A class of high-throughput proteomics labeling reagents based on secondary mass spectrometry quantification

Abstract

This invention discloses a class of high-throughput proteomics labeling reagents based on secondary mass spectrometry (MS / MS). The structure comprises: a reporter group – a balancing group – an activating group, wherein the activated gene can label the polypeptide through a specific reaction with different functional groups contained in the polypeptide. This invention discloses two different sets of chemical structures, each containing up to 16 chemically identical labeling reagents (named IBT-16PLEX labeling reagents), but each labeling reagent contains a 13C or 15N isotope at a different position. This invention discloses the chemical structure of the IBT-16PLEX labeling reagent, the position of the stable isotope in the labeling reagent, the synthetic route of the labeling reagent, and the specific application of the labeling reagent in proteomics. The IBT-16PLEX in this invention employs a novel chemical structure, reducing the difficulty of preparation, increasing the yield, thereby reducing product costs and expanding the application range of the IBT-16PLEX labeling reagent.

Description

Technical Field

[0001] This invention relates to the field of biomolecular analytical reagents, particularly for quantitative analysis in proteomics, specifically the preparation and application of an isotopic labeling reagent (IBT-16PLEX) capable of simultaneously quantifying the proteomics of 16 samples. Background Technology

[0002] In recent years, the field of proteomics has been continuously developing, especially in protein identification and quantitative analysis. This progress will help to deepen our understanding of the relationship between the presence of specific proteins in cells and their biological functions. Due to its high throughput and high specificity, biomolecular mass spectrometry is the most widely used tool in proteomics research. However, because mass spectrometry signals are affected by multiple factors such as instrumentation, interferences, and matrix effects, quantitative analysis of proteomics using mass spectrometry requires specialized sample and data processing methods, mainly including label-free methods and stable isotope labeling methods. Compared with label-free methods, stable isotope labeling methods have many advantages. Since different samples labeled with stable isotope reagents are pre-mixed before liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis, all samples can be analyzed in a single LC-MS / MS analysis, effectively reducing systematic errors of different samples in different LC-MS / MS analyses. It also has lower requirements for instrument reproducibility, thus minimizing the differences between different samples in different measurements and solving the reproducibility problems that are unavoidable in label-free methods. At the same time, because multiple samples can be analyzed qualitatively and quantitatively at once, the analysis time for large cohorts of samples can be greatly reduced.

[0003] Stable isotope labeling methods are further divided into methods based on mass spectrometry (MS1) and mass spectrometry (MS2). For example, SILAC (Stable isotope labeling with amino acids in cell culture) is an MS1-based protein labeling method, which involves adding amino acids containing stable isotope labels to the cell culture medium. Isobaric labeling is an MS2-based analytical method, including iTRAQ (isobaric tag for relative and absolute quantification), TMT (tandem mass tag), and IBT (isobaric tag).

[0004] Compared to quantitative methods based on primary mass spectrometry, such as SILAC, isotopic labeling, when multiple samples are mixed, combines the same polypeptide contained in different samples, effectively increasing the sample concentration. This results in stronger MS / MS fragment signals and significantly improved detection sensitivity. Simultaneously, it reduces the complexity of the mass spectrometry signal, making it more suitable for comparative analysis of multiple samples. Furthermore, SILAC-labeled quantitative methods rely on cells taking up stable isotopes of arginine or lysine to synthesize proteins, which then contain these labeled amino acids. This method is only feasible in cultured cells and is difficult to apply to the analysis of animal tissues and human samples. Isotopic labeling, however, is not limited by sample source and can be widely applied to various clinical samples.

[0005] Currently, various isotopic labeling reagents are available on the market, including iTRAQ-4PLEX, iTRAQ-8PLEX, TMT-10PLEX, and IBT-10PLEX. Recently, Thermo Fisher Scientific launched the TMTpro-16PLEX kit, which can simultaneously label 16 samples. However, TMTpro-16PLEX is not only expensive, but it also identifies far fewer proteins than TMT-10PLEX and IBT-10PLEX, significantly limiting its application in quantitative proteomics. Summary of the Invention

[0006] To address the aforementioned issues, this invention discloses an isotopic labeling technique (IBT-16PLEX) capable of simultaneously performing quantitative analysis on 16 samples. The invention includes the chemical structure of the IBT-16PLEX labeling reagent, the position of the stable isotope within the labeling reagent, the synthetic route of the labeling reagent, and the specific applications of the labeling reagent in proteomics.

[0007] This invention provides the following technical solution:

[0008] A new class of high-throughput proteomics labeling reagents based on secondary mass spectrometry (MS / MS) has been developed, named IBT-16PLEX reagents. IBT-16PLEX reagents comprise a group of 16 labeling reagents with identical chemical structures and molecular weights, but containing different stable isotopes at different positions. Their structure consists of three parts: a reporter group, a balancing group, and an activating group. The activating group labels the peptide through a specific reaction with functional groups (N-terminal amino groups, lysine amino groups, cysteine ​​thiol groups, etc.). The total molecular weight of the reporter and balancing groups is identical. However, the reporter and balancing groups are linked by an MS / MS cleavable bond. Thus, in MS / MS, IBT-16PLEX-labeled samples produce a set of characteristic reporter ions, and their relative peak heights are used to determine the relative abundance in the original sample.

[0009] This invention discloses the chemical structures of two different sets of IBT-16PLEX reagents (structure A and structure B). Each set of structures has slight differences and can be used interchangeably in specific applications.

[0010] The reaction of the IBT-16PLEX reagent (structure A) to label the peptide and the structure of the reporter group formed by the cleavage of the labeled peptide in tandem mass spectrometry are as follows.

[0011]

[0012] A set of IBT-16PLEX reagents comprises 16 chemically identical labeling reagents. Each labeling reagent contains either a 13C or 15N isotope at different positions, resulting in varying reporter group masses. However, these variations are compensated for by balancing groups. Thus, the total mass of the reporter group plus the balancing group is identical in each reagent. The reporter and balancing groups are linked by a shearable bond, which preferentially breaks during MS / MS, generating a series of reporter group ions with different molecular formulas and masses. The specific molecular formulas and precise masses of the reporter and balancing group ions are as follows:

[0013] Markup code Reporting group molecular formula Reporting group molecular weight Balanced group molecular formula Balanced group molecular weight Total molecular weight T114 <![CDATA[C7H 16 N + ]]> 114.1277 <![CDATA[ 13 C7H 11 15 NO2 + ]]> 149.0989 263.2266 T115N <![CDATA[C7H 16 15 N + ]]> 115.1248 <![CDATA[ 13 C7H 11 NO2 + ]]> 148.1019 263.2267 T115C <![CDATA[C6 13 CH 16 N + ]]> 115.1311 <![CDATA[C 13 C6H 11 15 NO2 + ]]> 148.0956 263.2267 T116N <![CDATA[C6 13 CH 16 15 N + ]]> 116.1281 <![CDATA[C 13 C6H 11 NO2 + ]]> 147.0986 263.2267 T116C <![CDATA[C5 13 C2H 16 N + ]]> 116.1344 <![CDATA[C2 13 C5H 11 15 NO2 + ]]> 147.0922 263.2266 T117N <![CDATA[C5 13 C2H 16 15 N + ]]> 117.1315 <![CDATA[C2 13 C5H 11 NO2 + ]]> 146.0952 263.2267 T117C <![CDATA[C4 13 C3H 16 N + ]]> 117.1378 <![CDATA[C3 13 C4H 11 15 NO2 + ]]> 146.0889 263.2267 T118N <![CDATA[C4 13 C3H 16 15 N + ]]> 118.1348 <![CDATA[C3 13 C4H 11 NO2 + ]]> 145.0918 263.2266 T118C <![CDATA[C3 13 C4H 16 N + ]]> 118.1411 <![CDATA[C4 13 C3H 11 15 NO2 + ]]> 145.0855 263.2266 T119N <![CDATA[C3 13 C4H 16 15 N + ]]> 119.1382 <![CDATA[C4 13 C3H 11 NO2 + ]]> 144.0885 263.2267 T119C <![CDATA[C2 13 C5H 16 N + ]]> 119.1445 <![CDATA[C5 13 C2H 11 15 NO2 + ]]> 144.0822 263.2267 T120N <![CDATA[C2 13 C5H 16 15 N + ]]> 120.1415 <![CDATA[C5 13 C2H 11 NO2 + ]]> 143.0851 263.2267 T120C <![CDATA[C 13 C6H 16 N + ]]> 120.1479 <![CDATA[C6 13 CH 11 15 NO2 + ]]> 143.0788 263.2266 T121N <![CDATA[C 13 C6H 16 15 N + ]]> 121.1449 <![CDATA[C6 13 CH 11 NO2 + ]]> 142.0818 263.2267 T121C <![CDATA[ 13 C7H 16 N + ]]> 121.1512 <![CDATA[C7H 11 15 NO2 + ]]> 142.0755 263.2267 T122 <![CDATA[ 13 C7H 16 15 N + ]]> 122.1482 <![CDATA[C7H 11 NO2 + ]]> 141.0784 263.2266

[0014] The corresponding chemical structures are as follows, where the asterisks represent the positions of the stable isotopes 15N and 13C in a group of IBT-16PLEX (structure A) reagents.

[0015]

[0016] By selecting different starting materials, the positions of 15N and 13C can be varied to any different positions within the reporter and balance groups without changing the molecular weights of the reporter and balance groups, rather than being limited to the isotopic positions shown above. Therefore, with a reporter group mass in the range of 114 to 122 Da, stable isotopes can be found in any feasible combination of positions while keeping the molecular formulas of the reporter and balance groups unchanged.

[0017] The IBT-16PLEX reagent can be used to identify and analyze up to 16 proteomic peptide samples via mass spectrometry. The identification and analysis steps are as follows:

[0018] 1) Liquid chromatography separation is performed using nano-reversed-phase columns; when needed, different two-dimensional chromatographic combinations can be used for high-precision separation (e.g., basic reversed-phase column + acidic reversed-phase column; cation exchange column + acidic reversed-phase column, etc.).

[0019] 2) The labeled peptides were identified and analyzed using an Orbitrap high-resolution mass spectrometer; the resolution was set to at least 30,000 to distinguish the 0.063 Da difference between a pair of reporter group ions.

[0020] 3) The signal intensity of the reporter group ion, as determined by quantitative analysis, should be at least 5% of the maximum peak value.

[0021] The synthetic route for structure A is as follows:

[0022]

[0023] The specific synthesis method is as follows:

[0024] 1) Compound 1 (β-Ala-OBzl.HCl) and acetone reacted with reducing agent NaCNBH3 to give compound 2;

[0025] 2) Compound 2 and compound 3 (Boc-Gly-OH) couple to form compound 4;

[0026] 3) Compound 4 loses its Boc group under acidic conditions to form compound 5;

[0027] 4) Compound 5 reacts sequentially with acetone and propionaldehyde in the presence of reducing agent NaCNBH3 to form compound 6;

[0028] 5) Compound 6 reacts with hydrogen under the catalysis of palladium on carbon to remove the Bzl protecting group and form compound 7;

[0029] 6) Compound 7 reacts with TSTU to form activated compound 8 (IBT-NHS);

[0030] The reaction of the IBT-16PLEX reagent (structure B) to label the peptide and the structure of the reporter group formed by the cleavage of the labeled peptide in tandem mass spectrometry are as follows.

[0031]

[0032] A set of IBT-16PLEX reagents comprises 16 chemically identical labeling reagents. Each labeling reagent contains either a 13C or 15N isotope at different positions, resulting in varying reporter group masses. However, these variations are compensated for by balancing groups. Thus, the total mass of the reporter group plus the balancing group is identical in each reagent. The reporter and balancing groups are linked by a shearable bond, which preferentially breaks during MS / MS, generating a series of reporter group ions with different molecular formulas and masses. The specific molecular formulas and precise masses of the reporter and balancing group ions are as follows:

[0033] Markup code Reporting group molecular formula Reporting group molecular weight Balanced group molecular formula Balanced group molecular weight Total molecular weight T114 <![CDATA[C7H 16 N + ]]> 114.1277 <![CDATA[ 13 C6H8N 15 N2O3 + ]]> 164.0671 278.1948 T115N <![CDATA[C7H 16 15 N + ]]> 115.1248 <![CDATA[ 13 C6H8N 15 NO3 + ]]> 163.0701 278.1949 T115C <![CDATA[C6 13 CH 16 N + ]]> 115.1311 <![CDATA[C 13 C5H8 15 N2O3 + ]]> 163.0638 278.1949 T116N <![CDATA[C6 13 CH 16 15 N + ]]> 116.1281 <![CDATA[C 13 C5H8N 15 NO3 + ]]> 162.0668 278.1949 T116C <![CDATA[C5 13 C2H 16 N + ]]> 116.1344 <![CDATA[C2 13 C4H8 15 N2O3 + ]]> 162.0604 278.1948 T117N <![CDATA[C5 13 C2H 16 15 N + ]]> 117.1315 <![CDATA[C2 13 C4H8N 15 NO3 + ]]> 161.0634 278.1949 T117C <![CDATA[C4 13 C3H 16 N + ]]> 117.1378 <![CDATA[C3 13 C3H8 15 N2O3 + ]]> 161.0571 278.1949 T118N <![CDATA[C4 13 C3H 16 15 N + ]]> 118.1348 <![CDATA[C3 13 C3H8N 15 NO3 + ]]> 160.0600 278.1948 T118C <![CDATA[C3 13 C4H 16 N + ]]> 118.1411 <![CDATA[C4 13 C2H8 15 N2O3 + ]]> 160.0537 278.1948 T119N <![CDATA[C3 13 C4H 16 15 N + ]]> 119.1382 <![CDATA[C4 13 C2H8N 15 NO3 + ]]> 159.0567 278.1949 T119C <![CDATA[C2 13 C5H 16 N + ]]> 119.1445 <![CDATA[C4 13 C2H8N 15 NO3 + ]]> 159.0567 278.2012 T120N <![CDATA[C2 13 C5H 16 15 N + ]]> 120.1415 <![CDATA[C4 13 C2H8N2O3 + ]]> 158.0597 278.2012 T120C <![CDATA[C 13 C6H 16 N + ]]> 120.1479 <![CDATA[C5 13 CH8N 15 NO3 + ]]> 158.0533 278.2012 T121N <![CDATA[C 13 C6H 16 15 N + ]]> 121.1449 <![CDATA[C5 13 CH8N2O3 + ]]> 157.0563 278.2012 T121C <![CDATA[ 13 C7H 16 N + ]]> 121.1512 <![CDATA[C6H8N 15 NO3 + ]]> 157.0500 278.2012 T122 <![CDATA[ 13 C7H 16 15 N + ]]> 122.1482 <![CDATA[C6H8N2O3 + ]]> 156.0529 278.2011

[0034] The corresponding chemical structures are as follows, where the asterisks represent the positions of the stable isotopes 15N and 13C in a group of IBT-16PLEX (structure B) reagents.

[0035]

[0036] By selecting different starting materials, the positions of 15N and 13C can be varied to any different positions within the reporter and balance groups without changing the molecular weights of the reporter and balance groups, rather than being limited to the isotopic positions shown above. Therefore, with a reporter group mass in the range of 114 to 122 Da, stable isotopes can be found in any feasible combination of positions while keeping the molecular formulas of the reporter and balance groups unchanged.

[0037] The IBT-16PLEX reagent can be used to identify and analyze up to 16 proteomic peptide samples via mass spectrometry. The identification and analysis steps are as follows:

[0038] 1) Liquid chromatography separation is performed using nano-reversed-phase columns; when needed, different two-dimensional chromatographic combinations can be used for high-precision separation (e.g., basic reversed-phase column + acidic reversed-phase column; cation exchange column + acidic reversed-phase column, etc.).

[0039] 2) The labeled peptides were identified and analyzed using an Orbitrap high-resolution mass spectrometer; the resolution was set to at least 30,000 to distinguish the 0.063 Da difference between a pair of reporter group ions.

[0040] 3) The signal intensity of the reporter group ion, as determined by quantitative analysis, should be at least 5% of the maximum peak value.

[0041] The synthesis path of structure B is as follows:

[0042]

[0043] The specific synthesis method is as follows:

[0044] 1) Compound 1 (β-Ala-OBzl.HCl) and compound 2 (Boc-Gly-OH) were coupled to form compound 3;

[0045] 2) Compound 3 loses its Boc group under acidic conditions to form compound 4;

[0046] 3) Compound 4 and compound 5 (Boc-Gly-OH) couple to form compound 6;

[0047] 4) Compound 6 loses its Boc group under acidic conditions to form compound 7;

[0048] 5) Compound 7 reacts sequentially with acetone and propionaldehyde in the presence of reducing agent NaCNBH3 to form compound 8;

[0049] 6) Compound 8 reacts with hydrogen under the catalysis of palladium on carbon to remove the Bzl protecting group and form compound 9;

[0050] 7) Compound 9 reacts with TSTU to form activated compound 10 (IBT-NHS).

[0051] The IBT-16PLEX reagent can directly label 16 peptide samples. After labeling, the samples are mixed for LC-MS / MS analysis. Since the reporter and balancing groups are linked by a cleavable bond in the MS / MS mass spectrometry, the different masses of the reporter ions formed during tandem mass spectrometry analysis can be analyzed by high-resolution mass spectrometry for quantification. Furthermore, because IBT-16PLEX contains only 13C and 15N isotopes and not the other commonly used isotope deuterium, it avoids deuterium-induced chromatographic shifts, ensuring quantitative accuracy.

[0052] This invention also discloses the application of the above-mentioned labeling reagent, which is used in peptide quantitative analysis methods, specifically the application of a class of high-throughput proteomics quantification isotopic labeling reagents (IBT-16PLEX) applicable to peptide quantitative analysis of 16 samples.

[0053] When using the IBT-16PLEX reagent for peptide quantification, the main steps include labeling, molecular fragmentation, and quantification. Up to 16 peptide samples can be labeled separately with the IBT-16PLEX reagent (structure A or structure B), mixed together, and then separated on liquid chromatography (LC). Characteristic fragment peaks and reporter groups are then generated in MS / MS, and the peptides are qualitatively and quantitatively analyzed based on the intensity of these peaks and reporter groups.

[0054] The polypeptides are obtained through the following methods:

[0055] (1) Proteins extracted from cells

[0056] (2) Digested by trypsin

[0057] (3) Desalting to obtain a polypeptide mixture

[0058] The peptides are obtained from cell cultures or other samples. Sample preparation can be derived from protein components extracted from cultured cells or other biological samples.

[0059] The present invention has the following beneficial effects:

[0060] The IBT-16PLEX in this invention employs a novel chemical structure, which reduces the difficulty of preparation, increases the yield, thereby reducing product costs and expanding the application scope of isotopic labeling.

[0061] This invention possesses the inherent high efficiency, high throughput, and high sensitivity of all isotropic and heterotropic reagents, while improving upon the shortcomings of other similar reagents and offering low cost.

[0062] The reagent of this invention contains 16 labels, which can simultaneously label 16 different samples. It can be applied to peptide quantitative analysis in various proteomics fields, and is particularly suitable for thermal proteome profiling in pharmaceutical analysis. Attached Figure Description

[0063] Figure 1 The synthesis route diagram of Embodiment 1 of the present invention.

[0064] Figure 2 The synthesis route diagram of Embodiment 2 of the present invention.

[0065] Figure 3 The synthesis route diagram of Embodiment 3 of the present invention.

[0066] Figure 4 Graph showing the accuracy of quantification using IBT-16PLEX reagent.

[0067] Figure 5 Dynamic range diagram of IBT-16PLEX reagent quantification. Detailed Implementation

[0068] The present invention will be further illustrated below with reference to the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0069] This invention discloses a class of high-throughput proteomics labeling reagents based on secondary mass spectrometry quantification, comprising two sets of isotopic labeling reagents, IBT-16PLEX reagents (structure A and structure B). Each set of IBT-16PLEX reagents contains 16 chemically identical labeling reagents, each containing a 13C or 15N isotope at different positions. The isotopic labeling reagents include: T-114, T-115N, T-115C, T-116N, T-116C, T-117N, T-118N, T-117C, T-118C, T-119N, T-119C, T-120N, T-120C, T-121N, T-121C, and T-122. The isotope-labeled reagent comprises an MS / MS cleavable bond and an isotope-labeled group capable of reacting with a peptide, for labeling and quantifying the peptide. The structure of the isotope-labeled reagent includes the following formula: reporter group – equilibrator group – activator group, wherein the reporter group and equilibrator group are linked by an MS / MS cleavable bond, and the activator group reacts with the amino group of the peptide to form a stable amide bond. The mass of the reporter group + equilibrator group in each reagent is identical. While maintaining the molecular formulas of the reporter group and equilibrator group, the stable isotope can include any combination of positions. Different positions in each labeling reagent contain either 13C or 15N isotopes, maintaining a consistent total molecular weight between the reporter group and equilibrator group. The labeling reagent generates the reporter group in secondary mass spectrometry through the position of the cleavage bond. The mass of the reporter group in a group of reagents is in the range of 114 to 122 Da, and can be independently selected from different combinations of the number and positions of isotopic atoms of 13C and / or 15N.

[0070] Compared with existing technologies, IBT-16PLEX has three major innovations: 1) By changing the chemical structure of the balancing group, the range of balancing group compensation for the reporter group mass is expanded, increasing the reporter group mass of IBT-16PLEX from 114-119 Da in IBT-10PLEX to 114-122 Da; 2) In the preparation process of IBT-16PLEX reagent, it is necessary to... 13 C3-propionaldehyde, this invention has developed from commercial products 13 Starting with C3-propionic acid (Sigma-Aldrich), only two steps are required. 13A novel and efficient synthetic method for C3-propionaldehyde; 3) In the process of synthesizing the IBT-16PLEX activating group, a novel synthetic method using reagent TSTU (2-Succinimido-1,1,3,3-tetra-methyluronium tetrafluor, 2-succinimido-1,1,3,3-tetramethyluronium tetrafluoroborate) was developed, which improved the yield and purity of the IBT-16PLEX reagent, greatly extended the stable storage time of the reagent, and reduced the amount of peptide-labeled reagent used.

[0071] Example 1: Synthesis route of IBT-16PLEX (structure A)

[0072]

[0073] like Figure 1 The diagram shows the synthetic route for IBT-16PLEX reagents (structure A). Using this route, a complete set of 16 IBT-16PLEX reagents can be synthesized from starting materials containing different isotopes (compound 1, acetone, compound 3, propionaldehyde).

[0074] Compound 1 (β-Ala-OBzl.HCl, 215 mg, 1 mmol) was dissolved in 20 mL of methanol, and acetone (225 μL, 180 mg) was added, followed by the addition of 95 mg of sodium cyanoborohydride (1.5 mmol). The reaction proceeded for 2 hours. Methanol was removed by rotary evaporation, and the mixture was extracted three times with 20 mL of saturated sodium bicarbonate solution and 20 mL of dichloromethane. The organic phase was then removed by rotary evaporation after combining the organic solutions. 5 mL of dioxane solution (4 M) in HCl was added, followed by the addition of 25 mL of diethyl ether. The mixture was filtered to obtain 195 mg of white solid compound 2 (0.88 mmol, 88% yield). Compound 2 (195 mg) and compound 3 (Boc-Gly-OH, 155 mg, 0.88 mmol) were dissolved in 20 mL of dichloromethane. EDC·HCl (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, 200 mg, 1.05 mmol) was added, and the reaction was carried out under argon protection for 20 hours. The reaction solution was washed three times with 30 mL of 25 mM dilute hydrochloric acid. The organic phase was dried and removed by rotary evaporation to obtain 240 mg of oily compound 4 (0.63 mmol, 72% yield). Compound 4 (240 mg) was dissolved in 20 mL of methanol. Acetone (145 μL, 115 mg) was added, followed by 60 mg of sodium cyanoborohydride, and the reaction was carried out for 2 hours. Methanol was removed by rotary evaporation, and propionaldehyde (145 μL, 115 mg) was added, followed by 30 mg of sodium cyanoborohydride, and the reaction was carried out for 2 hours. Methanol was removed by rotary evaporation. The crude compound was purified by silica gel column chromatography with dichloromethane:ethyl acetate (9:1) to give 174 mg of an oily compound 6 (0.50 mmol, 80% yield). Compound 6 was dissolved in 20 mL of methanol, and after adding 20 mg of Pd / C, the reaction was carried out for one hour under the influence of a hydrogen balloon. The solvent was removed to give 125 mg of a white solid compound 7 (0.46 mmol, 92% yield). Compound 7 (125 mg) was dissolved in 20 mL of dichloromethane, and TSTU (166 mg, 0.55 mmol) and DIPEA (diisopropylethylamine, 1.38 mmol, 178 mg, 227 μL) were added. The reaction was carried out under argon protection for 10 hours. The reaction solution was washed three times with 30 mL of 20 mM sodium bicarbonate solution and once with 30 mL of saturated brine. After drying the organic phase, the solution was evaporated to dryness to give a white oily compound 8, which was further dissolved in acetonitrile and freeze-dried to give 127 mg of a white solid (0.36 mmol, 78% yield).Analysis data: NMR (400MHz, CDCl3): 8.05 (s, 1H), 6.54 (s, 1H), 3.80 (t, 1H), 3.58 (t, 2H), 3.08 (s, 2H), 2.95 (m, 1H), 2.74 (s, 4H), 2.60 (t, 2H), 2.45 (t, 2H), 1.47 (m, 2H), 1.26 (d, 6H), 1.04 (d, 6H), 0.94 (t, 3H). ESI-MS (M+H). + (Calculated) 369.22, (actual) 369.03.

[0075] The reaction of the IBT-16PLEX reagent (structure A) to label the peptide and the structure of the reporter group formed by the cleavage of the labeled peptide in tandem mass spectrometry are as follows.

[0076]

[0077] A set of IBT-16PLEX reagents comprises 16 chemically identical labeling reagents (named: T114, T115N, T115C, T116N, T116C, T117N, T117C, T118N, T118C, T119N, T119C, T120N, T120C, T121N, T121C, T122). Each labeling reagent contains either a 13C or 15N isotope at different positions, resulting in varying reporter group masses. However, these variations are compensated for by balancing groups. Thus, the total mass of the reporter group plus the balancing group is identical in each reagent. The reporter and balancing groups are linked by a shearable bond, which is preferentially broken in MS / MS, generating a series of reporter ions with different molecular formulas and masses. The specific molecular formulas and precise masses of the reporter and balancing group ions are as follows:

[0078] Markup code Reporting group molecular formula Reporting group molecular weight Balanced group molecular formula Balanced group molecular weight Total molecular weight T114 <![CDATA[C7H 16 N + ]]> 114.1277 <![CDATA[ 13 C7H 11 15 NO2 + ]]> 149.0989 263.2266 T115N <![CDATA[C7H 16 15 N + ]]> 115.1248 <![CDATA[ 13 C7H 11 NO2 + ]]> 148.1019 263.2267 T115C <![CDATA[C6 13 CH 16 N + ]]> 115.1311 <![CDATA[C 13 C6H 11 15 NO2 + ]]> 148.0956 263.2267 T116N <![CDATA[C6 13 CH 16 15 N + ]]> 116.1281 <![CDATA[C 13 C6H 11 NO2 + ]]> 147.0986 263.2267 T116C <![CDATA[C5 13 C2H 16 N + ]]> 116.1344 <![CDATA[C2 13 C5H 11 15 NO2 + ]]> 147.0922 263.2266 T117N <![CDATA[C5 13 C2H 16 15 N + ]]> 117.1315 <![CDATA[C2 13 C5H 11 NO2 + ]]> 146.0952 263.2267 T117C <![CDATA[C4 13 C3H 16 N + ]]> 117.1378 <![CDATA[C3 13 C4H 11 15 NO2 + ]]> 146.0889 263.2267 T118N <![CDATA[C4 13 C3H 16 15 N + ]]> 118.1348 <![CDATA[C3 13 C4H 11 NO2 + ]]> 145.0918 263.2266 T118C <![CDATA[C3 13 C4H 16 N + ]]> 118.1411 <![CDATA[C4 13 C3H 11 15 NO2 + ]]> 145.0855 263.2266 T119N <![CDATA[C3 13 C4H 16 15 N + ]]> 119.1382 <![CDATA[C4 13 C3H 11 NO2 + ]]> 144.0885 263.2267 T119C <![CDATA[C2 13 C5H 16 N + ]]> 119.1445 <![CDATA[C5 13 C2H 11 15 NO2 + ]]> 144.0822 263.2267 T120N <![CDATA[C2 13 C5H 16 15 N + ]]> 120.1415 <![CDATA[C5 13 C2H 11 NO2 + ]]> 143.0851 263.2267 T120C <![CDATA[C 13 C6H 16 N + ]]> 120.1479 <![CDATA[C6 13 CH 11 15 NO2 + ]]> 143.0788 263.2266 T121N <![CDATA[C 13 C6H 16 15 N + ]]> 121.1449 <![CDATA[C6 13 CH 11 NO2 + ]]> 142.0818 263.2267 T121C <![CDATA[ 13 C7H 16 N + ]]> 121.1512 <![CDATA[C7H 11 15 NO2 + ]]> 142.0755 263.2267 T122 <![CDATA[ 13 C7H 16 15 N + ]]> 122.1482 <![CDATA[C7H 11 NO2 + ]]> 141.0784 263.2266

[0079] The corresponding chemical structures are as follows, where the asterisks represent the positions of the stable isotopes 15N and 13C in a group of IBT-16PLEX (structure A) reagents.

[0080]

[0081] By selecting different starting materials, the positions of 15N and 13C can be varied to any different location within the reporter and equilibrium groups, without changing the molecular weights of the reporter and equilibrium groups, rather than being limited to the isotopic positions shown above. Therefore, the reporter group mass, ranging from 114 to 122 Da, can be independently selected from different combinations of the number and positions of isotopic atoms of 13C and / or 15N. The IBT-16PLEX reagent can be used for identification and analysis of up to 16 proteomic peptide samples after labeling.

[0082] The identification and analysis steps are as follows:

[0083] 1) Liquid chromatography separation is performed using nano-reversed-phase columns; when needed, different two-dimensional chromatographic combinations can be used for high-precision separation (e.g., basic reversed-phase column + acidic reversed-phase column; cation exchange column + acidic reversed-phase column, etc.).

[0084] 2) The labeled peptides were identified and analyzed using an Orbitrap high-resolution mass spectrometer; the resolution was set to at least 30,000 to distinguish the 0.063 Da difference between a pair of reporter group ions.

[0085] 3) The signal intensity of the reporter group ion, as determined by quantitative analysis, should be at least 5% of the maximum peak value.

[0086] Example 2: Synthesis route of IBT-16PLEX (structure B)

[0087]

[0088] like Figure 2 The diagram shows the synthetic route for IBT-16PLEX reagent (structure B). Using this route, a complete set of 16 IBT-16PLEX reagents can be synthesized from starting materials containing different isotopes (compound 1, compound 2, compound 5, acetone, and propionaldehyde).

[0089] Compound 1 (β-Ala-OBzl.HCl, 215 mg, 1 mmol) and compound 2 (Boc-Gly-OH, 175 mg, 1 mmol) were dissolved in 20 mL of dichloromethane. EDC.HCl (229 mg, 1.2 mmol) was added, and the reaction was carried out under argon protection for 20 hours. The reaction solution was washed three times with 30 mL of 25 mM dilute hydrochloric acid. The organic phase was dried and removed by rotary evaporation to obtain 260 mg of oily compound 3 (0.77 mmol, 77% yield). Compound 3 (260 mg) was added to 50 mL of dioxane solution (4 M) in HCl and stirred for 1 hour. The resulting white precipitate was filtered and collected to give 194 mg of white solid compound 4 (0.71 mmol, 92% yield). Compound 4 (194 mg, 0.71 mmol) and compound 5 (Boc-Gly-OH, 125 mg, 0.71 mmol) were dissolved in 20 mL of dichloromethane. EDC·HCl (165 mg, 0.85 mmol) was added, and the reaction was carried out under argon protection for 20 hours. The reaction solution was washed three times with 30 mL of 25 mM dilute hydrochloric acid. The organic phase was dried and removed by rotary evaporation to obtain 210 mg of oily compound 6 (0.53 mmol, 75% yield). Compound 6 (210 mg) was added to 50 mL of dioxane solution (4 M) in HCl and stirred for 1 hour. The resulting white precipitate was filtered and collected to give 158 mg of white solid compound 7 (0.48 mmol, 90% yield). Compound 7 was dissolved in 20 mL of methanol, acetone (110 μL, 90 mg) was added, and then 45 mg of sodium cyanoborohydride (162 μL, 130 mg) was added and reacted for 2 hours. Methanol was removed using a rotary evaporator. Propanal (110 μL, 90 mg) was added, followed by the addition of 30 mg sodium cyanoborohydride, and the reaction proceeded for 2 hours. Methanol was removed again using a rotary evaporator. The crude product was purified by dichloromethane:ethyl acetate (9:1) on a silica gel column to give 136 mg of oily compound 8 (0.36 mmol, 75% yield). Compound 8 was dissolved in 20 mL of methanol, and after the addition of 20 mg of Pd / C, the reaction proceeded for one hour under the influence of a hydrogen balloon. The solvent was removed to give 98 mg of white solid compound 9 (0.34 mmol, 95% yield). Compound 9 (98 mg) was dissolved in 20 mL of dichloromethane, and TSTU (120 mg, 0.4 mmol) and DIPEA (diisopropylethylamine, 0.85 mmol, 110 mg, 140 μL) were added. The reaction proceeded for 10 hours under argon protection. The reaction solution was washed three times with 30 mL of 20 mM sodium bicarbonate solution and once with 30 mL of saturated brine. After drying the organic phase, the solution was evaporated to dryness to give a white solid compound 10 (IBT-16PLEX without stable isotopes, structure B, 99 mg, 76% yield).Analysis data: NMR (400MHz, CDCl3): 8.05(s, 1H), 6.54(s, 1H), 4.00(s, 2H), 3.58(t, 2H), 3.08(s, 2H), 2.95(m, 1H), 2.78(s, 4H), 2.60(t, 2H), 2.45(t, 2H), 1.47(m, 2H), 1.04(d, 6H), 0.94(t, 3H). ESI-MS (M+H). + (Calculated) 384.20, (Measured) 384.53.

[0090] The reaction of the IBT-16PLEX reagent (structure B) to label the peptide and the structure of the reporter group formed by the cleavage of the labeled peptide in tandem mass spectrometry are as follows.

[0091]

[0092] A set of IBT-16PLEX reagents comprises 16 chemically identical labeling reagents. Each labeling reagent contains either a 13C or 15N isotope at different positions, resulting in varying reporter group masses. However, these variations are compensated for by balancing groups. Thus, the total molecular weight of the reporter group plus the balancing group is identical in each reagent. The reporter and balancing groups are linked by shearable bonds, which preferentially break in MS / MS, generating a series of reporter group ions with different molecular formulas and masses. The specific molecular formulas and precise masses of the reporter and balancing group ions are as follows:

[0093] Markup code Reporting group molecular formula Reporting group molecular weight Balanced group molecular formula Balanced group molecular weight Total molecular weight T114 <![CDATA[C7H 16 N + ]]> 114.1277 <![CDATA[ 13 C6H8N 15 N2O3 + ]]> 164.0671 278.1948 T115N <![CDATA[C7H 16 15 N + ]]> 115.1248 <![CDATA[ 13 C6H8N 15 NO3 + ]]> 163.0701 278.1949 T115C <![CDATA[C6 13 CH 16 N + ]]> 115.1311 <![CDATA[C 13 C5H8 15 N2O3 + ]]> 163.0638 278.1949 T116N <![CDATA[C6 13 CH 16 15 N + ]]> 116.1281 <![CDATA[C 13 C5H8N 15 NO3 + ]]> 162.0668 278.1949 T116C <![CDATA[C5 13 C2H 16 N + ]]> 116.1344 <![CDATA[C2 13 C4H8 15 N2O3 + ]]> 162.0604 278.1948 T117N <![CDATA[C5 13 C2H 16 15 N + ]]> 117.1315 <![CDATA[C2 13 C4H8N 15 NO3 + ]]> 161.0634 278.1949 T117C <![CDATA[C4 13 C3H 16 N + ]]> 117.1378 <![CDATA[C3 13 C3H8 15 N2O3 + ]]> 161.0571 278.1949 T118N <![CDATA[C4 13 C3H 16 15 N + ]]> 118.1348 <![CDATA[C3 13 C3H8N 15 NO3 + ]]> 160.0600 278.1948 T118C <![CDATA[C3 13 C4H 16 N + ]]> 118.1411 <![CDATA[C4 13 C2H8 15 N2O3 + ]]> 160.0537 278.1948 T119N <![CDATA[C3 13 C4H 16 15 N + ]]> 119.1382 <![CDATA[C4 13 C2H8N 15 NO3 + ]]> 159.0567 278.1949 T119C <![CDATA[C2 13 C5H 16 N + ]]> 119.1445 <![CDATA[C4 13 C2H8N 15 NO3 + ]]> 159.0567 278.2012 T120N <![CDATA[C2 13 C5H 16 15 N + ]]> 120.1415 <![CDATA[C4 13 C2H8N2O3 + ]]> 158.0597 278.2012 T120C <![CDATA[C 13 C6H 16 N + ]]> 120.1479 <![CDATA[C5 13 CH8N 15 NO3 + ]]> 158.0533 278.2012 T121N <![CDATA[C 13 C6H 16 15 N + ]]> 121.1449 <![CDATA[C5 13 CH8N2O3 + ]]> 157.0563 278.2012 T121C <![CDATA[ 13 C7H 16 N + ]]> 121.1512 <![CDATA[C6H8N 15 NO3 + ]]> 157.0500 278.2012 T122 <![CDATA[ 13 C7H 16 15 N + ]]> 122.1482 <![CDATA[C6H8N2O3 + ]]> 156.0529 278.2011

[0094] The corresponding chemical structures are as follows, where the asterisks represent the positions of the stable isotopes 15N and 13C in a group of IBT-16PLEX (structure B) reagents.

[0095]

[0096] By selecting different starting materials, the positions of 15N and 13C can be varied to any different location within the reporter and equilibrium groups, without changing the molecular weights of the reporter and equilibrium groups, rather than being limited to the isotopic positions shown above. Therefore, the reporter group mass, ranging from 114 to 122 Da, can be independently selected from different combinations of the number and positions of isotopic atoms of 13C and / or 15N. The IBT-16PLEX reagent can be used for identification and analysis of up to 16 proteomic peptide samples after labeling.

[0097] The identification and analysis steps are as follows:

[0098] 1) Liquid chromatography separation is performed using nano-reversed-phase columns; when needed, different two-dimensional chromatographic combinations can be used for high-precision separation (e.g., basic reversed-phase column + acidic reversed-phase column; cation exchange column + acidic reversed-phase column, etc.).

[0099] 2) The labeled peptides were identified and analyzed using an Orbitrap high-resolution mass spectrometer; the resolution was set to at least 30,000 to distinguish the 0.063 Da difference between a pair of reporter group ions.

[0100] 3) The signal intensity of the reporter group ion, as determined by quantitative analysis, should be at least 5% of the maximum peak value.

[0101] Example 3: 13 Synthesis of C3-propionaldehyde

[0102]

[0103] like Figure 3 : 13 Two-step synthetic route for C3-propionaldehyde: the route required in Examples 1 and 2 13 C3-propionaldehyde was prepared using the method described in this embodiment.

[0104] 13 C3-propionic acid (4 g, 54 mmol) was added to 200 mL of dichloromethane, followed by N,O-dimethylhydroxylamine hydrochloride (59.4 mmol, 5.76 g) and EDC.HCl (59.4 mmol, 11.4 g). The reaction was carried out under nitrogen protection for 20 hours. After washing three times with water (50 mL) and saturated brine (50 mL), the organic phase was dried and then evaporated to dryness to give 5.4 g (46 mmol, 85% yield) of a white solid. This solid was dissolved in 50 mL of anhydrous methanol, and 0.875 g of lithium aluminum hydride powder (23 mmol) was slowly added. After reacting in the dark for 1 hour, methanol was removed by rotary evaporation. Excess lithium aluminum hydride and the intermediate from the hydrolysis of the Weinerb reaction were slowly added to the residual solid. The resulting propionaldehyde was subjected to liquid-liquid continuous extraction with 100 mL of diethyl ether, followed by distillation at atmospheric pressure to obtain the final product. 13 C3-propanal (1.35 g, 22.5 mmol, 49% yield) NMR (400 MHz, CD3Cl) 9.96 (m, 0.5 H), 9.62 (m, 0.5 H), 2.63 (m, 1 H), 2.29 (m, 1 H), 1.28 (m, 1.5 H), 0.94 (m, 1.5 H).

[0105] Example 4: Comparison of IBT-16PLEX (Structure B) and TMTpro-16PLEX

[0106] Bovine serum albumin (10 μg) was dissolved in 1 mL of denaturing solution (8 M urea, 50 mM sodium tetraborate, pH 8.3), 5 mM TCEP (tris(2-carboxyethyl)phosphine), and incubated at 37 °C for 30 min. Then, bromoacetamide (20 mM) was added to alkylate free cysteine ​​in the protein. After purification by methanol / chloroform precipitation, the protein was dissolved in 100 μL of buffer (200 mM sodium tetraborate, pH 8.3, 0.8 M urea), digested with trypsin (10 μg), and incubated at 37 °C for 8 h. 1 mg IBT-16PLEX (or 1 mg TMTpro) was dissolved in 200 μL of acetonitrile and reacted at room temperature for 2 h. After rotary evaporation, the solution was desalted using a C18 solid-phase column. The solution was redissolved in 20 μL of 0.1% formic acid, 10% acetonitrile solution and analyzed using a QE-HF Orbitrap mass spectrometer. Mass spectrometry analysis conditions: First-stage MS1 scan range: 350 to 2000 m / z, scan resolution: 70,000 m / s, minimum signal intensity: 20,000 m / s. Second-stage MS / MS scan resolution: 35,000 m / s, minimum signal-to-noise ratio: 1.5. Database search: Protein identification and quantification were performed using iQuant software with iPeak search. Protein identification utilized the SwissProt database (species Bovine) and decoy sequences. Specific parameter settings were: protein and peptide identification error rate (FDR) less than 1%; trypsin was selected as the specific enzyme, with a maximum of one erroneous cleavage per peptide; fixed modifications included IBT-16PLEX (N-term), IBT-16PLEX (K), carbamoyl methylation (C), and variable modifications included oxidation (M), deamination (N, Q). MS1 mass error value: 20 ppm; MS2 fragment mass error: 0.1 Da.

[0107] HeLa proteome was also analyzed using IBT-16PLEX and TMTpro-16PLEX. HeLa cells were added to 1 mL of cell lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 1 mM Na3VO4), centrifuged at high speed, and the supernatant was collected. 2 mL of acetone was added, and the cells were centrifuged again at high speed. The precipitated proteins were collected and dissolved in 1 mL of denaturing solution (8 M urea, 50 mM sodium tetraborate, pH 8.3, 5 mM TCEP, and tris(2-carboxyethyl) phosphine), and incubated at 37°C for 30 minutes. Then, bromoacetamide (20 mM) was added to alkylate free cysteine ​​residues in the proteins. Proteins were purified by methanol / chloroform precipitation, dissolved in 100 μL buffer (200 mM sodium tetraborate, pH 8.3, 0.8 M urea), digested with trypsin (100 μg), and kept at 37°C for 8 hours. 1 mg IBT-16PLEX (or 1 mg TMTpro) was dissolved in 200 μL acetonitrile and reacted at room temperature for 2 hours. After rotary evaporation, the solution was desalted using a C18 solid-phase column. The protein was redissolved in 20 μL of 0.1% formic acid and 10% acetonitrile solution and analyzed using a QE-HF Orbitrap mass spectrometer. Mass spectrometry conditions: MS1 scan range 350 to 2000 m / z, scan resolution 70000, minimum signal intensity 20000. MS / MS scan resolution 35000, minimum signal-to-noise ratio set to 1.5. Database search: Protein identification and quantification were performed using iQuant software with iPeak search. Protein identification utilized the SwissProt database (Species Human) and decoy sequences. Specific parameters were set as follows: the error rate (FDR) for protein and peptide identification was set to be less than 1%; trypsin was selected as the specific enzyme, with a maximum of one erroneous cleavage per peptide; fixed modifications included IBT-16PLEX (N-term), IBT-16PLEX (K), carbamoyl methylation (C), and variable modifications included oxidation (M), deamination (N, Q). The MS1 fragment quality error was 20 ppm, and the MS2 fragment quality error was 0.1 Da.

[0108] Data shows that labeling the HeLa proteome with IBT-16PLEX can identify approximately 30% more peptides (19141 / 14350–133%) and 13% more proteins (3372 / 2992–113%) than TMTpro, demonstrating a significant advantage.

[0109] Table 1: Comparison of the number of peptides and proteins identified after IBT-16PLEX and TMTpro labeling

[0110]

[0111] Example 5: IBT-16PLEX for quantitative proteomics analysis in HeLa cells

[0112] The HeLa proteomic peptides prepared in Example 4 were divided into 16 equal portions (1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1), labeled with IBT-16PLEX reagent, and then mixed. Mass spectrometry analysis was then performed following the steps in Example 4. After repeating the independent experiments three times, the data were statistically analyzed. The results showed that, using the sample labeled with a molecular marker of mass 114 as a control, the proportions of the other 15 molecular markers labeled were all close to 1.0.

[0113] like Figure 4 The figure shows the accuracy of IBT-16PLEX reagent quantification. The Y-axis represents the measured proportions of different IBT-16PLEX labels against the 114 label. The measured mean and error range were obtained from three independent experiments.

[0114] To determine the quantitative dynamic range of IBT-16PLEX, the HeLa proteomic peptides prepared in Example 4 were reacted with 16 reagents of IBT-16PLEX in the following ratio (1:10:10:10:5:5:5:1:1:1:0.2:0.2:0.2:0.1:0.1:0.1), and then mass spectrometry analysis was performed according to the steps in Example 4. After repeating the independent experiment three times, the data were statistically analyzed. The results showed that, using the sample labeled with a molecular marker of mass 114 as a control, the proportions of the other 15 molecular markers were close to the theoretical values.

[0115] like Figure 5 As shown: Dynamic range of IBT-16PLEX reagent quantification. The Y-axis is the logarithm (LogR) of the measured proportions of different IBT-16PLEX labels against the 114 label. The measured mean and error range were obtained from three independent experiments.

[0116] Example 6

[0117] This invention also discloses an IBT-16PLEX reagent from Examples 1 or 2 above for peptide quantitative analysis. The main steps include labeling, molecular fragmentation, and quantification. Up to 16 peptide samples can be labeled separately with the IBT-16PLEX reagent (structure A or structure B), mixed together, and then separated on liquid chromatography (LC). Characteristic fragment peaks and a reporter group are then generated in MS / MS. The peptides are qualitatively and quantitatively analyzed based on the intensity of the characteristic peaks and the reporter group.

[0118] The polypeptides are obtained through the following methods:

[0119] (1) Proteins extracted from cells

[0120] (2) Digested by trypsin

[0121] (3) Desalting to obtain a polypeptide mixture

[0122] The peptides are obtained from cell cultures or other samples. Sample preparation can be derived from protein components extracted from cultured cells or other biological samples.

[0123] The specific quantitative analysis methods are as described in Examples 4 and 5 above.

[0124] Example 7

[0125] The present invention also discloses the application of the labeling reagents of Examples 1 and 2 above. The labeling reagents are used in peptide quantitative analysis methods, specifically the application of a class of isotopic labeling reagents (IBT-16PLEX) applicable to high-throughput proteomics quantification of 16 samples in peptide quantitative analysis.

[0126] The technical means disclosed in this invention are not limited to those disclosed in the above embodiments, but also include technical solutions composed of any combination of the above technical features. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications are also considered within the scope of protection of this invention.

Claims

1. A class of high-throughput proteomics labeling reagents based on secondary mass spectrometry quantification, characterized in that: The reagent comprises a set of 16 labeling reagents with identical chemical structures and molecular weights, each containing a different stable isotope at different positions. The structure consists of three parts: a reporter group, a balancing group, and an activating group. The activating group labels the polypeptide through a specific reaction with different functional groups contained in the polypeptide. The reporter group and the balancing group have the same total mass and are linked by an MS / MS cleavable bond. Specifically, the reagent set comprises 16 labeling reagents with identical chemical structures, named: T114, T115N, T115C, T116N, T116C, T117N, T117C, T118N, T118C, T119N, T119C, T120N, T120C, T121N, T121C. T122; Each labeled reagent contains 13C or 15N isotopes at different positions to maintain the consistency of the total molecular weight of the reporter and equilibrium groups; the labeled reagents generate reporter groups in secondary mass spectrometry through the position of cleavage bonds; the reporter groups of a group of reagents have a mass range of 114 to 122 Da and can be independently selected from different combinations of isotopic atoms of 13C and / or 15N in different numbers and positions; the specific structural formula is as follows: 。 2. The high-throughput proteomics labeling reagent based on secondary mass spectrometry quantification as described in claim 1, characterized in that: The chemical structures corresponding to the reporter group and the equilibrium group ions are shown below, where the asterisks represent the positions of the stable isotopes 15N and 13C in a group of reagents; 。 3. The high-throughput proteomics labeling reagent based on secondary mass spectrometry quantification as described in claim 1, characterized in that: The reagent has the following structure: while keeping the molecular formulas of the reporter group and the equilibrium group unchanged, the stable isotope can include any combination of positions.

4. The high-throughput proteomics labeling reagent based on secondary mass spectrometry quantification as described in claim 1, characterized in that: The reagent, after labeling up to 16 proteomic peptide samples, is used for identification and analysis by mass spectrometry; the identification and analysis steps are as follows: 1) Liquid chromatography separation was performed using a one-dimensional nano-reversed-phase chromatography column; 2) High-precision separation is achieved using different two-dimensional chromatographic combinations: basic reversed-phase column + acidic reversed-phase column; cation exchange column + acidic reversed-phase column; 3) The labeled peptides were identified and analyzed using an Orbitrap high-resolution mass spectrometer; the resolution was set to at least 30,000 to distinguish the difference of 0.063 Da between a pair of reporter group ions; 4) The signal intensity of the reporter group ion, as determined by quantitative analysis, should be at least 5% of the maximum peak value.

5. The high-throughput proteomics labeling reagent based on secondary mass spectrometry quantification as described in claim 1, characterized in that: The synthetic route of the reagent is as follows: ， The specific synthesis method is as follows: 1) Compound 1 and compound 2 couple to form compound 3; 2) Compound 3 loses its Boc group under acidic conditions to form compound 4; 3) Compound 4 and compound 5 are coupled to form compound 6; 4) Compound 6 loses its Boc group under acidic conditions to form compound 7; 5) Compound 7 reacts sequentially with acetone and propionaldehyde in the presence of reducing agent NaCNBH3 to form compound 8; 6) Compound 8 reacts with hydrogen under the catalysis of palladium on carbon to remove the Bzl protecting group and form compound 9; 7) Compound 9 reacts with TSTU to form activated compound 10.

6. The application of any one of the high-throughput proteomics labeling reagents based on secondary mass spectrometry quantification as described in claims 1-5, characterized in that: The labeling reagent described herein is used in a method for quantitative analysis of peptides.

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