13C, 15N stable isotope labeled polypeptide abundance determination method based on high-resolution mass spectrum

Abstract

The invention provides a 13C and 15N stable isotope labeled polypeptide abundance determination method based on a high-resolution mass spectrum, and belongs to the technical field of high-resolution mass spectrum determination. The determination method comprises the following steps: selecting a certified standard substance to calculate the system deviation delta 1 of the high-resolution mass spectrometer; calculating a theoretical relative deviation delta 2 of 13C and 15N isotopes in the 13C and 15N stable isotope labeled polypeptide according to a formula 2; collecting and recording a mass spectrum signal of a to-be-measured sample, and calculating to obtain an actual measurement relative deviation delta 3 of the to-be-measured sample; and when delta2 > delta3 > delta1, calculating the abundance value of the corresponding isotope label according to a formula 3 and a formula 4. According to the method disclosed by the invention, the flow injection sample is directly detected by adopting the high-resolution mass spectrometry, so that the detection speed is high, the high resolution is ensured, and the detection time is saved; only the response intensity and the m / z value of a target ion peak are needed for the analysis of a mass spectrum, and the operation is simple and convenient.

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 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 peptides is increasing. Stable isotope labeled peptides are one of them.

[0003] Stable isotope labeled peptides are new labeled peptides produced by replacing some or all of the amino acids in the peptide with isotope atoms. They are compounds with important applications in drug development and clinical pathology research, and play an important role in clinical proteomics quantitative analysis, drug metabolism research, food safety testing, and peptide product quality control. With the rapid development of the peptide industry, the demand for peptide product quality is also increasing. For stable isotope labeled peptides, purity and labeling abundance are the key parameters for quality control. Therefore, it is necessary and urgent to develop a technology for the determination of the abundance of stable isotope labeled peptides. The detection of isotope distribution and abundance is a key indicator for the quality evaluation of isotope labeled peptide products. At present, the development of new mass spectrometry technologies such as electrostatic field orbital traps and ion mobility and programming languages ​​has made isotope distribution and abundance analysis more and more diverse. Organic mass spectrometry is an important tool for isotope distribution and abundance analysis at this stage, especially high-resolution mass spectrometry. In recent years, the advantages of high-resolution mass spectrometry in isotope abundance calculation have become more and more obvious. Because of its ultra-high resolution, it can achieve the separation of multiple isotope labeling mode peaks and provide higher accuracy and stability.

[0004] Gas isotope mass spectrometry can be used to measure the abundance of stable isotope labeled polypeptides. This method is to convert stable isotopes into corresponding gases through high-temperature combustion, and then introduce the gas to be tested into the mass spectrometer for detection. The scope of application of this method is small. 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. The triple quadrupole mass spectrometry has a low resolution. When the molecular weight of the fully labeled analyte is close to that of the unlabeled complete analyte, it is impossible to completely separate and identify it, and all mass overlapping 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), confirming the relative content of all isotopologues of the analyte, and finally calculating the abundance result. Quantitative nuclear magnetic resonance has the advantages of high analytical sensitivity, good reliability, non-destructiveness, and no sample pretreatment. However, when quantitative nuclear magnetic resonance is used to analyze polypeptides, a complex spectrum analysis process is required, and the amount of sample required for analysis during the test is large. For stable isotope labeled polypeptide products, the test 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 double-labeled compounds using a high-resolution liquid chromatography-mass spectrometer (CN116482256A), using a high-resolution liquid chromatography-mass spectrometer, through front-end chromatographic separation, and then to the mass spectrometry analysis mode, to calculate the isotopic abundance value of the double-labeled compound. On the one hand, this method uses a liquid-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 double-labeled small molecule compounds, and does not involve the abundance determination of macromolecules 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 object 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 is intended to solve the technical problems of complex operation and slow detection speed in existing technologies, and to 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:

[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, and a mass spectrum signal of the standard substance is obtained;

[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, in atom%; represent 15 The isotope abundance value of the N element is 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 value.

[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 establish a peptide product quality control system based on abundance spectrum to improve 13 C. 15 N stable isotope labeled polypeptide synthesis quality; in addition, the method of the present invention uses high-resolution mass spectrometry to directly detect the flow injection sample, which not only ensures high resolution, but also does not require liquid phase separation time and has a fast detection speed; by flow injection, the sample demand is also small; after injection, mass spectrometry data collection is performed. For the analysis of the mass spectrum, only the response intensity and m / z value of the target ion peak are needed, and the isotope labeling abundance value of the polypeptide can be calculated based on the data without complex spectrum analysis. The operation is simple and the detection speed is fast. 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, and a mass spectrum signal of the standard substance is obtained;

[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, in atom%; represent 15 The isotope abundance value of the N element is 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 value.

[0057] In the present invention, unless otherwise specified, the required raw materials 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 in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0059] Example 1

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

[0061] (1) Using a 1 / 10000 balance, 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 spray voltage 3.5 kV, capillary temperature 250 °C, auxiliary gas temperature 400 °C, full scan mode, resolution 120000 (FWHM), and scanning range m / z 50-1500;

[0063] (3) After the flow injection signal is stable, collect mass spectra for at least 2 min, and extract 10 mass spectra 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 of N isotopes, δ2, are all greater than 5.8 ppm, and the calculated results are shown in Table 1;

[0066] Table 1δ2 calculation table

[0067]

[0068] (6) Repeat steps (1) to (4) to prepare the polypeptide 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 the abundance calculation;

[0075] (9) Calculation based on mass spectrum 13 C6, 15 N-MFLSFPTTK isotopic 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 in the N-LEMYCAPLKPAK peptide

[0079] (1) Using a 1 / 10000 balance, 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 spray voltage 3.5 kV, capillary temperature 250 °C, auxiliary gas temperature 400 °C, full scan mode, resolution 120000 (FWHM), and scanning range m / z 50-1500;

[0081] (3) After the flow injection signal is stable, collect mass spectra for at least 2 min, and extract 10 mass spectra 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 of N isotopes, δ2, are all greater than 4.6 ppm, and the calculated results are shown in Table 3;

[0084] Table 3δ2 calculation table

[0085]

[0086]

[0087] (6) Repeat steps (1) to (4) to prepare the polypeptide 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 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 1 / 10000 balance, 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 spray voltage 3.5 kV, capillary temperature 250 °C, auxiliary gas temperature 400 °C, full scan mode, resolution 120000 (FWHM), and scanning range m / z 50-1500;

[0100] (3) After the flow injection signal is stable, collect mass spectra for at least 2 min, and extract 10 mass spectra 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 of N isotopes, δ2, are all greater than 3.4 ppm, and the calculated results are shown in Table 5;

[0103] Table 5δ2 calculation table

[0104]

[0105]

[0106] (6) Repeat steps (1) to (4) to prepare the polypeptide 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 are 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 1 / 10000 balance, 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 spray voltage 3.5 kV, capillary temperature 250 °C, auxiliary gas temperature 400 °C, full scan mode, resolution 120000 (FWHM), and scanning range m / z 50-1500;

[0118] (3) After the flow injection signal is stable, collect mass spectra for at least 2 min, and extract 10 mass spectra 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 of N isotopes, δ2, are all greater than 4.8 ppm, and the calculated results are shown in Table 7;

[0121] Table 7δ2 calculation table

[0122]

[0123]

[0124] (6) Repeat steps (1) to (4) to prepare the polypeptide 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 are 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 principle of the present invention. These improvements and modifications should also be regarded as 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.

2. The method according to claim 1 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 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, and a mass spectrum signal of the standard substance is obtained; 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; The measurement error of the high-resolution mass spectrometer itself is calculated according to Formula 1.

3. The method according to claim 2 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 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.

4. The method according to claim 1 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 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.

5. The method according to claim 1 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 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.

6. The method according to claim 1 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 formula 4 is: in, represent 13 C element isotope abundance value, in atom%; represent 15 The isotope abundance value of the N element is in atom%.

7. Use of the determination method according to any one of claims 1 to 6 in the determination of isotope labeling abundance values.

Images