A method for quantitative analysis of intact proteins based on mass spectrometric tags

Abstract

The application belongs to the field of chemical analysis, and particularly relates to a method for quantitatively analyzing intact proteins based on mass spectrometry tags. The specific technical scheme comprises the following steps: labeling a protein to be measured; separating the labeled protein by using a size exclusion chromatography-mass spectrometry combined technology; and quantitatively analyzing the protein to be measured by using a mass spectrometry mode of in-source fragmentation combined with multiple reaction monitoring. The application realizes the top-down high-sensitivity absolute quantification of intact proteins on a low-cost QqQ-MS by using a chemical labeling-in-source fragmentation-MRM combined strategy. The method for quantitatively analyzing proteins provided by the application has strong universality and good instrument compatibility.

Description

Technical Field

[0001] This invention belongs to the field of chemical analysis, specifically relating to a method for quantitative analysis of intact proteins based on mass spectrometry tags. Background Technology

[0002] Quantitative analysis of proteins plays a vital role in many fields. Currently, the mainstream methods for quantitative protein analysis can be divided into two categories: immunological methods (such as ELISA and immunoturbidimetry) and mass spectrometry (MS). However, both types of methods have significant limitations.

[0003] Limitations of immunological methods include: antibody specificity dependence: antibodies are prone to cross-contamination and batch-to-batch variations, leading to false positives / false negatives; narrow dynamic range: the linear range of traditional ELISA usually only covers 2-3 orders of magnitude, making it difficult to cover the concentration range of proteins in clinical samples (such as the concentration of certain biomarkers in serum spanning 5 orders of magnitude); and weak multiplexing capability: independent antibodies need to be developed for each target, resulting in high costs and low throughput.

[0004] In comparison, mass spectrometry (MS) is considered an ideal detection method due to its inherent high sensitivity, wide dynamic range, low background interference, and high throughput. However, mass spectrometry also has significant drawbacks: bottom-up methods suffer from enzyme-dependent digestion, uncontrollable digestion processes, and increased operational complexity and error risks due to multi-step pretreatment (reductive alkylation, desalting); while top-down methods are limited by the need for expensive detection equipment, resulting in high detection costs.

[0005] Specifically, depending on the target analyte, mass spectrometry can be performed based on mass tags or direct methods. ESI and MALDI are two "soft" ionization methods for detecting protein macromolecules. For ESI-MS, the presence of proteins in different charge states, charge dilution of proteins with low ionization efficiency, and the formation of protein adducts induced by non-volatile buffer environments typically reduce detection sensitivity and selectivity. MALDI-MS analysis of biomolecules usually requires a special matrix and yields less structural information.

[0006] Triple quadrupole mass spectrometry (QqQ-MS) possesses excellent sensitivity and specificity and is commonly used for quantitative analysis. Compared to traditional immunological, HPLC, and colorimetric methods, QqQ-MS exhibits significant advantages in accuracy, precision, selectivity, sensitivity, and quantitative dynamic range. However, current methods for protein quantification using QqQ-MS are limited by the instrument's detection range and require protein digestion before analysis—a so-called bottom-up approach. In this method, intact proteins in the sample are digested into small peptide fragments by enzymes (such as trypsin); then, QqQ-MS quantifies the constituent peptides to determine the concentration of intact proteins in the sample. However, this digestion step is influenced by numerous factors, and digestion efficiency cannot be guaranteed to be 100%, potentially leading to errors in quantitative analysis.

[0007] To avoid errors introduced by the digestion step, a top-down approach can be used, which involves directly analyzing intact proteins using a high-resolution mass spectrometer. However, this method requires more expensive mass spectrometers, such as time-of-flight, orbital trap, and Fourier transform ion cyclotron resonance (FTIR) instruments. Furthermore, the detection sensitivity may not meet requirements due to the influence of protein ionization efficiency.

[0008] Therefore, if a more direct and accurate protein quantification method can be provided based on existing common and widely used QqQ-MS detection equipment, it will have significant research and application value. Summary of the Invention

[0009] The purpose of this invention is to provide a method for quantitative analysis of intact proteins based on mass spectrometry tags.

[0010] To achieve the above-mentioned objectives, the technical solution adopted by this invention is: a mass spectrometry tag for labeling proteins, wherein the general structural formula of the mass spectrometry tag is:

[0011] R1 is a mass spectrometry signal molecule, which is a group with mass spectrometry sensitization capability and a molecular weight between 50 and 1000 Da, and can be any one of quaternary ammonium, pyridinyl, quinolinyl, or alkyl-substituted amino groups; R2 and R3 are linker groups, which are groups that can react with amino acids on the protein to be labeled, and can be any one of hydrogen, N-hydroxysuccinimide ester, sulfonic acid-N-hydroxysuccinimide ester, sulfonyl fluoride, sulfonylimide fluoride, and hydroxylamine.

[0012] Preferably, the structure of R1 is as shown in Formula I or Formula II:

[0013] Formula I:

[0014] In Formula I, x is an integer from 1 to 8, and R4, R5 and R6 are short-chain alkanes from C1 to C4, respectively;

[0015] Formula II:

[0016] In Equation II, R7, R8, R9 and R 10 They are either hydrogen or short-chain alkanes of C1 to C4, R 11 and R 12 This indicates the presence of one or more substituents, which are hydrogen or short-chain alkanes from C1 to C4, R. 13 It can be hydroxyl or methoxy.

[0017] Preferably, when R3 is hydrogen, the structure of R2 is as shown in Formula III or Formula IV:

[0018] Formula III:

[0019] In Formula III, R 14 It can be a benzene ring, an alkyl-substituted benzene ring, or a halo-substituted benzene ring;

[0020] Formula IV: y is an integer from 1 to 15.

[0021] Preferably, when R3 is not hydrogen, the R2-based structure is any one of the following structures:

[0022]

[0023] Where m is an integer from 1 to 15, and n is an integer from 1 to 100.

[0024] Accordingly, a mass spectrometry tag for labeling proteins includes a linker and a mass spectrometry signal compound, wherein the linker is any one of the following compounds:

[0025]

[0026] Where y and m are integers from 1 to 15, and n is an integer from 1 to 100;

[0027] The structural formula of the mass spectrometry signal compound is shown in formula V or formula VI:

[0028] Formula V:

[0029] In equation V, x is an integer from 1 to 8, and R 15 R 16 and R 17 These are short-chain alkanes, ranging from C1 to C4.

[0030] Formula VI:

[0031] In Equation VI, R 18 R 19 R20 and R 21 They are either hydrogen or short-chain alkanes of C1 to C4, R 22 and R 23 This indicates the presence of one or more substituents, which are hydrogen or short-chain alkanes from C1 to C4, R. 24 It can be hydroxyl or methoxy.

[0032] Accordingly, a method for labeling proteins using the mass spectrometry tag includes the following steps:

[0033] (1) Dissolve the protein to be labeled to obtain a protein solution;

[0034] (2) Dissolve the mass spectrometry tag in the solution to obtain the mass spectrometry tag solution;

[0035] (3) Add the protein solution to the mass spectrometry tag solution, followed by acetonitrile and formic acid, and react for 1-4 hours.

[0036] Accordingly, a method for labeling proteins using the mass spectrometry tag includes the following steps:

[0037] (1) Process the sample containing the protein to be labeled to obtain a protein solution;

[0038] (2) Dissolve the mass spectrometry signal compound in a solution to obtain a mass spectrometry signal compound solution;

[0039] (3) Dissolve the linker in a solution to obtain a linker solution;

[0040] (4) Add the protein solution to be labeled to the linker solution. After the two react, add the mass spectrometry signal compound solution, followed by acetonitrile and formic acid. React for 1-4 hours.

[0041] Preferably, the molar ratio of the protein to be labeled to the linker is 1:5 to 1:100; and the reaction temperature is 4℃ to 50℃.

[0042] Accordingly, a method for quantitative analysis of proteins includes the following steps:

[0043] (1) Label the protein to be tested using the method described above;

[0044] (2) Separate the labeled proteins using chromatography-mass spectrometry;

[0045] (3) Quantitative analysis of the target protein was performed using the mass spectrometry mode of source cleavage combined with multiple reaction monitoring.

[0046] The present invention has the following beneficial effects:

[0047] In-source dissociation (ISD) refers to the fragmentation of an analyte during the initial ionization process within an ion source. For example, using MALDI-TOF in ISD mode to analyze intact antibodies, semi-quantitative analysis is achieved by detecting the Fc fragment (m / z = 25 kDa); or a high voltage (>6000 V) is applied within an ESI source to cleave intact proteins into peptides, which are then quantified by QqQ-MS. This method suffers from uncontrollable cleavage sites, poor fragment reproducibility, and a lack of specific marker ions, making it difficult to distinguish homologous proteins. Furthermore, if QqQ-MS is used for direct quantification of intact proteins, such as using reversed-phase chromatography (RP-LC) to separate intact proteins, proteins with a molecular weight >50 kDa are outside the mass-to-charge ratio detection range of QqQ-MS (m / z < 4000).

[0048] Therefore, this invention achieves high-sensitivity, top-down absolute quantification of intact proteins on low-cost QqQ-MS through a chemical labeling-intra-source cleavage-MRM combined strategy. Specifically:

[0049] Bottom-up protein analysis involves complex, tedious, and time-consuming digestion procedures, which can easily lead to inaccurate quantitative results. Therefore, this invention employs a top-down quantitative method to avoid the digestion step. However, if whole proteins are used directly for mass spectrometry analysis, the molecular weight of the proteins often exceeds the detection range of mass spectrometry, and the ionization efficiency is low, making it difficult to detect low concentrations of proteins. Therefore, this invention labels the proteins by introducing mass spectrometry tags with high ionization efficiency. These tags can easily undergo directional fragmentation during source fragmentation, and combined with multiple reaction monitoring (MRM) mass spectrometry detection, absolute quantitative analysis of labeled proteins can be achieved from top to bottom.

[0050] Furthermore, this invention features targeted selection of mass spectrometry tags. Small molecule mass spectrometry tags are covalently linked to proteins via chemical labeling, leveraging the tags' high ionization efficiency to enhance the signal. Existing techniques for signal enhancement based on mass spectrometry tags rely on high-resolution mass spectrometry (such as Orbitrap) to resolve complex fragment spectra, making them unsuitable for low-cost QqQ-MS. In this invention, however, the synergistic effect of bifunctional groups is utilized: the R1 group contains high ionization efficiency groups such as quaternary ammonium and pyridyl groups, which improve the mass spectrometry response; the R2 group employs highly reactive groups such as N-hydroxysuccinimide ester (NHS) and sulfonamide fluoride, which enhance labeling efficiency; in addition, the special linker chemical chains readily break directionally during source fragmentation, facilitating subsequent mass spectrometry detection.

[0051] In the source fragmentation-MRM coupled method, this invention precisely controls the fragmentation parameters. By optimizing the DP voltage and CE, the mass spectrometer tag is directionally broken during ionization to generate stable fragment ions. A specific MRM channel is used to monitor only characteristic fragment ions (m / z = 386.2), avoiding interference from complex matrices (the signal-to-noise ratio is improved by 20 times compared with the traditional full scan mode).

[0052] This invention also optimizes the compatibility of QqQ-MS. This includes: chromatographic separation innovation: using SEC chromatography instead of traditional reversed-phase chromatography to avoid irreversible adsorption of large protein molecules on the reversed-phase column.

[0053] The quantitative protein analysis method provided by this invention has strong universality (covering secretory proteins (such as BSA), membrane proteins (such as CD20), and antibodies (such as IgG), and good instrument compatibility (compatible with multiple brands of QqQ-MS such as Agilent 6495 and Sciex QTRAP 6500+, without requiring hardware modifications to the equipment). It supports the quantification of proteins in complex matrices such as serum and tissue lysates, providing reliable data support for subsequent research. Attached Figure Description

[0054] Figure 1 This is a schematic diagram illustrating the process of binding proteins to mass spectrometry tags.

[0055] Figure 2 This is a schematic diagram of the protein-mass spectrometry tag binding process in Example 1;

[0056] Figure 3 This is a schematic diagram of the detection results of bovine serum albumin using the two-step method in Example 1;

[0057] Figure 4 This is a schematic diagram of the working solution results for the SEC-MS detection of the standard curve in the two-step method of Example 1;

[0058] Figure 5 This is a schematic diagram illustrating the detection results of bovine serum albumin using the one-step method in Example 1.

[0059] Figure 6 This is a schematic diagram showing the results of Example 3 on bovine serum albumin;

[0060] Figure 7 This is a schematic diagram of the detection results after the mass spectrometry label was changed for the control example.

[0061] Figure 8 This is a schematic diagram of the test results after the connector was replaced, as shown in the control example. Detailed Implementation

[0062] This invention provides a method for the quantitative analysis of intact proteins. The principle is as follows: amino acids on the protein bind to linkers, then undergo a click chemical reaction with mass spectrometry signal molecules, thereby binding a mass spectrometry tag to the protein. Subsequently, liquid chromatography-triple quadrupole tandem mass spectrometry (LC-MS / MS) is used to separate and quantitatively analyze the analyte. The mass spectrometry acquisition mode is in-source fragmentation combined with multiple reaction monitoring (MRM). The mass spectrometry tag on the protein undergoes in-source fragmentation at the ion source, producing a specific fragment ion with m / z = 718.3, and a characteristic fragment with m / z = 386.2 generated by collision-induced fragmentation of this fragment ion, enabling quantitative analysis. The protein labeling method (binding a mass spectrometry tag to the protein) includes a "one-step" and a "two-step" method, the process of which is as follows: Figure 1 As shown, the "one-step method" refers to a mass spectrometry tag being a single compound, with both the mass spectrometry signal molecule and the linker being groups on the mass spectrometry tag. The protein reacts with both the mass spectrometry signal molecule and the linker simultaneously. The "two-step method" refers to a mass spectrometry tag consisting of two independent compounds: a mass spectrometry signal compound and a linker. The protein reacts with the linker first, and then the mass spectrometry signal compound is added.

[0063] The specific analysis method of the one-step method includes the following steps:

[0064] (1) A chemical labeling method is used to bind a mass spectrometry tag to the target protein; specifically: the sample containing the target protein is processed to obtain a target protein solution. The mass spectrometry tag is dissolved in DMSO solution to obtain a mass spectrometry tag solution. The target protein solution is added to the mass spectrometry tag solution, and the molar ratio of the target protein to the linker is controlled at 1:5 to 1:100. Acetonitrile and formic acid are then added, and the concentration of acetonitrile in the reaction system is controlled at 10% and the concentration of formic acid is 1%. The reaction is carried out at 4℃ to 50℃ for 1 to 4 h; preferably at room temperature for 2 h. After the reaction is completed, the mixture is centrifuged. Crystal violet is added to the centrifuged solution as an internal standard solution.

[0065] (2) Labeled proteins were separated using size exclusion chromatography-mass spectrometry (SEC-MS);

[0066] (3) Quantitative analysis was performed using mass spectrometry mode combining source fragmentation with multiple reaction monitoring (MRM), specifically through the following ion signals: specific fragment ions generated by source fragmentation (m / z = 718.3); characteristic ions generated after collision-induced dissociation of the fragment ions (m / z = 386.2).

[0067] The general structural formula of the mass spectrometry tag is:

[0068] R1 is a mass spectrometry signal molecule, which is a group with mass spectrometry sensitization capability and a molecular weight between 50 and 1000 Da, and can be any one of quaternary ammonium, pyridinyl, quinolinyl, or alkyl-substituted amino groups; R2 and R3 are linker groups, which are groups that can react with amino acids on the protein to be labeled, and can be any one of hydrogen, N-hydroxysuccinimide ester, sulfonic acid-N-hydroxysuccinimide ester, sulfonyl fluoride, sulfonylimide fluoride, and hydroxylamine.

[0069] The preferred embodiment is that the structure of R1 is as shown in Equation I or Equation II:

[0070] Formula I:

[0071] In Formula I, the wavy line represents the connecting bond to the general formula, x is any integer from 1 to 8, and R4, R5 and R6 are short-chain alkanes from C1 to C4, respectively.

[0072] Formula II:

[0073] In Equation II, R7, R8, R9 and R 10 They are either hydrogen or short-chain alkanes of C1 to C4, R 11 and R 12 This indicates the presence of one or more substituents, which are hydrogen or short-chain alkanes from C1 to C4, R. 13 It can be hydroxyl or methoxy.

[0074] Preferably, when R3 is hydrogen, i.e., when the click chemistry reaction of azide cycloalkyne addition occurs, the structure of R2 is as shown in Formula III or Formula IV:

[0075] Formula III: R 14 It can be a benzene ring, an alkyl-substituted benzene ring, or a halosubstituted benzene ring.

[0076] Formula IV: y is an integer from 1 to 15.

[0077] Preferably, when R3 is not hydrogen, the SPAAC reaction occurs, and the R2-based structure is any one of the following structures:

[0078]

[0079] In other words, if the linker structure contains octyne, the SPAAC reaction occurs, in which R2 and R3 form a ring with octyne; if the linker structure is a straight-chain alkyne, the azide reaction occurs, not a cyclization reaction, and only R2 is attached to the corresponding group.

[0080] A more preferred embodiment is that the R1 group is selected from any one of the following groups:

[0081]

[0082] The two-step analysis method includes the following steps:

[0083] (1) A chemical labeling method is used to bind the mass spectrometry tag to the target protein; specifically: the sample containing the target protein is processed to obtain a target protein solution. The linker is dissolved in DMSO solution to obtain a linker solution. The mass spectrometry signal compound is dissolved in DMSO solution to obtain a mass spectrometry signal compound solution. The target protein solution is added to the linker solution, and the molar ratio of the target protein to the linker is controlled at 1:5 to 1:100. Acetonitrile and formic acid are then added, and the concentration of acetonitrile in the reaction system is controlled at 10% and the concentration of formic acid is 1%. The reaction is carried out at 4℃ to 50℃ for 1 to 4 h; preferably at room temperature for 2 h. Then the mass spectrometry signal compound solution is added, and the reaction is carried out at room temperature for 0.5 h. After the reaction is completed, the mixture is centrifuged. Crystal violet is added to the centrifuged solution as an internal standard solution.

[0084] (2) Labeled proteins were separated using size exclusion chromatography-mass spectrometry (SEC-MS);

[0085] (3) Quantitative analysis was performed using mass spectrometry mode combining source fragmentation with multiple reaction monitoring (MRM), specifically through the following ion signals: specific fragment ions generated by source fragmentation (m / z = 718.3); characteristic ions generated after collision-induced dissociation of the fragment ions (m / z = 386.2).

[0086] The general structural formula of the mass spectrometry signal compound is shown in formula V or formula VI:

[0087] Formula V:

[0088] In equation V, x is any integer from 1 to 8, and R 15 R 16 and R 17 These are short-chain alkanes, ranging from C1 to C4.

[0089] Formula VI:

[0090] In Equation VI, R 18 R 19 R 20 and R 21 They are either hydrogen or short-chain alkanes of C1 to C4, R 22 and R 23 This indicates the presence of one or more substituents, which are hydrogen or short-chain alkanes from C1 to C4, R. 24 It can be hydroxyl or methoxy.

[0091] The preferred scheme is as follows: the mass spectrometry signal compound is selected from the following compounds:

[0092]

[0093] The preferred embodiment is that the linker is selected from the following compounds:

[0094]

[0095] Where y and m are any integers from 1 to 15, and n is any integer from 1 to 100.

[0096] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. All obtained data are average values ​​obtained after at least three repetitions, and each repetition yields valid data.

[0097] Example 1

[0098] The reaction principle of this embodiment is as follows: Figure 2 As shown, lysine residues on the protein react with DBCO-CONH-SS-NHS, followed by a ring-strain-promoted cycloaddition reaction (SPAAC) with 5-carboxytetramethylazidorhodamine attached to an azide group, thereby constructing a mass spectrometry tag on the protein to be analyzed. Bovine serum albumin (BSA) was used for relevant tests in all examples. This is because BSA has a well-defined molecular weight (approximately 66 kDa) and a stable structure; moreover, BSA is often added as a standard to serum or tissue lysates, and its experimental data directly reflect the method's tolerance to complex biological matrices.

[0099] 1. Two-step method

[0100] (1) Protein labeling

[0101] 1 mg of bovine serum albumin was dissolved in 10 mL of ultrapure water to obtain a 0.1 mg / mL bovine serum albumin solution. 1 mg of DBCO-CONH-SS-NHS (linker) was dissolved in 176.8 μL of DMSO solution to obtain a 10 mM DBCO-CONH-SS-NHS solution; 1 mg of 5-4TAMRA (mass spectrometry signal compound) was dissolved in 195.1 μL of DMSO solution to obtain a 10 mM 5-4TAMRA solution.

[0102] DBCO-CONH-SS-NHS:

[0103] Take 50 μL of 0.1 mg / mL bovine serum albumin solution, add 2 μL of 10 mM DBCO-CONH-SS-NHS solution, then add 10 μL of acetonitrile and 1 μL of formic acid, and bring the solution to 100 μL with ultrapure water. React at room temperature for 2 hours. Then add 2.4 μL of 10 mM 5-4TAMRA solution to the above reaction solution and react at room temperature for 0.5 hours. Subsequently, centrifuge using a 10 kDa ultrafiltration centrifuge tube. Add crystal violet as an internal standard solution (final concentration of crystal violet is 10 ng / mL) to the ultrafiltration solution for instrumental analysis.

[0104] (2) On-machine testing

[0105] Liquid chromatography conditions: Column: ACQUITY Protein BEH SEC Column, 1.7 μm, 4.6 × 150 mm; flow rate: 0.3 mL / min; mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: acetonitrile solution; isocratic elution program: 25% mobile phase B, run for 12 minutes.

[0106] Mass spectrometry conditions: Ion source: electrospray ionization source, positive ion mode; capillary voltage: 5500V; ion source temperature (TEM): 450℃; ion source nebulizer gas (GS1): 50psi; ion source heating auxiliary gas (GS2): 50psi; curtain gas (CUR): 35psi; collision gas (CAD): 7psi; scan mode: MRM; MRM parameter settings are shown in Table 1. In Table 1, Q1 is the parent ion; Q3 is the daughter ion; DP is the declustering voltage; CE is the collision voltage.

[0107] Table 1 MRM Parameter Settings

[0108] name Q1 Q3 DP CE Mass spectrometry label 718.3 386.2 280 55 Internal Standard 372.0 356.0 260 45

[0109] Labeled bovine serum albumin standard solutions were detected using in-source lysis combined with multiple reaction monitoring (MRM). The results are as follows: Figure 3 As shown in the figure. The results show that the mass spectrometry tag on bovine serum albumin was successfully fragmented within the source and detected by multiple reaction monitoring (MRM). The retention time was 3.03 min.

[0110] DBCO-CONH-SS-NHS and 5-4TAMRA were used as labeling reagents, and the working solution of the standard curve was detected by SEC-MS. Linear fitting was performed with BSA concentration as the x-axis and the peak area ratio of BSA to the internal standard as the y-axis. Results are as follows: Figure 4As shown, the linear correlation coefficient is greater than 0.99 in the range of bovine serum albumin concentration from 1 μg / mL to 32 μg / mL, indicating that this method can be used for absolute quantitative analysis of intact proteins.

[0111] 2. One-step method

[0112] Weigh 1 mg of DBCO-CONH-SS-NHS (linker) and dissolve it in 176.8 μL of DMSO solution to obtain a 10 mM DBCO-CONH-SS-NHS solution; weigh 1 mg of 5-4TAMRA (mass spectrometry signal compound) and dissolve it in 195.1 μL of DMSO solution to obtain a 10 mM 5-4TAMRA solution. Add the DBCO-CONH-SS-NHS solution to the 5-4TAMRA solution and react at room temperature to obtain the mass spectrometry tag.

[0113]

[0114] Take 50 μL of 0.1 mg / mL bovine serum albumin solution and add it to the above mass spectrometry tag solution. Then add 10 μL of acetonitrile and 1 μL of formic acid, and bring the solution to 100 μL with ultrapure water. React at room temperature for 2 hours. Then centrifuge using a 10 kDa ultrafiltration centrifuge tube. Add crystal violet as an internal standard solution (final concentration of crystal violet is 10 ng / mL) to the ultrafiltration solution for analysis. All other conditions are exactly the same as the one-step method.

[0115] The results are as follows Figure 5 As shown in the figure. The results show that the peak area measured by the one-step method is 1.307E5, and the peak area measured by the two-step method is 1.164E5, with the peak areas of the two methods being close. This indicates that both the one-step and two-step methods can successfully label the protein.

[0116] Example 2

[0117] The two-step method of Example 1 was used to determine different concentrations of protein:

[0118] 1. A BSA solution with a concentration of 2 μg / mL was prepared, and the BSA solution was detected using the mass spectrometry tagging method described in Example 1. The concentration of the BSA solution was found to be 2.01 μg / mL.

[0119] 2. A BSA solution with a concentration of 32 μg / mL was prepared, and the BSA solution was detected using the mass spectrometry tagging method described in Example 1. The concentration of the BSA solution was found to be 31.6 μg / mL.

[0120] The results show that the method of the present invention can accurately quantify proteins of different concentrations.

[0121] Example 3

[0122] Weigh 1 mg of bovine serum albumin and dissolve it in 1 mL of ultrapure water to obtain a 1 mg / mL bovine serum albumin solution. Weigh 1 mg of DBCO-Sulfo-NHS ester sodium (linker) and dissolve it in 188 μL of DMSO solution to obtain a 10 mM DBCO-Sulfo-NHS ester sodium solution; weigh 1 mg of 5-4TAMRA (mass spectrometry signal compound) and dissolve it in 195.1 μL of DMSO solution to obtain a 10 mM 5-4TAMRA solution.

[0123]

[0124] Take 50 μL of 1 mg / mL bovine serum albumin solution, add 2 μL of 10 mM DBCO-Sulfo-NHS estersodium solution, then add 10 μL of acetonitrile and 1 μL of formic acid, and bring the solution to 100 μL with ultrapure water. React at room temperature for 2 hours.

[0125] Then, 2.4 μL of 10 mM 5-4 TAMRA solution was added to the above reaction solution, and the reaction was carried out at room temperature for 0.5 h. Subsequently, the solution was centrifuged using a 10 kDa ultrafiltration centrifuge tube. Crystal violet was added to the ultrafiltration solution as an internal standard (final concentration of crystal violet was 10 ng / mL) for instrumental analysis.

[0126] The results are as follows Figure 6 As shown in the figure. The results show that the measured peak area is 1.04E7.

[0127] Comparison Example

[0128] 1. Replace other mass spectrometry labels: Replace DBCO-CONH-SS-NHS and 5-4TAMRA in the two-step method of Example 1 with 3-(2-pyridinedithio)propionic acid N-hydroxysuccinimide ester (mass spectrometry label is a complete compound), and keep all other conditions exactly the same.

[0129] The results of the computer test are as follows Figure 7 As shown. The theoretical fragment ion: m / z = 111.01372, but no corresponding fragment ion peak appeared in the spectrum. This may be because the tag is too small, making it difficult to break down at the ion source after reacting with the protein.

[0130] 2. Replace with other connectors: Replace DBCO-CONH-SS-NHS in the two-step method of Example 1 with:

[0131]

[0132] The theoretical fragmentation was the same as in the example, with the labeled protein concentration at 1 mg / mL, and all other conditions were identical. The results were as follows: Figure 8 As shown. Figure 8 The peak area is 7.65E4. This may be due to the short linker strand and the large steric hindrance of the protein, leading to a sharp decrease in labeling efficiency.

[0133] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, alterations, substitutions, or variations made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.

Claims

1. A mass spectrometry tag for labeling proteins, characterized in that: The general structural formula of the mass spectrometry tag is: ; R1 is a mass spectrometry signal molecule, which is a group with mass spectrometry sensitization capability and a molecular weight of 50 to 1000 Da. The structure of R1 is shown in Equation I or Equation II. Formula I: , In Formula I, x is an integer from 1 to 8, and R4, R5 and R6 are short-chain alkanes from C1 to C4, respectively; Formula II: , In Equation II, R7, R8, R9 and R 10 They are either hydrogen or short-chain alkanes of C1-C4, R 11 and R 12 This indicates the presence of one or more substituents, which are hydrogen or short-chain alkanes (C1-C4). R 13 It is either hydroxyl or methoxy; R2 and R3 are linker groups, which are groups that can react with amino acids on the protein to be labeled, and can be any one of hydrogen, N-hydroxysuccinimide ester, sulfonic acid-N-hydroxysuccinimide ester, sulfonyl fluoride, sulfonyl fluoride, and hydroxylamine. When R3 is hydrogen, the structure of R2 is as shown in Formula III or Formula IV: Formula III: R 14 It can be a benzene ring, an alkyl-substituted benzene ring, or a halo-substituted benzene ring; Formula IV: y is an integer from 1 to 15; When R3 is not hydrogen, the structure of R2 can be any one of the following structures: 。 2. A method for labeling proteins using the mass spectrometry tag as described in claim 1, characterized in that: The method includes the following steps: (1) Process the sample containing the protein to be labeled to obtain a protein solution; (2) Dissolve the mass spectrometry tag in the solution to obtain the mass spectrometry tag solution; (3) Add the protein solution to the mass spectrometry tag solution, followed by acetonitrile and formic acid, and react for 1-4 hours.

3. A method for quantitative analysis of proteins, characterized in that: The method includes the following steps: (1) Labeling the protein to be tested using the method described in claim 2; (2) Separate the labeled proteins using chromatography-mass spectrometry; (3) Quantitative analysis of the target protein was performed using the mass spectrometry mode of source cleavage combined with multiple reaction monitoring.

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