Detection method for measuring isotope abundance of 13C and 15N double-labeled amino acids by using temperature gradient release technology

Abstract

The invention relates to a detection method for measuring isotope abundance of 13C and 15N double-labeled amino acids by using a temperature gradient release technology, which comprises the following steps: oxidizing 13C and 15N double-labeled amino acids by using an oxidant to generate 15N labeled nitrogen and 13C labeled carbon dioxide at the same time, and sequentially releasing the 15N labeled nitrogen and the 13C labeled carbon dioxide by combining the temperature gradient release technology to realize isotope abundance of 13C and 15N double-labeled amino acids. And testing the abundance of the < 15 > N isotope. Compared with the prior art, the method has the advantages that the temperature gradient release technology is utilized, the isotope abundance of < 13 > C and < 15 > N can be obtained at the same time through one-time pretreatment, and the method has the advantages of being good in repeatability, high in accuracy, efficient and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of stable isotope abundance detection and relates to a method for detecting 13 C, 15 Method for detecting isotope abundance of N-doubly labeled amino acids. Background Art

[0002] Stable isotope labeling by aminoacids in cell culture (SILAC) is a simple, stable and powerful method in quantitative proteomics using mass spectrometry (MS). SILAC technology has the advantages of stable and reliable stable isotope labeling rate, high throughput in application, dynamic study of changes in different proteomes, high stable isotope labeling efficiency and high test sensitivity. It is also used for tracer determination and tracking of various dynamic parameters in amino acid metabolic pathways, synthesis rate and structure identification in proteomics. SILAC technology requires the in vivo metabolism of amino acids. 13 C, 15 N-double-labeled amino acids are incorporated into proteins, which are then rapidly and comprehensively identified, characterized, and quantified by mass spectrometry (MS). 13 C, 15 In addition to proteomics, N-doped amino acids are also widely used in metabolomics and life sciences, playing an important role. 13 C, 15 The isotopic abundance of N-doubly labeled amino acids is directly involved in the calculation or has an impact on the experiment.

[0003] Existing similar detection methods mainly include high performance liquid chromatography-mass spectrometry, high resolution mass spectrometry, gas isotope mass spectrometry, etc. For example, Chinese patent application CN104122339 A provides a D, 13 C or 15The isotope abundance detection method of N-labeled organic compounds adopts high performance liquid chromatography-mass spectrometry to obtain mass spectrum data, and uses the "mass cluster" classification algorithm to calculate the isotope abundance; in addition, as Chinese patent application CN116482256A provides a method for determining the isotope abundance of double-labeled compounds using a high-resolution liquid chromatography-mass spectrometer, using a computing instrument to detect the required resolution of the isotope abundance value of the double-labeled compound to be tested, and determine whether it meets the analysis requirements, according to the mass spectrum response of the compound, select a sample of appropriate concentration for high-resolution liquid chromatography-mass spectrometry detection, repeat the injection 6-8 times, calculate the instrument detection deviation value IDD and the relative isotope deviation value RID according to the liquid chromatography-mass spectrometer results, and when IDD is less than RID, the abundance calculation can be performed, and the appropriate mass spectrum scanning range is selected according to the extracted ion flow diagram to calculate the isotope abundance value of the double-labeled compound to be tested; and as Chinese patent application CN111983007A provides a method for determining nitric acid or nitrate 15 Method and device for measuring N isotope abundance, which can be used to measure 15 The N-labeled nitric acid or nitrate sample, the oxide and the reducing agent are placed in a reaction tube, which is evacuated and sealed, and the reaction tube is heated to generate the reaction. 15 N is marked with nitrogen. After the reaction tube has cooled to room temperature, it is placed in the injection tube. Then, the injection tube and the injection line of the gas isotope mass spectrometer connected to it are evacuated to break the reaction tube so that the inside 15 N-labeled nitrogen was introduced into the gas isotope mass spectrometer to detect and record the relative intensities of mass numbers 28, 29, and 30, and the relative intensities of the samples were calculated. 15 N isotope abundance.

[0004] The “quality cluster” classification algorithm requires data processing such as deconvolution, is suitable for single markers, and cannot be measured 13 C, 15 N double labeling 13 C or 15 The high-resolution liquid-mass spectrometer mainly uses a very high resolution, and the abundance calculation can be performed only after the instrument detection deviation value IDD is less than the relative isotope deviation value RID. The existing gas isotope mass spectrometry method requires two pre-processing methods to obtain 13 C, 15 The N isotope abundance is high, and the pre-treatment process is complicated and the cycle is long. The present invention is proposed based on this. Summary of the invention

[0005] The purpose of the present invention is to provide a method for measuring the release of 13 C, 15 Method for detecting isotope abundance of N-doubly labeled amino acids.

[0006] The present invention utilizes gas isotope mass spectrometry to first 13 C, 15 N-double-labeled amino acid redox production 15 N marks nitrogen and 13 C labeled carbon dioxide, since the ion source of the gas isotope mass spectrometry is an electron impact ion source (EI), 10% 13 C-labeled carbon dioxide is broken down in the ion source to produce 13 C represents carbon monoxide, 13 The presence of C-labeled carbon monoxide 15 N-labeled nitrogen causes interference and cannot be accurately measured 15 N isotope abundance, therefore, through the temperature gradient release technique, 15 N marks nitrogen and 13 C-labeled carbon dioxide is released in sequence for testing, achieving one-time pretreatment to obtain 13 C, 15 N isotope abundance.

[0007] The purpose of the present invention can be achieved by the following technical solutions:

[0008] A method for measuring the release of 13 C, 15 The method for detecting the isotope abundance of N-doubly labeled amino acids first uses an oxidant to 13 C, 15 N-double-labeled amino acids are simultaneously produced by oxidation 15 N marks nitrogen and 13 C labeled carbon dioxide, combined with temperature gradient release technology, makes 15 N marks nitrogen and 13 C-labeled carbon dioxide is released in sequence, achieving 13 C, 15 Test of N isotope abundance.

[0009] Furthermore, the temperature gradient release technology is specifically: using different temperature environments to generate 15 N marks nitrogen and 13 C-labeled carbon dioxide is processed to release different gases.

[0010] Furthermore, the refrigerants used to provide different temperature environments are liquid nitrogen and cold water, wherein liquid nitrogen is used to 13 C marks the freezing of carbon dioxide and makes 15 N-labeled nitrogen release is used for 15 N isotope abundance test; cold water is used to freeze 13 C marks the thawing of carbon dioxide, so that 13 C labeled carbon dioxide is released for 13C isotope abundance test.

[0011] Furthermore, the oxidant is chromium trioxide.

[0012] Furthermore, the oxidant and 13 C, 15 The ratio of the added amount of N-double-labeled amino acid is (30-40): (5-10). Meanwhile, the oxidation temperature is 450-550° C., preferably 500° C., and the time is 1.5-2.5 h, preferably 2 h.

[0013] Furthermore, the 13 C, 15 N double labeled amino acids 13 C, 15 N-double-labeled lysine hydrochloride can also be used for other 13 C, 15 N double-labeled amino acids, the types of isotope-labeled amino acids here do not belong to the innovative points of the present invention and will not be described in detail here. 13 C. 15 N will exist in natural abundance, therefore, the 13 C, 15 N-divalently labeled amino acids can also be replaced by 15 N mark or 13 C amino acids.

[0014] Furthermore, the oxidant is chromium trioxide, or other oxidants that can oxidize isotope-labeled amino acids to 5 N marks nitrogen and 13 C marks the oxidant of carbon dioxide and will not be described in detail.

[0015] Further, released 15 N marks nitrogen and 13 C-labeled carbon dioxide was sequentially introduced into the gas isotope mass spectrometer for isotope abundance determination.

[0016] Furthermore, during the determination of isotope abundance, liquid nitrogen is used to 13 C marks the freezing of carbon dioxide and makes 15 N-labeled nitrogen release is used for 15 N isotope abundance test; after the test is completed, the remaining gas is completely pumped out through the vacuum system of the instrument. Remove the liquid nitrogen and use cold water to freeze the 13 C marks carbon dioxide thawing, which lasts for two or three minutes. The main function of cold water is to thaw the frozen 13 C labeled carbon dioxide is slowly released for 13There are two reasons why hot water with a higher temperature is not used in the test of C isotope abundance. First, the large temperature difference between liquid nitrogen freezing (-196℃) and hot water can easily cause the glass tube to break. Second, hot water can cause the water generated in the reaction to be released. 13 C-labeled carbon dioxide enters the mass spectrometer together, causing the vacuum degree of the instrument to decrease, affecting the test.

[0017] Furthermore, the temperature of the cold water is 2-8°C.

[0018] Compared with the prior art, the present invention can release the isotope-labeled amino acids produced by oxidation through temperature gradient release technology. 15 N marks nitrogen and 13 C labeled carbon dioxide is released sequentially, avoiding interference with the test results when both exist at the same time, and achieving simultaneous acquisition of 13 C, 15 N isotope abundance, the method has good repeatability, high accuracy and high efficiency, etc. DETAILED DESCRIPTION

[0019] The present invention is described in detail below in conjunction with specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and a specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0020] In the following examples, unless otherwise specified, raw materials or processing techniques are conventional commercially available raw materials or conventional processing techniques in the art.

[0021] Example 1

[0022] 15 N-labeled lysine hydrochloride (product of CIL, USA, NLM-1683-1, 99.0 atom% 15 N, 1.1atom% 13 C, of ​​which 1.1atom% 13 C is natural abundance)

[0023] The pretreatment mainly takes 40mg chromium trioxide, 10mg 13 C, 15 N-double-labeled amino acids are oxidized and generated simultaneously under vacuum 15 N marks nitrogen and 13 C labeled carbon dioxide, reaction temperature 500 ℃, reaction time 2h, combined with temperature gradient release technology, using liquid nitrogen 13 C marks carbon dioxide freezing, used for 15 After the N isotope abundance test is completed, the remaining gas is completely pumped out through the vacuum system of the instrument. Remove the liquid nitrogen and use cold water to 13C labeled carbon dioxide thawing, used for 13 C isotope abundance test.

[0024] Instrument test conditions: Nu evolution gas isotope mass spectrometer, electron impact (EI) ion source; electron energy 100eV; electron multiplier voltage 2.1KV, ion source temperature 140°C; high voltage 6KV; collector slit 600, mass scan m / z18~50.

[0025] The abundance test result is 99.0atom% 15 N, 1.1atom% 13 C.

[0026] The measured data and the indicated value result are 99.0atom% 15 N, 1.1atom% 13 C, the deviation is only 0atom% 15 N, 0atom% 13 C, the results are consistent, it can be seen that the present invention 13 C, 15 The N isotope abundance determination method also has a very high measurement accuracy.

[0027] Example 2

[0028] 13 C-labeled lysine hydrochloride (product of CIL, USA, 0.36 atom% 15 N, 99.0atom% 13 C, of ​​which 0.36atom% 15 N is natural abundance)

[0029] The pretreatment mainly takes 30mg chromium trioxide, 10mg 13 C, 15 N-double-labeled amino acids are oxidized and generated simultaneously under vacuum 15 N marks nitrogen and 13 C labeled carbon dioxide, reaction temperature 500 ℃, reaction time 2h, combined with temperature gradient release technology, using liquid nitrogen 13 C marks carbon dioxide freezing, used for 15 The test of N isotope abundance uses cold water to 13 C labeled carbon dioxide thawing, used for 13 C isotope abundance test.

[0030] Nu evolution gas isotope mass spectrometer, test conditions: electron impact (EI) ion source; electron energy 100eV; electron multiplier voltage 2.1KV, ion source temperature 140℃; high voltage 6KV; collector slit 600, mass scan m / z18~50.

[0031] The abundance test result is 0.37atom% 15 N, 99.1atom% 13 C.

[0032] The measured data and the indicated value result 0.36atom% 15 N, 99.0atom% 13 C, the deviation is only 0.01atom% 15 N, 0.1atom% 13C The results are basically consistent, which shows that the present invention 13 C, 15 The N isotope abundance determination method also has a very high measurement accuracy.

[0033] Example 3

[0034] 13 C, 15 N double-labeled lysine hydrochloride (product of CIL, USA, average isotopic abundance 99.2atom%, 13 C, 15 N does not provide a separate identifier for specific values)

[0035] The pretreatment mainly takes 30mg chromium trioxide, 10mg 13 C, 15 N-double-labeled amino acids are oxidized and generated simultaneously under vacuum 15 N marks nitrogen and 13 C labeled carbon dioxide, reaction temperature 500 ℃, reaction time 2h, combined with temperature gradient release technology, using liquid nitrogen 13 C marks carbon dioxide freezing, used for 15 The test of N isotope abundance uses cold water to 13 C labeled carbon dioxide thawing, used for 13 C isotope abundance test.

[0036] Nu evolution gas isotope mass spectrometer, test conditions: electron impact (EI) ion source; electron energy 100eV; electron multiplier voltage 2.1KV, ion source temperature 140℃; high voltage 6KV; collector slit 600, mass scan m / z18~50.

[0037] The abundance test result is 99.1atom% 15 N, 99.2atom% 13 C, 13 C, 15 N double labeled lysine hydrochloride has 6 13 C and 2 15N, the average isotopic abundance calculated by the enhancement method is 99.2atom%.

[0038] The measured data were compared with the indicated value result, the average isotope abundance of 99.2atom%, and the results were consistent. 13 C, 15 The N isotope abundance determination method also has a very high measurement accuracy.

[0039] Comparative Example 1:

[0040] 13 C, 15 N double-labeled lysine hydrochloride (product of CIL, USA, average isotopic abundance 99.2atom%, 13 C, 15 N does not provide a separate identifier for specific values)

[0041] The pretreatment mainly uses chromium trioxide to 13 C, 15 N-double-labeled amino acids are simultaneously produced by oxidation 15 N marks nitrogen and 13 C marks carbon dioxide.

[0042] The Nu evolution gas isotope mass spectrometer was used for direct testing. The test conditions were: electron impact (EI) ion source; electron energy 100 eV; electron multiplier voltage 2.1 KV, ion source temperature 140°C; high voltage 6 KV; collector slit 600, mass scan m / z 18-50.

[0043] The abundance test result is 16.7atom% 15 N, 98.9atom% 13 C, 13 C, 15 N double labeled lysine hydrochloride has 6 13 C and 2 15 N, the average isotope abundance calculated by the enhancement method is 78.4atom%. The main reason for the abundance skewness is 13 C-labeled carbon dioxide is produced by electron bombardment at an electron energy of 100 eV 13 The mass-to-charge ratio of CO is m / z 29. 15 N 14 The mass-to-charge ratio of N is consistent with m / z 29, and the generated 13 More CO leads to 15 The low N isotope abundance further leads to a decrease in the average isotope abundance, and the test results are obviously different.

[0044] Comparative Example 2:

[0045] The test method of Example 1 in the invention patent CN116482256A can only test 15 N isotope abundance, undetectable 13 Compared with comparative example 2, Example 1 of the present invention uses the temperature gradient release technology to obtain 13 C isotope abundance and 15 The two key indicators of N isotope abundance show that it has innovative advantages in technology.

[0046] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A method for measuring the release of 13 C, 15 The method for detecting the isotope abundance of N-doubly labeled amino acids is characterized in that: First, use an oxidant 13 C, 15 N-double-labeled amino acids are simultaneously produced by oxidation 15 N marks nitrogen and 13 C labeled carbon dioxide, combined with temperature gradient release technology, makes 15 N marks nitrogen and 13 C-labeled carbon dioxide is released in sequence, achieving 13 C, 15 Test of N isotope abundance.

2. A method according to claim 1 for determining the release of the product by using a temperature gradient release technique 13 C, 15 The method for detecting the isotope abundance of N-doubly labeled amino acids is characterized in that: The oxidant is chromium trioxide.

3. A method according to claim 1 for determining the release of the product by using a temperature gradient release technique 13 C, 15 The method for detecting the isotope abundance of N-doubly labeled amino acids is characterized in that: The oxidant and 13 C, 15 The ratio of the added amount of N-double-labeled amino acids is (30-40):(5-10).

4. A method according to claim 1 for determining the release of the product by using a temperature gradient release technique 13 C, 15 The method for detecting the isotope abundance of N-doubly labeled amino acids is characterized in that: The oxidation temperature is 450-550°C and the time is 1.5-2.5h.

5. A method according to claim 1 for determining the release of the product by using a temperature gradient release technique 13 C, 15 The method for detecting the isotope abundance of N-doubly labeled amino acids is characterized in that: The temperature gradient release technology is specifically: using different temperature environments to generate 15 N marks nitrogen and 13 C-labeled carbon dioxide is processed to release different gases.

6. A method according to claim 5 for determining the release of the product by using a temperature gradient release technique 13 C, 15 The method for detecting the isotope abundance of N-doubly labeled amino acids is characterized in that: The refrigerants used to provide different temperature environments are liquid nitrogen and cold water.

7. A method according to claim 6 for determining the release of the product by using a temperature gradient release technique 13 C, 15 The method for detecting the isotope abundance of N-doubly labeled amino acids is characterized in that: The liquid nitrogen is used to 13 C marks the freezing of carbon dioxide and makes 15 N-labeled nitrogen release is used 15 N isotope abundance test; cold water is used to freeze 13 C marks the thawing of carbon dioxide, so that 13 C labeled carbon dioxide is released for 13 C isotope abundance test.

8. A method according to claim 6 for determining the release of the product by using a temperature gradient release technique 13 C, 15 The method for detecting the isotope abundance of N-doubly labeled amino acids is characterized in that: The temperature of the cold water is 2-8°C.

9. A method according to claim 1 for determining the release of a substance using a temperature gradient release technique 13 C, 15 The method for detecting the isotope abundance of N-doubly labeled amino acids is characterized in that: Released 15 N marks nitrogen and 13 C-labeled carbon dioxide was sequentially introduced into the gas isotope mass spectrometer for isotope abundance determination.

10. A method of measuring the release of the product by using a temperature gradient release technique according to claim 1 13 C, 15 The method for detecting the isotope abundance of N-doubly labeled amino acids is characterized in that: Said 13 C, 15 N double labeled amino acids 13 C, 15 N double labeled lysine hydrochloride, 13 C-labeled lysine hydrochloride or 15 N-labeled lysine hydrochloride.