A high-resolution mass spectrometry-based 13 C. 15 Method for determining the abundance of N-stable isotope-labeled peptides

Abstract

The present invention provides a method based on high resolution mass spectrometry. 13 C. 15 The method for determining the abundance of N-stable isotope labeled polypeptides belongs to the field of high-resolution mass spectrometry. The determination method of the present invention comprises the following steps: selecting a certified reference material to calculate the high-resolution mass spectrometer system deviation δ1; calculating according to formula 2 13 C. 15 N stable isotope labeled peptides 13 C. 15 The theoretical relative deviation of the N isotope is δ2; the mass spectrum signal of the sample to be tested is collected and recorded, and the actual measured relative deviation δ3 of the sample to be tested is calculated; when δ2>δ3>δ1, the corresponding isotope labeling abundance value is calculated according to Formula 3 and Formula 4. The method of the present invention uses high-resolution mass spectrometry to directly detect the sample by flow injection, which has a fast detection speed, ensures high resolution, and saves detection time. The analysis of the mass spectrum only requires the response intensity and m / z value of the target ion peak, and the operation is simple.

Description

Technical Field

[0001] The present invention relates to the technical field of high-resolution mass spectrometry, and in particular to a method based on high-resolution mass spectrometry. 13 C. 15 Method for determining the abundance of N stable isotope labeled peptides. Background Art

[0002] Peptides are condensation products of amino acids and are also enzymatic hydrolysis products of proteins. In the body, peptides are involved in a variety of physiological functions such as hormones, nerves, cell growth and reproduction, and are often used in disease diagnosis and treatment, peptide drugs, drug carriers, antibody research, and nutritional foods. The molecular size of peptides is between small organic molecules and large protein molecules. Compared with small molecule drugs, peptides are more easily metabolized in the body; compared with large molecule protein drugs, peptides are more stable. With the increasingly extensive and in-depth research on peptides in the field of biomedicine, the application of peptide products in the pharmaceutical industry has made great progress, and the demand for labeled and modified peptide types is increasing. Stable isotope labeled peptides are one of them.

[0003] Stable isotope-labeled peptides, generated by replacing some or all of the amino acids in a peptide with isotopic atoms, are compounds with important applications in drug discovery and clinical pathology research. They play a crucial role in clinical proteomics quantitative analysis, drug metabolism research, food safety testing, and peptide product quality control. The rapid development of the peptide industry has led to increasingly stringent demands for peptide product quality. For stable isotope-labeled peptides, purity and label abundance are key quality control parameters. Therefore, the development of technologies for determining the abundance of stable isotope-labeled peptides is essential and urgent. Isotope distribution and abundance are key indicators for evaluating the quality of isotope-labeled peptide products. New mass spectrometry technologies such as electrostatic field orbital traps and ion mobility spectroscopy, as well as advances in programming languages, have enabled increasingly diverse isotope distribution and abundance analysis. Organic mass spectrometry, particularly high-resolution mass spectrometry, is a key tool for isotope distribution and abundance analysis. Its advantages in isotope abundance calculation have become increasingly evident in recent years. Its ultra-high resolution allows for the separation of peaks from multiple isotope labeling patterns, while offering high accuracy and stability.

[0004] Gas isotope mass spectrometry can be used to determine the abundance of stable isotope-labeled polypeptides. This method converts stable isotopes into corresponding gases through high-temperature combustion, and then introduces the test gas into the mass spectrometer for detection. This method has a small scope of application. The measurement results of labeled high-abundance samples are greatly affected by the purity of the carrier gas and the labeled sample, and the repeatability is poor. Triple quadrupole mass spectrometry has a low resolution. When the molecular weight of the fully labeled analyte and the unlabeled analyte are close, it is impossible to completely separate and identify them, and all overlapping mass peaks need to be analyzed. Sheng Liyan et al. invented a method for determining the abundance of stable isotopes based on triple quadrupole mass spectrometry (CN118549558A), which confirms the relative content of all isotopologues of the analyte and ultimately calculates the abundance result. Quantitative nuclear magnetic resonance has the advantages of high analytical sensitivity, good reliability, non-destructiveness, and no need for sample pretreatment. However, when used to analyze polypeptides, quantitative nuclear magnetic resonance requires a complex spectrum analysis process, and the sample amount required for analysis during the test is large. For stable isotope-labeled polypeptide products, the testing cost is high.

[0005] The method based on high-resolution mass spectrometry is currently the most commonly used abundance determination method. This method has high sensitivity, fast detection speed, high resolution and is applicable to a variety of complex compounds, but it needs to be used in conjunction with a variety of separation tools, such as liquid chromatography, capillary electrophoresis, etc., and the operation has a certain degree of complexity. Lei Wen et al. invented a method for determining the isotopic abundance of doubly labeled compounds using a high-resolution liquid chromatography-mass spectrometer (CN116482256A). A high-resolution liquid chromatography-mass spectrometer was used to calculate the isotopic abundance value of the doubly labeled compound through front-end chromatographic separation and then mass spectrometry analysis mode. On the one hand, this method uses a liquid chromatography-mass spectrometry method, and the experimental time is relatively long in the liquid phase separation part; on the other hand, the invention is mainly aimed at the abundance determination of doubly labeled small molecule compounds, and does not involve the abundance determination of large molecules such as polypeptides.

[0006] Therefore, a method based on high-resolution mass spectrometry with simple operation and fast detection is provided. 13 C. 15 The method of determining the abundance of N stable isotope labeled peptides has important practical significance. Summary of the Invention

[0007] The purpose of the present invention is to provide a method based on high resolution mass spectrometry. 13 C. 15 The method for determining the abundance of N-stable isotope-labeled peptides aims to solve the technical problems of complex operation and slow detection speed in existing technologies, and provide support for the detection of macromolecular compounds such as stable isotope-labeled peptides.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a method based on high resolution mass spectrometry. 13 C. 15 The method for determining the abundance of N stable isotope labeled polypeptides comprises the following steps:

[0010] Select certified reference materials to calculate the high-resolution mass spectrometer system deviation δ1;

[0011] Calculate according to formula 2 13 C. 15 N stable isotope labeled peptides 13 C. 15 The theoretical relative deviation of N isotopes is δ2;

[0012] Collecting and recording the mass spectrometry signal of the sample to be tested, and calculating the actual measurement relative deviation δ3 of the sample to be tested;

[0013] When δ2>δ3>δ1, the corresponding isotope labeling abundance value is calculated according to Formula 3 and Formula 4.

[0014] Furthermore, the calculation of the high-resolution mass spectrometer system deviation δ1 is specifically as follows:

[0015] Use a 1 / 10000 balance to accurately weigh the standard substance and prepare it into working concentration to obtain the standard substance working solution;

[0016] The standard substance working solution is injected by flow injection, and its molecular weight is measured by high-resolution mass spectrometry. The scanning range is determined according to the molecular weight of the actual sample to obtain the standard substance mass spectrum signal;

[0017] After the mass spectrometry signal of the standard substance is stable, a mass spectrum of at least 2 minutes is collected, and 10 mass spectra are extracted for analysis at the same time interval;

[0018] The measurement error of the high-resolution mass spectrometer itself is calculated according to Formula 1.

[0019] Furthermore, the formula 1 is:

[0020]

[0021] Among them, M theory is the theoretical molecular weight of the standard substance, M measure is the average of ten repeated measurements.

[0022] Furthermore, the formula 2 is:

[0023]

[0024] Where M is the m / z value of the target peptide, a and b are 13 C and 15 N is the number of markers.

[0025] Furthermore, the formula 3 is:

[0026]

[0027] Among them, A i is the relative normalized peak intensity ratio of the compound, n refers to the number of stable isotope labels; Ii is the peak response intensity of the sample in the mass spectrum.

[0028] Furthermore, the formula 4 is:

[0029]

[0030] in, represent 13 C element isotope abundance value, unit is atom%; represent 15 The isotope abundance value of the element N is expressed in atom%.

[0031] The present invention also provides the application of the determination method described in the above technical solution in the determination of isotope labeling abundance values.

[0032] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0033] The high-resolution mass spectrometry method established by the present invention 13 C. 15 N stable isotope labeled peptide abundance determination technology can effectively evaluate the quality of stable isotope labeled peptide products and evaluate the detection 13 C. 15 The effectiveness and accuracy of N stable isotope labeled peptide technology, and then the quality control system of peptide products based on abundance spectrum was established to improve 13 C. 15 The synthesis quality of N-stable isotope-labeled polypeptides can be determined. Furthermore, the method of the present invention utilizes high-resolution mass spectrometry to directly detect flow-injected samples, ensuring high resolution while eliminating the need for liquid phase separation and facilitating rapid detection. Flow injection also requires minimal sample volume. Following injection, mass spectrometry data acquisition is performed. Analysis of the mass spectrum requires only the response intensity and m / z value of the target ion peak, eliminating the need for complex spectral analysis to calculate the isotope abundance of the polypeptide. This allows for simple operation and rapid detection. DETAILED DESCRIPTION

[0034] The present invention provides a method based on high resolution mass spectrometry. 13 C. 15 The method for determining the abundance of N stable isotope labeled polypeptides comprises the following steps:

[0035] Select certified reference materials to calculate the high-resolution mass spectrometer system deviation δ1;

[0036] Calculate according to formula 2 13 C. 15 N stable isotope labeled peptides 13 C. 15 The theoretical relative deviation of N isotopes is δ2;

[0037] Collecting and recording the mass spectrometry signal of the sample to be tested, and calculating the actual measurement relative deviation δ3 of the sample to be tested;

[0038] When δ2>δ3>δ1, the corresponding isotope labeling abundance value is calculated according to Formula 3 and Formula 4.

[0039] In the present invention, the calculation of the high-resolution mass spectrometer system deviation δ1 is specifically:

[0040] Use a 1 / 10000 balance to accurately weigh the standard substance and prepare it into working concentration to obtain the standard substance working solution;

[0041] The standard substance working solution is injected by flow injection, and its molecular weight is measured by high-resolution mass spectrometry. The scanning range is determined according to the molecular weight of the actual sample to obtain the standard substance mass spectrum signal;

[0042] After the mass spectrometry signal of the standard substance is stable, a mass spectrum of at least 2 minutes is collected, and 10 mass spectra are extracted for analysis at the same time interval;

[0043] The measurement error of the high-resolution mass spectrometer itself is calculated according to Formula 1.

[0044] In the present invention, the formula 1 is:

[0045]

[0046] Among them, M theory is the theoretical molecular weight of the standard substance, M measure is the average of ten repeated measurements.

[0047] In the present invention, the formula 2 is:

[0048]

[0049] Where M is the m / z value of the target peptide, a and b are 13 C and 15 N is the number of markers.

[0050] In the present invention, the formula 3 is:

[0051]

[0052] Among them, A i is the relative normalized peak intensity ratio of the compound, n refers to the number of stable isotope labels; Ii is the peak response intensity of the sample in the mass spectrum.

[0053] In the present invention, the formula 4 is:

[0054]

[0055] in, represent 13 C element isotope abundance value, unit is atom%; represent 15 The isotope abundance value of the element N is expressed in atom%.

[0056] The present invention also provides the application of the determination method described in the above technical solution in the determination of isotope labeling abundance values.

[0057] In the present invention, unless otherwise specified, the raw materials required for preparation are all commercially available products well known to those skilled in the art.

[0058] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0059] Example 1

[0060] Determination 13 C6, 15 Abundance of 1+ ions of the N-MFLSFPTTK peptide

[0061] (1) Using a microbalance, accurately weigh 0.2 mg of reserpine and prepare a working solution with a concentration of 0.1 mg / ml;

[0062] (2) The prepared reserpine working solution was injected by flow injection, and its molecular weight was determined by high-resolution mass spectrometry. The mass spectrometry parameters were optimized, and the mass spectrometry parameters were as follows: spray voltage 3.5 kV, capillary temperature 250 °C, auxiliary gas temperature 400 °C, full scan mode, resolution 120,000 (FWHM), and scanning range m / z 50-1500;

[0063] (3) After the flow injection signal stabilized, mass spectra were collected for at least 2 min, and 10 mass spectra were extracted for analysis at the same time interval;

[0064] (4) Reserpine standard substance was used to calculate the high-resolution mass spectrometer system deviation δ1, and the average value of 10 measurements was δ1 = 0.8 ppm;

[0065] (5) Calculation13 C. 15 N stable isotope labeled peptides 13 C. 15 The theoretical relative deviations δ2 of N isotopes are all greater than 5.8 ppm, and the calculated results are shown in Table 1;

[0066] Table 1 δ2 calculation table

[0067]

[0068] (6) Repeat steps (1) to (4) to prepare the peptide sample to be tested and perform the measurement;

[0069] (7) Calculate the relative deviation δ3 of the actual measurement of the peptide sample;

[0070] The following table is 13 C6, 15 The measurement results of N-MFLSFPTTK are all less than 5ppm;

[0071] Table 2 13 C6, 15 N-MFLSFPTTK measurement results table

[0072]

[0073]

[0074] (8) From this, we can see that δ2>δ3>δ1=5.8ppm>5ppm>0.8ppm. We can continue with the abundance calculation;

[0075] (9) Calculation based on mass spectrum 13 C6, 15 N-MFLSFPTTK isotope abundance value;

[0076] (10) Measured 13 C. 15 The isotopic abundance of N is 99.36atom% 13 C, 95.90atom% 15 N.

[0077] Example 2

[0078] Determination 13 C6, 15 Abundance of 2+ ions of the N-LEMYCAPLKPAK peptide

[0079] (1) Using a microbalance, accurately weigh 0.2 mg of reserpine and prepare a working solution with a concentration of 0.1 mg / ml;

[0080] (2) The prepared reserpine working solution was injected by flow injection, and its molecular weight was determined by high-resolution mass spectrometry. The mass spectrometry parameters were optimized, and the mass spectrometry parameters were as follows: spray voltage 3.5 kV, capillary temperature 250 °C, auxiliary gas temperature 400 °C, full scan mode, resolution 120,000 (FWHM), and scanning range m / z 50-1500;

[0081] (3) After the flow injection signal stabilized, mass spectra were collected for at least 2 min, and 10 mass spectra were extracted for analysis at the same time interval;

[0082] (4) Reserpine standard substance was used to calculate the high-resolution mass spectrometer system deviation δ1, and the average value of 10 measurements was δ1 = 0.8 ppm;

[0083] (5) Calculation 13 C. 15 N stable isotope labeled peptides 13 C. 15 The theoretical relative deviations δ2 of N isotopes are all greater than 4.6 ppm, and the calculated results are shown in Table 3;

[0084] Table 3 δ2 calculation table

[0085]

[0086]

[0087] (6) Repeat steps (1) to (4) to prepare the peptide sample to be tested and perform the measurement;

[0088] (7) Calculate the relative deviation δ3 of the actual measurement of the peptide sample;

[0089] The following table is 13 C6, 15 The measurement results of N-LEMYCAPLKPAK are all less than 4ppm;

[0090] Table 4 13 C6, 15 N-LEMYCAPLKPAK measurement results table

[0091]

[0092]

[0093] (8) From this, we can see that δ2>δ3>δ1=4.6ppm>4.0ppm>0.8ppm. We can continue with the abundance calculation;

[0094] (9) Calculation based on mass spectrum 13 C6, 15N-LEMYCAPLKPAK isotope abundance value;

[0095] (10) Measured 13 C. 15 The isotopic abundance of N is 99.19atom% 13 C, 99.70atom% 15 N.

[0096] Example 3

[0097] Determination 13 C6, 15 Abundance of 3+ ions in the N-TYFPHFDLSHGSAQVK peptide

[0098] (1) Using a microbalance, accurately weigh 0.2 mg of reserpine and prepare a working solution with a concentration of 0.1 mg / ml;

[0099] (2) The prepared reserpine working solution was injected by flow injection, and its molecular weight was determined by high-resolution mass spectrometry. The mass spectrometry parameters were optimized, and the mass spectrometry parameters were as follows: spray voltage 3.5 kV, capillary temperature 250 °C, auxiliary gas temperature 400 °C, full scan mode, resolution 120,000 (FWHM), and scanning range m / z 50-1500;

[0100] (3) After the flow injection signal stabilized, mass spectra were collected for at least 2 min, and 10 mass spectra were extracted for analysis at the same time interval;

[0101] (4) Reserpine standard substance was used to calculate the high-resolution mass spectrometer system deviation δ1, and the average value of 10 measurements was δ1 = 0.8 ppm;

[0102] (5) Calculation 13 C. 15 N stable isotope labeled peptides 13 C. 15 The theoretical relative deviations δ2 of N isotopes are all greater than 3.4 ppm, and the calculated results are shown in Table 5;

[0103] Table 5 δ2 calculation table

[0104]

[0105]

[0106] (6) Repeat steps (1) to (4) to prepare the peptide sample to be tested and perform the measurement;

[0107] (7) Calculate the relative deviation δ3 of the actual measurement of the peptide sample;

[0108] The following table is13 C6, 15 The measurement results of N-TYFPHFDLSHGSAQVK were all less than 3.1ppm;

[0109] Table 6 13 C6, 15 Measurement results table of N-TYFPHFDLSHGSAQVK

[0110]

[0111] (8) From this, we can see that δ2>δ3>δ1=3.4ppm>3.1ppm>0.8ppm. We can continue with the abundance calculation;

[0112] (9) Calculation based on mass spectrum 13 C6, 15 N-TYFPHFDLSHGSAQVK isotope abundance value;

[0113] (10) Measured 13 C. 15 The isotopic abundance of N is 99.29atom% 13 C, 95.71atom% 15 N.

[0114] Example 4

[0115] Determination 13 C6, 15 Abundance of 4+ ions in the N-DRVYIHPFHL peptide

[0116] (1) Using a microbalance, accurately weigh 0.2 mg of reserpine and prepare a working solution with a concentration of 0.1 mg / ml;

[0117] (2) The prepared reserpine working solution was injected by flow injection, and its molecular weight was determined by high-resolution mass spectrometry. The mass spectrometry parameters were optimized, and the mass spectrometry parameters were as follows: spray voltage 3.5 kV, capillary temperature 250 °C, auxiliary gas temperature 400 °C, full scan mode, resolution 120,000 (FWHM), and scanning range m / z 50-1500;

[0118] (3) After the flow injection signal stabilized, mass spectra were collected for at least 2 min, and 10 mass spectra were extracted for analysis at the same time interval;

[0119] (4) Reserpine standard substance was used to calculate the high-resolution mass spectrometer system deviation δ1, and the average value of 10 measurements was δ1 = 0.8 ppm;

[0120] (5) Calculation 13 C. 15N stable isotope labeled peptides 13 C. 15 The theoretical relative deviations δ2 of N isotopes are all greater than 4.8 ppm, and the calculated results are shown in Table 7;

[0121] Table 7 δ2 calculation table

[0122]

[0123]

[0124] (6) Repeat steps (1) to (4) to prepare the peptide sample to be tested and perform the measurement;

[0125] (7) Calculate the relative deviation δ3 of the actual measurement of the peptide sample;

[0126] The following table is 13 C6, 15 The measurement results of N-DRVYIHPFHL were all less than 1.5ppm;

[0127] Table 8 13 C6, 15 N-DRVYIHPFHL measurement results table

[0128] (8) From this, we can see that δ2>δ3>δ1=4.8ppm>1.5ppm>0.8ppm. We can continue with the abundance calculation;

[0129] (9) Calculation based on mass spectrum 13 C6, 15 N-DRVYIHPFHL isotope abundance value;

[0130] (10) Measured 13 C. 15 The isotopic abundance of N is 98.98atom% 13 C, 99.47atom% 15 N.

[0131] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method based on high-resolution mass spectrometry 13 C. 15 The method for determining the abundance of N stable isotope labeled polypeptides is characterized by: The following steps are involved: Select certified reference materials to calculate the high-resolution mass spectrometer system deviation δ1; Calculate according to formula 2 13 C. 15 N stable isotope labeled peptides 13 C. 15 The theoretical relative deviation of N isotopes is δ2; Collecting and recording the mass spectrometry signal of the sample to be tested, and calculating the actual measurement relative deviation δ3 of the sample to be tested; When δ2>δ3>δ1, the corresponding isotope labeling abundance value is calculated according to Formula 3 and Formula 4; The calculation of the high-resolution mass spectrometer system deviation δ1 is specifically: Use a 1 / 10000 balance to accurately weigh the standard substance and prepare it into working concentration to obtain the standard substance working solution; The standard substance working solution is injected by flow injection, and its molecular weight is measured by high-resolution mass spectrometry. The scanning range is determined according to the molecular weight of the actual sample to obtain the standard substance mass spectrum signal; After the mass spectrometry signal of the standard substance is stable, collect a mass spectrum for at least 2 minutes, and extract 10 mass spectra for analysis at the same time interval; Calculate the measurement error of the high-resolution mass spectrometer itself according to formula 1; The formula 1 is: Among them, M theory is the theoretical molecular weight of the standard substance, M measure is the average of ten repeated measurements; The formula 2 is: Where M is the m / z value of the target peptide, a and b are 13 C and 15 N is the number of markers; The formula 3 is: Among them, A i is the relative normalized peak intensity ratio of the compound, n refers to the number of stable isotope labels; Ii is the peak response intensity of the sample in the mass spectrum; The formula 4 is: in, represent 13 C element isotope abundance value, unit is atom%; represent 15 The isotope abundance value of the element N is expressed in atom%.

2. Use of the determination method according to claim 1 in determination of isotope labeling abundance values.

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