A high resolution mass spectrometry-based intact 15 Method for determining protein abundance using n stable isotope labeling

Abstract

The application relates to the technical field of high-resolution mass spectrometry, and discloses a complete N stable isotope labeling protein abundance determination method based on high-resolution mass spectrometry 15 A method for determining the abundance of N stable isotope labeled protein comprises the following steps: collecting and calculating the mass spectrum signal of a sample to be measured to obtain the actual measured relative isotope peak intensity of the sample at m / z=i; calculating the predicted relative isotope peak intensity of m / z=i under different N isotope abundances; calculating the correlation coefficient r under different N isotope abundances according to formula 1; and comparing the correlation coefficients r calculated under different N isotope abundances, wherein the r value closest to 1 is the N stable isotope labeling protein abundance value. 15 15 15 15 The application has the advantages of small sample consumption, no need for pretreatment such as derivatization, simple operation, high resolution, small calculation workload, good stability and accuracy.​​​

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of high-resolution mass spectrometry, in particular to a method for determining the abundance of intact N-stable isotope labeled proteins based on high-resolution mass spectrometry. 15 The present application relates to the technical field of high-resolution mass spectrometry, in particular to a method for determining the abundance of intact N-stable isotope labeled proteins based on high-resolution mass spectrometry. BACKGROUND

[0002] Proteins are important components of living organisms, are organic macromolecules, and are the basic organic substances that constitute cells. In general, proteins in living organisms are composed of 20 natural amino acids and are involved in almost all life activities. From the structural point of view, proteins have complex and diverse spatial conformations. The primary structure is the linear arrangement order of amino acids, and the secondary structure such as alpha-helix and beta-fold is further folded to form the tertiary structure, and multiple subunits are combined to form the quaternary structure. This precise structure endows proteins with specific functions. With the development of science and technology, biologists and chemists have continuously deepened the research in the field of life sciences, and the role of stable isotope labeled proteins in scientific research has become increasingly prominent. Stable isotope labeled protein macromolecule technology provides a powerful tool for in-depth understanding of the structure, function and metabolic pathway of proteins. Stable isotope labeling technology can reveal the conformational changes of proteins under different conditions by tracking the position and dynamic changes of labeled elements in the protein structure. The flow of labeled elements in the protein metabolic pathway can be tracked to obtain valuable information about the function of proteins. Therefore, the importance of stable isotope labeled proteins in scientific research is self-evident.

[0003] Among them, the isotopic abundance value is an important quality control index of stable isotope labeling reagents. Stable isotope abundance detection provides accurate data basis for scientific research. In the fields of environmental science, ecology, earth science and other fields, stable isotopes are used as tracers. By measuring the relative abundance changes of isotopes in different substances, key information in the material cycle and environmental process can be revealed.

[0004] Currently, the abundance detection method is only for labeled small molecule compounds, and there is no report on the abundance detection of macromolecules. For small molecule compounds, the detection methods of isotope abundance mainly include nuclear magnetic resonance spectroscopy and mass spectrometry.

[0005] Nuclear magnetic resonance spectroscopy is mainly used for the abundance detection of deuterium labeled compounds. Xie et al. developed a method for determining the deuterium isotope abundance of deuterium labeled compounds by using nuclear magnetic hydrogen spectrum or deuterium spectrum (CN112305007A). The content of hydrogen in the deuterium labeled compound is measured by the peak area of the internal standard method, and then the isotope abundance of the deuterium labeled compound is obtained, and the information of the deuterium labeling site can also be obtained. However, the quantitative nuclear magnetic method is not suitable for the determination of the abundance of stable isotope labeled proteins when analyzing biological macromolecule samples, because the spectral analysis process is complex and a large amount of sample is required.

[0006] Low-resolution mass spectrometry mainly includes quadrupole mass spectrometry. Sheng Liyan et al. invented a method for determining the abundance of stable isotopes based on triple quadrupole mass spectrometry (CN118549558A), which uses the selected reaction monitoring mode of triple quadrupole mass spectrometry to obtain the relative content of isotope number bodies in the measured substance, and finally calculates the abundance of the measured substance. However, low-resolution mass spectrometry is limited by instrument resolution, which leads to ineffective separation of mass spectrometry peaks with similar mass-to-charge ratios, and large errors. At the same time, the deconvolution calculation method is used in the calculation of isotopic abundance, which is difficult.

[0007] High-resolution mass spectrometry mainly includes Fourier transform mass spectrometry, time-of-flight mass spectrometry, and orbit ion trap mass spectrometry, which has the characteristics of high sensitivity, rapid stability, and high accuracy. Li Xiujun et al. invented a method for detecting the isotopic distribution and abundance of deuterium-labeled compounds (CN112630345B), which detects deuterium-labeled compounds by Q-Orbitrap quadrupole orbit trap high-resolution mass spectrometry, and collects multiple Full-MS mass spectra to obtain an isotopic deviation value greater than the instrument mass deviation value. Based on mass spectrometry detection, the isotopic distribution and abundance of the deuterium-labeled compound are obtained. High-resolution mass spectrometry can achieve accurate mass number determination, which not only significantly reduces the workload of calculation results, but also helps to ensure the accuracy of the results, and is especially suitable for the detection of isotopic distribution and abundance. However, the invention mainly focuses on the determination of the isotopic abundance of deuterium-labeled compounds, and does not involve the determination of the abundance of protein biomacromolecules.

[0008] Proteins are large molecules with complex spatial structures obtained by processing and deformation of polypeptides, which are products of amino acid condensation. Currently, there are only abundance determination techniques for labeled amino acids and polypeptides, and there is no direct abundance determination and analysis method for intact proteins.

[0009] Therefore, a method for determining the abundance of intact stable 15 N isotopically labeled proteins based on high-resolution mass spectrometry is provided. SUMMARY

[0010] In view of this, the present application provides a method for determining the abundance of intact stable 15 N isotopically labeled proteins based on high-resolution mass spectrometry, which aims to solve at least one of the above background technical problems.

[0011] The present application provides a method for determining the abundance of intact stable 15 N isotopically labeled proteins based on high-resolution mass spectrometry, comprising the following steps:

[0012] Collecting and calculating the mass spectrometry signal of the measured sample to obtain the actual measured relative isotopic peak intensity of the sample at m / z = i ;

[0013] Computing different 15 m / z=i under different N isotope abundance

[0014] According to formula 1, respectively calculate the Pearson correlation coefficient r under different N isotope abundance 15

[0015] Compare the Pearson correlation coefficient r calculated under different N isotope abundance 15 The closer the r value is to 1, the higher the correlation between the predicted isotope distribution and the experimental measurement, and the r value closest to 1 corresponds to 15 N isotope abundance, which represents the 15N stable isotope labeled protein abundance value, unit: atom%;

[0016] The formula 1 is:

[0017]

[0018] Among them, is the actual measured relative isotope peak intensity of the compound, refers to the average value of the actual measured relative isotope peak intensity; is the predicted relative isotope peak intensity of the compound, refers to the average value of the predicted relative isotope peak intensity, n is the total number of isotope peaks calculated by the Pearson correlation coefficient r, and i corresponds to the isotope peak of different mass-to-charge ratio (m / z=i) in the mass spectrum.

[0019] Preferably, the method for obtaining the actual measured relative isotope peak intensity of the sample at m / z=i is specifically:

[0020] Accurately weigh 15 N stable isotope labeled protein, prepare into working concentration to obtain sample;

[0021] According to 15 the mass spectrum response of N stable isotope labeled protein, select the corresponding concentration sample for high-resolution liquid chromatography-mass spectrometry detection, determine the chromatographic conditions according to the sample properties, and repeat the sample injection;

[0022] Select a narrow m / z range at the peak top point for analysis to obtain the actual measured relative isotope peak intensity of the sample at m / z=i .

[0023] Preferably, the accurate weighing adopts a millionth balance.

[0024] Preferably, the number of repeated injections is 6-8 times. ​​​

[0025] Preferably, the narrow m / z range is specifically from the minimum m / z of the isotope distribution of the unlabeled protein to the maximum m / z of the 100% enriched isotope distribution.

[0026] Preferably, the different N isotope abundances are calculated separately. 15 In the Pearson correlation coefficient r under the N isotope abundance, the calculation is performed only using > 20% of the isotope peaks. > 20% of the isotope peaks.

[0027] Preferably, the different N isotope abundances are calculated separately. 15 In the Pearson correlation coefficient r under the N isotope abundance, the calculation is performed only using > 20% of the isotope peaks. > 20% of the isotope peaks.

[0028] The application also provides the use of the determination method described in the above technical solution in the isotope-labeled protein abundance.

[0029] Compared with the prior art, the application has the beneficial effects that:

[0030] The application provides a determination method for the abundance of intact stable isotope-labeled protein based on high-resolution mass spectrometry. The method uses a high-resolution liquid chromatograph-mass spectrometer to determine the abundance of intact stable isotope-labeled protein, so as to fill the blank of the determination method for the abundance of stable isotope-labeled protein. The method is directed to the direct analysis of intact protein, and does not need complex sample pretreatment such as enzyme cutting and hydrolysis on the protein sample; meanwhile, the sample consumption is small, and no pretreatment such as derivatization is needed, and the operation is simple; the resolution is high, the calculation workload is small, and the method has good stability and accuracy; and the method is suitable for the determination of the abundance of stable isotope-labeled protein.

[0031] In addition, the application can evaluate the effectiveness and accuracy of the detection 15 of N stable isotope-labeled protein, and then establish a protein product quality control system based on the abundance spectrum, and improve 15 the synthesis quality of N stable isotope-labeled protein. More comprehensive and reliable information is provided for elucidating the synthesis metabolic pathway of important biomolecules in organisms and the action mechanism research of important signal molecules. DETAILED DESCRIPTION

[0032] A number of illustrative embodiments of the application are described below in detail. The detailed description is not intended to be limiting of the application, but rather to provide a more complete understanding of certain aspects, features and embodiments of the application. It is understood that the terms used herein are merely descriptive, but are not intended to be limiting of the application.

[0033] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0034] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0035] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0036] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0037] This invention proposes a complete method based on high-resolution mass spectrometry. 15 The method for determining the abundance of nitrogen-stabilized isotope-labeled proteins includes the following steps:

[0038] The mass spectrometry signal of the sample to be tested is acquired and calculated to obtain the actual measured relative isotope peak intensity of the sample at m / z=i. ;

[0039] Different results obtained through computer programs 15 Predicted relative isotopic peak intensity for m / z=i under N isotopic abundance ;

[0040] Calculate the different values ​​according to Formula 1. 15 Pearson correlation coefficient r under N isotope abundance;

[0041] Compare the differences 15 The Pearson correlation coefficient r calculated for N isotope abundance is such that the closer the r value is to 1, the higher the correlation between the predicted isotope distribution and the experimental measurement. The r value closest to 1 corresponds to... 15 The abundance of nitrogen isotopes represents 15N stable isotope labeled protein abundance value, unit: atom %.

[0042] Preferably, the sample in m / z = i actual measurement relative to the isotope peak intensity The obtaining method is specifically:

[0043] Accurately weigh 15 N stable isotope labeled protein, prepare into working concentration, and obtain a sample;

[0044] According to 15 The mass spectrum response of the N stable isotope labeled protein, select the corresponding concentration sample for high-resolution liquid chromatography-mass spectrometry detection, determine the chromatographic conditions according to the sample properties, and repeat the sample injection 6-8 times;

[0045] Select a narrow m / z range at the peak top for analysis, the range is from the minimum m / z of the isotope distribution of the unlabeled protein to the maximum m / z of the 100% enriched isotope distribution;

[0046] Obtain the actual measurement relative to the isotope peak intensity of the sample in m / z = i .

[0047] In the present application, the formula 1 is:

[0048] ;

[0049] Among them, is the actual measurement relative to the isotope peak intensity of the compound, refers to the average value of the actual measurement relative to the isotope peak intensity; is the predicted relative isotope peak intensity of the compound, refers to the average value of the predicted relative isotope peak intensity.

[0050] In the present application, the pearson correlation coefficient r under different 15 N isotope abundance is calculated respectively. > 20%, > 20% of the isotope peak.

[0051] The present application also provides the application of the determination method in the isotope labeled protein abundance.

[0052] Example 1

[0053] Based on 5+ ion 15 N-IGF1 abundance detection

[0054] (1) Accurately weigh 15N-IGF1 protein solution was prepared into a working solution with a concentration of 1 mg / mL;

[0055] (2) Based on the mass spectrometry response of the compound, prepare the... 15 The N stable isotope labeled protein was detected by high-resolution liquid chromatography-mass spectrometry, with repeated injections 6-8 times.

[0056] (3) Chromatographic conditions: Column: Kinetex-C18 (2.6 μm, 2.1 x 150 mm); Mobile phase: A: 0.1% FA-H2O; B: 0.1% FA-ACN; Elution conditions: 15 min gradient elution; Flow rate: 0.3 mL / min; Injection volume: 2 μL;

[0057] (4) Mass spectrometry parameters: spray voltage 3.5kV, capillary temperature 250℃, auxiliary gas temperature 400℃, full scan mode, resolution 120000 (FWHM), scan range m / z 300-3500;

[0058] (5) Select a narrow range of m / z at the peak for analysis, ranging from the minimum m / z of the isotope distribution of unlabeled protein (1530.9339) to the maximum m / z of the 100% enriched isotope distribution (1554.5894);

[0059] (6) Obtain the actual measured relative isotope peak intensity of the sample at m / z=i ( >20%), and obtained through computer programs. 15 Predicted relative isotopic peak intensity of m / z =i under N isotopic abundance ( >20%), and the results are shown in Table 1;

[0060] Table 1 Differences 15 Table of predicted relative isotopic peak intensities for m / z =i under N isotopic abundance

[0061]

[0062] (7) Calculate the different 15 The Pearson correlation coefficients r for N isotope abundances are shown in Table 2. Only N isotope abundances were used in the analysis. >20%, >20% isotope peaks;

[0063] Table 2 Differences 15 Table of Pearson correlation coefficient r calculated based on N isotope abundance.

[0064] ;

[0065] (8) The Pearson correlation coefficient r, closest to 1, is 0.991, indicating the best correlation between the predicted and measured isotope distributions. The corresponding isotope abundance of 91.9 atom% is... 15 N-IGF1 protein abundance value.

[0066] Example 2

[0067] Based on 6+ ions 15 N-IGF1 abundance detection

[0068] (1) Use a balance with a ratio of 1 / 1,000,000 to accurately weigh. 15 N-IGF1 protein solution was prepared into a working solution with a concentration of 1 mg / ml;

[0069] (2) Based on the mass spectrometry response of the compound, prepare the... 15 The N stable isotope labeled protein was detected by high-resolution liquid chromatography-mass spectrometry, with repeated injections 6-8 times.

[0070] (3) Chromatographic conditions: Column: Kinetex-C18 (2.6 μm, 2.1 x 150 mm); Mobile phase: A: 0.1% FA-H2O; B: 0.1% FA-ACN; Elution conditions: 15 min gradient elution; Flow rate: 0.3 mL / min; Injection volume: 2 μL;

[0071] (4) Mass spectrometry parameters: spray voltage 3.5kV, capillary temperature 250℃, auxiliary gas temperature 400℃, full scan mode, resolution 120000 (FWHM), scan range m / z 300-3500;

[0072] (5) Select a narrow range of m / z at the peak for analysis, ranging from the minimum m / z of the isotope distribution of unlabeled protein (1275.9461) to the maximum m / z of the 100% enriched isotope distribution (1595.6590);

[0073] (6) Obtain the actual measured relative isotope peak intensity of the sample at m / z=i ( >20%), and obtained through computer programs. 15 Predicted relative isotopic peak intensity of m / z=i under N isotopic abundance ( >20%), and the results are shown in Table 3;

[0074] Table 3 Differences 15 Table of predicted relative isotopic peak intensities for m / z=i under N isotopic abundance

[0075] ;

[0076] (7) Calculate the different 15 The Pearson correlation coefficients r for N isotope abundances are shown in Table 4. Only N isotope abundances were used in the analysis. >20%, >20% isotope peaks;

[0077] Table 4 Differences 15 Table of Pearson correlation coefficient r calculated based on N isotope abundance.

[0078] ;

[0079] (8) The Pearson correlation coefficient r, closest to 1, is 0.987, indicating the best correlation between the predicted and measured isotope distributions. The corresponding isotope abundance of 91.8 atom% is... 15 N-IGF1 protein abundance value.

[0080] Example 3

[0081] Based on 7+ ions 15 N-IGF1 abundance detection

[0082] (1) Use a balance with a ratio of 1 / 1,000,000 to accurately weigh. 15 N-IGF1 protein solution was prepared into a working solution with a concentration of 1 mg / ml;

[0083] (2) Based on the mass spectrometry response of the compound, prepare the... 15 N-IGF1 protein was detected by high-resolution liquid chromatography-mass spectrometry, with repeated injections 6-8 times;

[0084] (3) Chromatographic conditions: Column: Kinetex-C18 (2.6 μm, 2.1 x 150 mm); Mobile phase: A: 0.1% FA-H2O; B: 0.1% FA-ACN; Elution conditions: 15 min gradient elution; Flow rate: 0.3 mL / min; Injection volume: 2 μL;

[0085] (4) Mass spectrometry parameters: spray voltage 3.5kV, capillary temperature 250℃, auxiliary gas temperature 400℃, full scan mode, resolution 120000 (FWHM), scan range m / z 300-3500;

[0086] (5) Select a narrow range of m / z at the peak for analysis, ranging from the minimum m / z of the isotope distribution of unlabeled protein (1093.8120) to the maximum m / z of the 100% enriched isotope distribution (1110.7088);

[0087] (6) Obtain the actual measured relative isotope peak intensity of the sample at m / z=i ( >20%), and the predicted relative isotopic peak intensity of m / z=i under different 15N isotopic abundance was obtained by computer program >20%), and the results are shown in Table 5;

[0088] Table 5 Predicted relative isotopic peak intensity data table of m / z=i under different 15N isotopic abundance 15 N isotopic abundance

[0089] ;

[0090] (7) The pearson correlation coefficient r under different 15N isotopic abundance was calculated respectively, and the results are shown in Table 6, and only the isotopic peaks with abundance >20% were used for analysis >20%, >20% of the isotopic peak;

[0091] Table 6 Calculation results table of pearson correlation coefficient r under different 15N isotopic abundance 15 N isotopic abundance

[0092] ;

[0093] (8) The r value closest to 1 is 0.992, which represents the best correlation between the predicted isotopic distribution and the measured isotopic distribution, and the corresponding isotopic abundance 91.9 atom% is the 15 N-IGF1 protein abundance value.

[0094] Example 4

[0095] Detection of 8+ ion based N-IGF1 abundance 15 N-IGF1 abundance

[0096] (1) Accurately weigh the N-IGF1 protein solution by using a millionth balance, and prepare a working solution with a concentration of 1 mg / ml; 15 N-IGF1 protein solution;

[0097] (2) According to the mass spectrometric response of the compound, the prepared N-IGF1 protein was detected by high-resolution liquid chromatography-mass spectrometry, and the sample was injected repeatedly for 6-8 times; 15 N-IGF1 protein;

[0098] (3) Chromatographic conditions: chromatographic column: Kinetex-C18 (2.6 μm, 2.1 x 150 mm); mobile phase: A: 0.1% FA-H2O; B: 0.1% FA-ACN; elution condition: 15 min gradient elution; flow rate: 0.3 mL / min; injection volume: 2 μL;

[0099] (4) Mass spectrometry parameters: spray voltage 3.5kV, capillary temperature 250℃, auxiliary gas temperature 400℃, full scan mode, resolution 120000 (FWHM), scan range m / z 300-3500;

[0100] (5) Select a narrow range of m / z at the peak for analysis, ranging from the minimum m / z of the isotope distribution of unlabeled protein (957.2114) to the maximum m / z of the 100% enriched isotope distribution (971.9961);

[0101] (6) Obtain the actual measured relative isotope peak intensity of the sample at m / z=i ( >20%), and obtained through computer programs. 15 Predicted relative isotopic peak intensity of m / z=i under N isotopic abundance ( >20%), and the results are shown in Table 7;

[0102] Table 7 Differences 15 Table of predicted relative isotopic peak intensities for m / z=i under N isotopic abundance

[0103] ;

[0104] (7) Calculate the different 15 The Pearson correlation coefficients r for N isotope abundances are shown in Table 8. Only N isotope abundances were used in the analysis. >20%, >20% isotope peaks;

[0105] Table 8 Differences 15 Table of Pearson correlation coefficient r calculated based on N isotope abundance.

[0106] ;

[0107] (8) The r-value closest to 1 is 0.990, which indicates the best correlation between the predicted isotope distribution and the measured isotope distribution. The corresponding isotope abundance of 91.9 atom% is... 15 N-IGF1 protein abundance value.

[0108] In summary, protein compounds, when ionized, primarily exhibit multiple charges. Therefore, this invention fully considers the multivalent ion forms of proteins, such as 5+, 6+, 7+, and 8+. High-resolution mass spectrometry, using a full-scan mode, yields the measured relative isotope peak intensities, and the abundance value of the labeled protein is calculated based on the aforementioned technical solution. As can be seen from the above, the protein labeled abundance measurement of this invention does not require complex sample pretreatment such as enzymatic digestion or hydrolysis.

[0109] It should be pointed out finally that the above embodiments are only used to illustrate the technical solutions of the present application but not to limit it. Although the present application has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that the specific embodiments of the present application can be modified or equivalently replaced without departing from the spirit and scope of the present application, and any modification or equivalent replacement should be covered within the protection scope of the claims of the present application.

Claims

1. A high resolution mass spectrometry based intact 15 A method for determining the abundance of N stable isotope labeled proteins, characterized in that, The method comprises the following steps: Collect and calculate the mass spectrum signal of the sample to be tested, and obtain the actual measured relative isotope peak intensity X of the sample at m / z=i i Calculate the different 15 N isotope abundance of m / z=i Predicted relative isotope peak intensity Y i ; According to Formula 1, the Pearson correlation coefficient r under different N isotopic abundances is calculated respectively 15 Pearson correlation coefficient r under N isotopic abundance Comparing different 15 The Pearson correlation coefficient r calculated under the N isotope abundance, the closer the r value is to 1, the highest correlation between the predicted isotope distribution and the experimental measurement, and the r value closest to 1 corresponds to 15 The N isotope abundance is the isotope abundance of the stable isotope labeled protein 15 The N stable isotope labeled protein abundance value is atom%. The formula 1 is as follows: wherein X i is the actual measured relative isotopic peak intensity of the compound, is the average of the actual measured relative isotopic peak intensity; Y i is the predicted relative isotopic peak intensity of the compound, is the average of the predicted relative isotopic peak intensity, n is the total number of isotopic peaks calculated from the pearson correlation coefficient r, corresponding to the isotopic peaks of different mass-to-charge ratios (m / z = i) in the mass spectrum; The sample is actually measured at m / z = i relative to the isotope peak intensity X i The method for obtaining the same is specifically: accurately weighed 15 N-stable isotope-labeled proteins are prepared to working concentrations to obtain samples; According to 15 The mass spectrum response of N stable isotope labeled protein is selected to carry out high-resolution liquid chromatograph-mass spectrometer detection, and the chromatographic conditions are determined according to the sample properties, and the sample is repeatedly injected. A narrow m / z range analysis at the peak top is selected, resulting in the actual measured relative isotopic peak intensity X of the sample at m / z = i i； The accurate weighing adopts a millionth balance; The number of times of repeated sampling is 6-8 times. The narrow m / z range is specifically from the minimum m / z of the isotope distribution of the unlabeled protein to the maximum m / z of the 100% enriched isotope distribution.

2. The high resolution mass spectrometry-based intact 15 A method for N stable isotope labeling protein abundance determination, characterized by, The different 15 In the Pearson correlation coefficient r at the N isotope abundance, X i Only use the isotope peaks with X > 20%.

3. The high resolution mass spectrometry-based intact 15 A method for N stable isotope labeling protein abundance determination, characterized by, The different 15 The Pearson correlation coefficient r in the N isotope abundance, Y i Only use Y>20% of the isotope peak.

4. Application of the determination method in any of claims 1-3 to the abundance of the isotopically labeled protein.

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