阅读说明：本技术 一种测定蛋白质或多肽相对含量的方法 (Method for determining relative content of protein or polypeptide ) 是由 赵海义 华权高 李立 周璀 陈亚运 鲁思琴 舒芹 于 2019-09-25 设计创作，主要内容包括：本发明提供一种测定蛋白质或多肽相对含量的方法,所述方法将样品经过前处理成多肽样品后,往多肽样品中加入参考肽,进行液相色谱-平行反应监测质谱测试,用参考肽做多肽样品定量输出值的归一化标准,得到所述多肽样品相对于所述参考肽的相对含量。本发明的有益效果在于(1)PRM相对定量实验流程简单易操作,能够普遍应用于常规的蛋白或多肽检测项目,不需要花费较长的优化步骤。(2)外加参考肽段不受样品内源性因素的干扰,且不受管家蛋白在不同实验处理条件下表达量不准确的影响以及多肽等样本实验设计的限制；(3)不需要额外合成同位素标记内参肽段,方法建立快速简便,投入小。(The invention provides a method for determining the relative content of protein or polypeptide, which comprises the steps of preprocessing a sample into a polypeptide sample, adding reference peptide into the polypeptide sample, carrying out liquid chromatography-parallel reaction monitoring mass spectrometry, and using the reference peptide as a normalization standard of a quantitative output value of the polypeptide sample to obtain the relative content of the polypeptide sample relative to the reference peptide. The invention has the beneficial effects that (1) the PRM is simple and easy to operate relative to a quantitative experimental process, can be generally applied to conventional protein or polypeptide detection items, and does not need to spend long optimization steps. (2) The additional reference peptide segment is not interfered by endogenous factors of the sample and is not influenced by inaccurate expression of housekeeping protein under different experimental treatment conditions and is not limited by experimental design of samples such as polypeptide and the like; (3) no need of additional synthesis of isotope labeled reference peptide segment, quick and simple method establishment and small investment.)

1. A method for determining the relative amount of a protein or polypeptide, said method comprising:

step one, adding a known amount of reference peptide or protein reagent containing the reference peptide to a polypeptide sample to obtain a processed sample, wherein the reference peptide response is stable and is not interfered by endogenous signals, and the reference peptide comprises one or more peptide sections;

step two, carrying out desalination treatment on the treated sample to obtain a detection sample;

step three, according to the sequence information of the target polypeptide of the detection sample, designing a parallel reaction monitoring mode detection method through skyline software, selecting the mass-to-charge ratio of one or more peptide segments of the reference peptide and the mass-to-charge ratio of the target peptide segment of the detection sample in the step one, and adding the selected mass-to-charge ratios into an inclusionlist to establish a mass spectrum collection method;

step four, performing liquid phase-mass spectrometry detection on the detection sample according to the mass spectrometry collection method in the step three, further screening protein peptide fragments according to a detection result, scanning the screened protein peptide fragments by adopting a parallel reaction monitoring mode, and smashing the screened protein peptide fragments to obtain a secondary ion spectrum, wherein the screened protein peptide fragments comprise one or more peptide fragments of the reference peptide in the step three;

step five: analyzing the data obtained in the fourth step, quantifying each peptide segment, outputting a graph of the relationship between the retention time and the peak intensity of the protein peptide segment of the detection sample, and using the graph area of one peptide segment of the reference peptide or the average value of the graph areas of a plurality of peptide segments in the fourth step as the normalization standard of the quantitative output value of the polypeptide sample to obtain the relative content of the polypeptide sample relative to the reference peptide;

wherein the reference peptide is selected from beta-galactosidase; the protein reagent containing the reference peptide is a beta-galactosidase reagent.

2. The method for determining the relative content of protein or polypeptide according to claim 1, wherein the first step further comprises the preparation of polypeptide sample, and the preparation of polypeptide sample comprises the following steps:

s1-1, performing protein brandford quantification or cell calculation quality control on an experimental sample, adding an equivalent or isovolumetric sample into a polypeptide extraction lysate for lysis, centrifuging, and selecting a supernatant;

s1-2, adding a reducing agent into the supernatant to perform a reduction reaction to obtain a reduced sample;

s1-3, adding the reduced sample into an alkylating reagent for alkylation reaction to obtain a primary sample;

s1-4, carrying out ultrafiltration machine centrifugation treatment on the primary sample to obtain the polypeptide sample.

3. The method for determining the relative content of a protein or polypeptide according to claim 1, wherein the reference peptide comprises one or more of a first reference peptide fragment, a second reference peptide fragment, a third reference peptide fragment, a fourth reference peptide fragment and a fifth reference peptide fragment, the first reference peptide fragment has a mass-to-charge ratio of 563.2784, the second reference peptide fragment has a mass-to-charge ratio of 719.3682, the third reference peptide fragment has a mass-to-charge ratio of 832.4523, the fourth reference peptide fragment has a mass-to-charge ratio of 1061.5222, and the fifth reference peptide fragment has a mass-to-charge ratio of 1176.5491.

4. The method of claim 3, wherein the reference peptide pattern area is an average of the pattern area of the first reference peptide fragment, the pattern area of the second reference peptide fragment, the pattern area of the third reference peptide fragment, the pattern area of the fourth reference peptide fragment, and the pattern area of the fifth reference peptide fragment.

5. The method for determining the relative content of protein or polypeptide according to claim 1, wherein the β -galactosidase reagent is AB Sciex LC/MS Peptide calibration kit.

6. The method for determining the relative content of protein or polypeptide according to claim 5, wherein the beta-galactosidase reagent is added in an amount of 15-30 fmol and in a volume of 1.2-2.2 uL.

7. The method for determining the relative content of protein or polypeptide according to claim 1, wherein in the second step, the processed sample is desalted by a C18 column to obtain the detection sample.

8. The method of claim 1, wherein in the fourth step, the sample is first introduced into a C18 capture column, and the eluent is subjected to gradient from the C18 analytical column at a flow rate of 250nL/min to 350nL/minEluting, wherein the water phase of the eluent comprises 1 to 2.5 mass percent of acetonitrile, 0.05 to 0.2 mass percent of formic acid and 98 to 98.5 mass percent of H₂O, wherein the organic phase of the eluent comprises 98-98.5 percent of acetonitrile, 0.05-0.2 percent of formic acid and 1-2.5 percent of H in percentage by mass₂And (C) O.

9. The method for determining the relative content of protein or polypeptide according to claim 1, wherein in the fourth step, the screening of the protein peptide fragment is performed by mass spectrometry information-dependent acquisition, and the mass spectrometry conditions are set as follows: scanning a primary mass spectrum with the ion accumulation time of 250MS, acquiring secondary mass spectra of 30 precursor ions with the ion accumulation time of 100MS, and performing MS in the range of 350-1500m/z¹Collecting and performing MS in the range of 100-1500m/z²The precursor ion dynamic exclusion time was set to 15s for the acquisition.

Technical Field

The field is the field of protein or polypeptide quantitative analysis, and particularly relates to a method for determining the relative content of protein or polypeptide.

Background

The HPLC-MS/MS targeted quantification technology mainly refers to a monitoring technology which sets instrument parameters in a targeted manner for collection according to a known or assumed target, records signals of ions meeting the setting, removes signal interference irrelevant to the target, and finally obtains a quantitative result through data arrangement and analysis.

Based on different detection conditions, experimental purposes and application ranges, the HPLC-MS/MS targeted quantitative technology mainly comprises a single reaction monitoring technology (SRM), a multi-reaction monitoring technology (MRM) and a parallel reaction monitoring technology (PRM) which is recently attracted gradually and is increasingly applied to the field of protein/polypeptide detection, and the traditional SRM/MRM technology is based on the low-resolution detection performance of a triple quadrupole detector, can only detect limited precursor ions and daughter ion pairs and has limited flux. The detection process requires a lot of time to optimize the detection conditions and the results can only be used for quantification.

In the conventional targeted quantitative technology, in order to obtain the standard for the normalization and correction of quantitative results, a common approach is to synthesize a stable isotope-labeled form of a target peptide fragment by chemical synthesis. Through label synthesis, stable isotope labels (such as 2H, 13C, 15N and 18O) are introduced into the peptide fragments, so that the polypeptides labeled with 'light' and 'heavy' isotopes in the same mass spectrum scan have the same chromatographic behavior and ionization efficiency, the relative intensity of mass spectrum peak signals appearing in pairs can accurately reflect the abundance ratio (protein ratio) of the polypeptides in a sample, and the quantitative reliability is good; the standard peptide fragment is obtained by a chemical synthesis method, although the quantitative accuracy is very high, the time is spent for searching the specific peptide fragment with good detection effect and high reproducibility, and the time period of the experimental process and the synthesis stage is usually very long. In addition, in one experiment, the isotope-labeled forms of a plurality of target peptide fragments are often required to be synthesized, and the research cost is increased due to the extra expense of gold.

In addition, when a sample is subjected to on-machine detection and quantitative analysis, a proper quality control means is often required for controlling in order to correct the difference of target expression caused by protein quantification, experiments and on-machine analysis in the experimental process. In the case of protein or polypeptide sample detection, quantitative quality control of protein or polypeptide is generally performed after pretreatment steps such as sample extraction. However, due to the technical defects of the quantitative method, the method usually selects a proper internal reference protein or peptide fragment to correct the sample amount in the experimental process so as to achieve the purpose of more accurate research, and different types of samples are researched, different experimental design requirements are met, and different limitations are met for the selection of the internal reference. The common approach is based on the fact that housekeeping proteins usually perform a large number of basic functions, and that high levels of expression in tissues and cells can be sustained to select peptide fragments specific for housekeeping proteins for correction. However, in some specific samples, such as the content detection of some polypeptide samples, it is difficult to correct the content of housekeeping proteins.

Disclosure of Invention

In order to solve the technical defects of small detection flux, high cost, long test time and difficult adaptation of the conventional internal reference protein or peptide fragment to all samples in the traditional detection method, the invention provides a method for determining the relative content of protein or polypeptide.

The specific technical scheme is as follows:

step one, adding a known amount of reference peptide or protein reagent containing the reference peptide to a polypeptide sample to obtain a processed sample, wherein the reference peptide response is stable and is not interfered by endogenous signals, and the reference peptide comprises one or more peptide sections;

step two, carrying out desalination treatment on the treated sample to obtain a detection sample;

step three, according to the sequence information of the target polypeptide of the detection sample, designing a parallel reaction monitoring mode detection method through skyline software, selecting the mass-to-charge ratio of one or more peptide segments of the reference peptide and the mass-to-charge ratio of the target peptide segment of the detection sample in the step one, and adding the selected mass-to-charge ratios into an inclusionlist to establish a mass spectrum collection method;

step four, performing liquid phase-mass spectrometry detection on the detection sample according to the mass spectrometry collection method in the step three, further screening protein peptide fragments according to a detection result, scanning the screened protein peptide fragments by adopting a parallel reaction monitoring mode, and smashing the screened protein peptide fragments to obtain a secondary ion spectrum, wherein the screened protein peptide fragments comprise one or more peptide fragments of the reference peptide in the step three;

step five: analyzing the data obtained in the fourth step, quantifying each peptide segment, outputting a graph of the relationship between the retention time and the peak intensity of the protein peptide segment of the detection sample, and using the graph area of one peptide segment of the reference peptide or the average value of the graph areas of a plurality of peptide segments in the fourth step as the normalization standard of the quantitative output value of the polypeptide sample to obtain the relative content of the polypeptide sample relative to the reference peptide;

wherein the reference peptide is selected from beta-galactosidase; the protein containing the reference peptide is a beta-galactosidase reagent.

In the above technical solution, the first step further comprises preparation of a polypeptide sample, and the preparation of the polypeptide sample comprises the following steps:

s1-1, performing protein brandford quantification or cell calculation quality control on an experimental sample, adding an equivalent or isovolumetric sample into a polypeptide extraction lysate for lysis, centrifuging, and selecting a supernatant;

s1-2, adding a reducing agent into the supernatant to perform a reduction reaction to obtain a reduced sample;

s1-3, adding the reduced sample into an alkylating reagent for alkylation reaction to obtain a primary sample;

s1-4, carrying out ultrafiltration machine centrifugation treatment on the primary sample to obtain the polypeptide sample.

In the above technical solution, the reference peptide includes one or more of a first reference peptide fragment, a second reference peptide fragment, a third reference peptide fragment, a fourth reference peptide fragment and a fifth reference peptide fragment, the first reference peptide fragment has a mass-to-charge ratio of 563.2784, the second reference peptide fragment has a mass-to-charge ratio of 719.3682, the third reference peptide fragment has a mass-to-charge ratio of 832.4523, the fourth reference peptide fragment has a mass-to-charge ratio of 1061.5222, and the fifth reference peptide fragment has a mass-to-charge ratio of 1176.5491.

In the above technical solution, the reference peptide pattern area is an average value of a pattern area of the first reference peptide fragment, a pattern area of the second reference peptide fragment, a pattern area of the third reference peptide fragment, an image area of the fourth reference peptide fragment, and a pattern area of the fifth reference peptide fragment.

In the technical scheme, the beta-galactosidase reagent is AB Sciex LC/MSpeptide Calibration Kit.

In the technical scheme, the addition amount of the beta-galactosidase reagent is 15-30 fmol, and the addition volume is 1.2-2.2 uL.

In the above technical solution, in the second step, the detection sample is obtained by desalting the treatment sample through a C18 column.

In the technical scheme, in the fourth step, the detection sample firstly enters a C18 capture column, the eluent is subjected to gradient elution from a C18 analytical column at the flow rate of 250 nL/min-350 nL/min, and the water phase of the eluent comprises 1-2.5% of acetonitrile, 0.05-0.2% of formic acid and 98-98.5% of H in percentage by mass₂O, wherein the organic phase of the eluent comprises 98-98.5 percent of acetonitrile, 0.05-0.2 percent of formic acid and 1-2.5 percent of H in percentage by mass₂And (C) O.

In the above technical solution, in the fourth step, the screening of the protein peptide fragment is obtained by mass spectrum information dependent acquisition, and the mass spectrum conditions are set as follows: scanning a primary mass spectrum with the ion accumulation time of 250MS, acquiring secondary mass spectra of 30 precursor ions with the ion accumulation time of 100MS, and performing MS in the range of 350-1500m/z¹Collecting and performing MS in the range of 100-1500m/z²The precursor ion dynamic exclusion time was set to 15s for the acquisition.

Compared with the prior art, the invention has the beneficial effects that (1) the PRM is simple and easy to operate relative to a quantitative experimental process, can be generally applied to conventional protein or polypeptide detection items, and does not need to spend long optimization steps. (2) The additional reference peptide segment is not interfered by endogenous factors of the sample and is not influenced by inaccurate expression of housekeeping protein under different experimental treatment conditions and is not limited by experimental design of samples such as polypeptide and the like; (3) no need of additional synthesis of isotope labeled reference peptide segment, quick and simple method establishment and small investment.

Drawings

The visualization result of octaskyline in the embodiment of fig. 1 is shown.

Detailed Description

The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.

The term "PRM (parallel reaction monitoring technology)" is based on Q-Orbitrap and Q-TOF as representative high resolution, high precision mass spectrum platform, using the selection ability of quadrupole mass analyzer, using Q1 to select the parent ion of the target peptide fragment, using Orbitrap or TOF high resolution mass spectrum analyzer to detect all fragment information in the selected parent ion window in the second mass spectrum, and can accurately and specifically analyze and quantify the target protein/peptide fragment in the complex sample. Due to the fact that the PRM has higher detection flux compared with the traditional SRM/MRM by means of the high-resolution mass spectrum analyzer, all the precursor ions of up to 100 in the list can be subjected to secondary fragmentation synchronously, and the detection result can be used for quantification and database retrieval to achieve quantification.