do not do quality control, and only after the mass spectrometer is on-line, the identification number and enrichment efficiency are used to determine whether the experiment is successful, but it cannot be determined whether the enrichment efficiency or the identification number is low due to degradation or enrichment process. SUMMARY

[0004] The present application provides a method for monitoring the enrichment process of phosphorylated peptides in phosphoproteomics.

[0005] Technical scheme: In order to achieve the above-mentioned application purpose, the present application adopts the following technical scheme:

[0006] A method for monitoring the enrichment process of phosphorylated peptides in phosphoproteomics, comprising the following steps:

[0007] 1) setting at least two groups of exogenous peptide reference products, each group of exogenous peptide reference product containing at least three peptides containing different phosphorylation modification sites, and for each peptide in each group, there is a corresponding peptide with the same sequence but different isotopic labels in other groups;

[0008] 2) adding the above different exogenous peptide reference products at different time points during sample processing, and the time points of adding any two groups of exogenous peptide reference products include the step of enriching phosphorylated peptides;

[0009] 3) Injection detection, extraction of processing data, quantification of exogenous peptide reference, dividing the abundance of the peptide of the exogenous peptide reference added before by the abundance of the same sequence peptide of the exogenous peptide reference added after, to obtain the yield ratio of different phosphorylation modification peptides, so as to evaluate whether the phosphorylation enrichment process between the two exogenous peptide reference addition time points is qualified.

[0010] The method is used to evaluate whether the phosphorylation enrichment efficiency and experimental process are qualified in the phosphorylation peptide enrichment process.

[0011] Preferably, in step 1), the different phosphorylation modification sites are selected from the phosphorylation modification sites distributed on threonine, serine, tyrosine, histidine, arginine, lysine, aspartic acid, glutamic acid and cysteine residues, preferably the phosphorylation modification sites distributed on threonine, serine and tyrosine residues.

[0012] Preferably, in step 1), each group of exogenous peptide references contains at least three peptides containing different phosphorylation modification sites, wherein the peptides in each group have the same or different sequences.

[0013] Preferably, in step 1), the isotope labeling is different, including: the peptides in one group are not isotope-labeled, and the peptides in other groups are isotope-labeled, but the isotopes used for labeling in each group are different; or the peptides in each group are isotope-labeled, but the isotopes used for labeling in each group are different.

[0014] Preferably, in step 1), the isotopes in the isotope labeling include: 12C, 13C, 14C, 14N, 15N, 16O, 18O, 1H, 2H, 32S, 34S, 30P, 32P.

[0015] Preferably, in step 2), the sample is selected from cells, tissues, plasma, serum, whole blood, urine, cerebrospinal fluid, saliva, lymph, pleural effusion, breast milk, tissue fluid, microorganisms or plants.

[0016] Preferably, in step 2), the exogenous peptide references are accurately quantified before being added, preferably added in consistent amounts, or added in proportion.

[0017] As a specific embodiment, the step of phosphorylation peptide enrichment includes all steps or part of steps of the phosphorylation peptide enrichment. That is, between the time points of the addition of the two groups of exogenous peptide references, some can include all steps of the phosphorylation peptide enrichment, and some can include part of the steps of the phosphorylation peptide enrichment.

[0018] Preferably, in step 3), the detection method is mass spectrometry detection, such as LC-MS / MS and MALDI-TOF.

[0019] Further, in step 3), if the exogenous peptide reference added in advance is added before the enrichment of the phosphorylated peptides or at the beginning of the enrichment, and the exogenous peptide reference added later is added at the end of the enrichment of the phosphorylated peptides or after the enrichment, the peptide abundance of the exogenous peptide reference added in advance is divided by the abundance of the peptide of the same sequence of the exogenous peptide reference added later, to obtain the yield ratio of different phosphorylated modified peptides, so as to evaluate whether the entire phosphorylated peptide enrichment process is qualified.

[0020] Further, in step 3), if the exogenous peptide reference added in advance is added before the enrichment of the phosphorylated peptides or at the beginning of the enrichment, and the exogenous peptide reference added later is added at the end of the enrichment of the phosphorylated peptides or after the enrichment, the peptide abundance of the exogenous peptide reference added in advance is divided by the abundance of the peptide of the same sequence of the exogenous peptide reference added later, to obtain the yield ratio of different phosphorylated modified peptides, so as to evaluate whether the entire phosphorylated peptide enrichment process is qualified.

[0021] Preferably, in step 3), the extracted and processed data can be used to extract qualitative and quantitative data of the peptide reference by using software, and the qualitative and quantitative data can be extracted by using a real-time identification method provided by part of the advanced mass spectrometry detection software or after obtaining the full data.

[0022] Preferably, in step 3), the extracted and processed data can be used to extract qualitative and quantitative data of the peptide reference by using software, and the qualitative and quantitative data can be extracted by using a real-time identification method provided by part of the advanced mass spectrometry detection software or after obtaining the full data.

[0023] As a preferred solution, when two groups of exogenous peptide references are set, the specific method is as follows:

[0024] 1) Prepare two groups of exogenous peptide references A and B, the group A of exogenous peptide references contains three peptides of different phosphorylated modification sites, which are used as detection markers, and the three peptides in the group B are the same as the sequence of the peptides in the group A, but are isotopically labeled, which are used as calibration markers, and the isotopic labels are arginine (R) non-heavy (12C) and 13C labels, and the distribution of the phosphorylated modification sites is: threonine, serine, and tyrosine residues.

[0025] 2) Use the two groups of references separately, add the detection marker peptide group A into the sample before the enrichment of the phosphorylated peptides or during the enrichment, and add the calibration marker peptide group B into the sample after the enrichment, and then perform sample injection and detection.

[0026] 3) using proteome analysis software Proteome Discoverer, exporting all qualitative and quantitative peptide data, manually or programmatically searching and determining the corresponding peptide abundance of the exported data, and calculating the yield according to the ratio of the detection standard peptide group A to the calibration standard peptide group B to evaluate whether the phosphorylated peptide enrichment process is qualified, and the calculation formula is: recovery rate = peptide intensity of detection standard peptide group A / peptide intensity of calibration standard peptide group B.

[0027] Beneficial effects: Compared with the prior art, the present application can effectively evaluate whether the phosphorylated peptide enrichment process is qualified, the present method can directly and quantitatively evaluate the influence of the phosphorylated peptide enrichment process on the sample, thereby evaluating the loss of phosphorylated peptides in the sample operation process, further guiding the improvement of the phosphorylated peptide enrichment process, and also being used for evaluating the enrichment effect of different phosphorylated peptide enrichment methods on the phosphorylated peptides in the sample. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 : Process schematic diagram of the evaluation method of the present application.

[0029] Figure 2 : Experimental process schematic diagram of Example 1. DETAILED DESCRIPTION

[0030] The present application is described below, and the described embodiments are the most preferred embodiments of the present application, but the present application is not limited to the following examples.

[0031] EMBODIMENT

[0032] 1. A set of peptides including 3 peptides: T(phos)PVISGGPYEYR, TPVIS(phos)GGPYEYR, TPVISGGPY(phos)EYR, corresponding to threonine, serine and tyrosine phosphorylation modification, respectively, and lysine (R)

[0033] Non-isotopically labeled (12C), named detection standard peptide group A;

[0034] 2. Corresponding isotopically labeled peptides (R labeled with 13C): T(phos)PVISGGPYEYR(13C6), TPVIS(phos)GGPYEYR(13C6), TPVISGGPY(phos)EYR(13C6), named calibration standard peptide group B;

[0035] 3. The detection standard peptide group A and the calibration standard peptide group B are each matched in a ratio of 1:1:1, and the detection standard peptide group A and the calibration standard peptide group B are each diluted to a concentration of 100 fmol / μL;

[0036] 4. After the completion of the proteome extraction of HEK293 cells using the kit, take 400 μg of the proteome sample in two groups, respectively add 2 μL of trypsin, and use 50 mM NH4HCO3 solution to make up to 20 μL of the system, and perform enzymolysis at 37 °C overnight;

[0037] 5. Take an SPE C18 1 mL desalination column, and add the following in sequence: 1 mL of methanol, 1 mL of 0.1% formic acid * 2 times, sample * 2 times, 1 mL of 0.1% formic acid * 3 times, and 1 mL of 70% acetonitrile to elute the sample;

[0038] 6. Perform centrifugal vacuum freeze-drying on the eluted sample;

[0039] 7. Add 5 μL of 100 fmol / μL detection standard peptide group A to the sample tube, respectively, and add 200 μL of Buffer 1 (80% acetonitrile, 5% trifluoroacetic acid, 1M lactic acid) to mix;

[0040] 8. Add 4 mg of silica beads to the sample tube, and perform room temperature 1,000 rpm shaking incubation for 30 min;

[0041] 9. After 2,000 g centrifugation for 3 min, remove the supernatant;

[0042] 10. Wash with Buffer 2 (80% acetonitrile, 5% trifluoroacetic acid) 4 times, each time add 500 μL to mix, and then perform room temperature 1,000 rpm shaking for 5 min, and then remove the supernatant after 2,000 g centrifugation;

[0043] 1,000 rpm shaking for 5 min, and then remove the supernatant after 2,000 g centrifugation;

[0044] 11. Add Buffer 3 (40% acetonitrile, pH = 7) to one of the beads, and add Buffer 4 (40% acetonitrile, 15% ammonia water, pH = 10) to the other beads, and perform room temperature 1,000 rpm shaking incubation for 5 min for elution, and then remove the supernatant to a new EP tube after 2,000 g centrifugation for 5 min, to obtain sample 1 and sample 2, respectively;

[0045] 12. Perform centrifugal vacuum freeze-drying on the eluted sample;

[0046] 12. Perform centrifugal vacuum freeze-drying on the eluted sample;

[0047] 13. To the sample, 5 μL of 100 fmol / μL calibration standard peptide segment group B was added, 15 μL of 0.1% formic acid water was added, mixed for 14. 15 μL was injected into liquid chromatography mass spectrometry for 150 min DDA data acquisition, liquid chromatography mass spectrometry was Thermo U3000+HF for detection; mobile phase A was 2% acetonitrile, 0.1% formic acid aqueous solution, mobile phase B was 80% acetonitrile and 0.1% formic acid aqueous solution; the gradient was set as 0-5 min, 3% B, 5-140 min, 3%-90% B, 140-150 min, 90%-3% B;

[0048] 15. The data was extracted by Proteome Discoverer software in this example, the alkylated modification of immobilized modification was cysteine, the variable modification was serine, threonine and tyrosine phosphorylation modification, the peptide qualitative and quantitative data table was derived, and two groups of peptides were extracted from the table as follows:

[0049] Table 1. Quantitative information of 3 peptide segment standards extracted from sample 1

[0050] Peptide segment Detection standard peptide segment A Calibration standard peptide segment B Ratio 1 T(phos)PVISGGPYEYR 1,045,335 23,506,900 0.04 2 TPVIS(phos)GGPYEYR 1,078,561 24,012,080 0.04 3 TPVISGGPY(phos)EYR 762,352 26,410,140 0.03

[0051] Table 2. Quantitative information of 3 peptide segment standards extracted from sample 2

[0052] Peptide segment Detection standard peptide segment A Calibration standard peptide segment B Ratio 1 T(phos)PVISGGPYEYR 20,758,352 23,509,600 0.88 2 TPVIS(phos)GGPYEYR 21,025,310 23,467,090 0.90 3 TPVISGGPY(phos)EYR 16,935,320 26,320,180 0.64

[0053] Table 3. Quantitative information of part of sample peptides extracted from sample 1 and 2

[0054] Peptide segment Sample 1 Sample 2 Ratio 1 NFS(phos)FMNPGMER 967,992 10,797,992 0.09 2 LPNGEPSPDPGGKGT(phos)PR 87,395 1,090,877 0.08 3 ISVY(phos)YNEAYGR 6,360 361,037 0.02

[0055] By analyzing the ratio data, it was found that the quantitative ratio of the three phosphorylated peptides in sample 1 was significantly small, and by comparing the quantitative ratio of the same peptides in sample 1 and sample 2, it was found that the recovery ratio of the phosphorylated peptides in sample 1 was also small, indicating that in the process of using titanium dioxide to enrich phosphorylated peptides, the effect of using low pH buffer to elute phosphorylated peptides was not as good as that of high pH buffer, and the pH of the buffer needed to be improved to increase the elution effect.

Claims

1. A method for monitoring the enrichment process of phosphorylated peptides in phosphoproteomics, characterized in that, The method comprises the following steps: 1) providing at least two groups of exogenous peptide segment references, each group of exogenous peptide segment references comprising at least three peptides with different phosphorylation modification sites, and for each peptide in each group, there is a corresponding peptide with the same sequence but different isotope labeling in other groups; 2) adding the different exogenous peptide segment references at different time points during sample processing, and between the time points of adding any two groups of exogenous peptide segment references, a step of enriching phosphorylated peptides is included; 3) injecting and detecting, extracting processing data, quantifying the exogenous peptide segment references, dividing the abundance of the peptide segments of the exogenous peptide segment references added earlier by the abundance of the peptide segments of the exogenous peptide segment references added later to obtain the yield ratio of the different phosphorylation modification peptides, so as to evaluate whether the phosphorylation enrichment process between the time points of adding the two groups of exogenous peptide segment references is qualified.

2. The method for monitoring the enrichment process of phosphorylated peptides in phosphoproteomics according to claim 1, characterized in that, In step 1), the different phosphorylation modification sites are selected from the phosphorylation modification sites distributed on threonine, serine, tyrosine, histidine, arginine, lysine, aspartic acid, glutamic acid and cysteine residues.

3. The method for monitoring the enrichment process of phosphorylated peptides in phosphoproteomics according to claim 2, characterized in that, The different phosphorylation modification sites are selected from the phosphorylation modification sites distributed on threonine, serine and tyrosine residues.

4. The method for monitoring the process of phosphopeptide enrichment in phosphoproteomics according to claim 1, characterized in that, In step 1), each group of exogenous peptide segment references comprises at least three peptides with different phosphorylation modification sites, and the peptides in each group have the same sequence or different sequences.

5. The method for monitoring the enrichment process of phosphorylated peptides in phosphoproteomics according to claim 1, characterized in that, In step 1), the different isotope labeling includes that the peptides in one group have no isotope labeling, the peptides in other groups have isotope labeling, but the isotopes used for labeling are different; or the peptides in each group have isotope labeling, but the isotopes used for labeling are different.

6. The method for monitoring the process of phosphopeptide enrichment in phosphoproteomics according to claim 1, wherein, In step 1), the isotopes in the isotope labeling include 12C, 13C, 14C, 14N, 15N, 16O, 18O, 1H, 2H, 32S, 34S, 30P and 32P.

7. The method for monitoring the process of phosphopeptide enrichment in phosphoproteomics according to claim 1, characterized in that, In step 2), the sample is selected from cells, tissues, whole blood or tissue fluid.

8. The method for monitoring the process of phosphopeptide enrichment in phosphoproteomics according to claim 1, characterized in that, In step 2), the exogenous peptide segment references are accurately quantified before being added; and the step of enriching phosphorylated peptides includes all steps or part of the steps of enriching phosphorylated peptides.

9. The method for monitoring the enrichment process of phosphorylated peptides in phosphoproteomics according to claim 8, characterized in that, In step 2), the exogenous peptide segment references are accurately quantified before being added, that is, the added amounts are consistent or the added amounts are in proportion.

10. The method for monitoring the process of phosphopeptide enrichment in phosphoproteomics according to claim 1, characterized in that, In step 3), the detection method is mass spectrometry.

11. The method for monitoring the process of phosphopeptide enrichment in phosphoproteomics according to claim 1, characterized in that, In step 3), if the exogenous peptide segment references added earlier are added before or at the beginning of the enrichment of phosphorylated peptides, and the exogenous peptide segment references added later are added at the end of or after the enrichment of phosphorylated peptides, the abundance of the peptide segments of the exogenous peptide segment references added earlier is divided by the abundance of the peptide segments of the exogenous peptide segment references added later to obtain the yield ratio of the different phosphorylation modification peptides, so as to evaluate whether the whole process of enriching phosphorylated peptides is qualified.

12. The method for monitoring the process of phosphopeptide enrichment in phosphoproteomics according to claim 1, characterized in that, In step 3), if the pre-added exogenous peptide segment reference and the post-added exogenous peptide segment reference only include part of the steps of the phosphorylated peptide segment enrichment process between the two time points of adding, the peptide segment abundance of the pre-added exogenous peptide segment reference is divided by the abundance of the peptide segment of the same sequence of the post-added exogenous peptide segment reference, to obtain the yield ratio of different phosphorylated modified peptide segments, so as to evaluate whether the part of the above phosphorylated peptide segment enrichment process is qualified.

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