Method for quantitatively analyzing complete protein based on mass spectrum label

Abstract

The invention belongs to the field of chemical analysis, and particularly relates to a method for quantitatively analyzing complete protein based on a mass spectrum tag. The specific technical scheme comprises the following steps: marking a to-be-detected protein; separating the marked protein by using a size exclusion chromatography-mass spectrometry technology; carrying out quantitative analysis on the protein to be detected by utilizing a mass spectrum mode of combining in-source cracking with multi-reaction monitoring. According to the invention, high-sensitivity absolute quantification of complete protein from top to bottom is realized on low-cost QqQ-MS through a chemical labeling-in-source cleavage-MRM combination strategy. The method for quantitatively analyzing the protein, provided by the invention, is high in universality and good in instrument compatibility.

Description

Technical Field

[0001] The present invention belongs to the field of chemical analysis, and particularly relates to a method for quantitative analysis of intact proteins based on mass spectrometry tags. Background Art

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

[0003] The limitations of immunological methods include: antibody specificity dependence: antibodies are prone to cross - contamination and batch differences, resulting in false positives / false negatives; narrow dynamic range: the linear range of traditional ELISA usually only involves 2 - 3 orders of magnitude, making it difficult to cover the protein concentration span in clinical samples (for example, the concentration of some biomarkers in serum spans 5 orders of magnitude); weak multiplex detection ability: independent antibodies need to be developed for each target, with high cost and low throughput.

[0004] In contrast, mass spectrometry (MS) is considered an ideal detection method due to its inherent high sensitivity, wide dynamic range, low background interference, and high - throughput detection ability. However, mass spectrometry also has obvious defects: the bottom - up method has problems such as enzyme digestion dependence, uncontrollability in the enzyme digestion process, and multi - step pretreatment (reduction alkylation, desalting) increasing operation complexity and error risk; while the top - down method is limited by the need to use expensive detection equipment and high detection costs.

[0005] Specifically, according to different detection objects, mass spectrometry can be measured based on mass tags or direct methods. ESI and MALDI are two "soft" ionization methods that can detect large protein molecules. For ESI - MS, the presence of proteins with different charge states, charge dilution of proteins with low ionization efficiency, and the formation of protein adducts induced by non - volatile buffer environments usually reduce detection sensitivity and selectivity. When using MALDI - MS to analyze biological macromolecules, special matrices are usually required, and less structural information is generated.

[0006] Triple quadrupole mass spectrometry (QqQ-MS) has excellent sensitivity and specificity and is usually used for quantitative analysis. Compared with traditional immunology, HPLC, colorimetry, etc., QqQ-MS shows greater advantages in terms of accuracy, precision, selectivity, sensitivity, and quantitative dynamic range. In the prior art, the method of using QqQ-MS for protein quantification is limited by the mass range detected by the instrument and requires protein digestion before analysis, that is, the so-called bottom-up method. In this method, the intact proteins in the sample are digested into small peptide fragments by enzymes (such as trypsin); then the component peptides are quantified by QqQ-MS to determine the concentration of intact proteins in the sample. However, the digestion step in this method is affected by many factors, and the digestion efficiency is difficult to ensure 100%, which may bring errors to quantitative analysis.

[0007] To avoid the errors caused by the digestion step, a top-down method can also be adopted, that is, using a high-resolution mass spectrometer to directly analyze intact proteins. However, this method requires the use of more expensive mass spectrometers, such as time-of-flight, orbitrap, and Fourier transform ion cyclotron resonance instruments. However, affected by the protein ionization efficiency, its detection sensitivity does not necessarily meet the requirements.

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

[0009] The object of the present invention is to provide a method for quantitatively analyzing intact proteins based on mass spectrometry tags.

[0010] To achieve the above object of the invention, the technical solution adopted by the present invention is: a mass spectrometry tag for labeling proteins, and the general structural formula of the mass spectrometry tag is:

[0011] Among them, R1 is a mass spectrometry signal molecule, which is a group with mass spectrometry sensitization ability and a molecular weight in the range of 50-1000 Da, and is any one of quaternary ammonium root, pyridyl, quinolinyl, and alkyl-substituted amino; R2 and R3 are linker groups, which are groups capable of reacting with amino acids on the protein to be labeled, and are any one of hydrogen, N-hydroxysuccinimide ester, sulfonic acid group-N-hydroxysuccinimide ester, sulfonyl fluoride, sulfimide fluoride, and hydroxylamine.

[0012] Preferably, the structure of R1 is 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 respectively short-chain alkanes with C1-C4.

[0015] Formula II:

[0016] In Formula II, R7, R8, R9 and R 10 are each hydrogen or a short-chain alkane having 1 to 4 carbon atoms, R 11 and R 12 represent the presence of one or more substituents, which are hydrogen or a short-chain alkane having 1 to 4 carbon atoms, and R 13 is a hydroxyl group or a methoxy group.

[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 is a benzene ring, an alkyl-substituted benzene ring, or a halogenated benzene ring;

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

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

[0022]

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

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

[0025]

[0026] wherein, 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 as shown in Formula V or Formula VI:

[0028] Formula V:

[0029] In Formula V, x is an integer from 1 to 8, and R 15 , R 16 and R 17 are each a short-chain alkane having 1 to 4 carbon atoms;

[0030] Formula VI:

[0031] In Formula VI, R 18 , R 19 , R20 and R 21 are each hydrogen or a C1-C4 short-chain alkane, and R 22 and R 23 represent the presence of one or more substituents, which are hydrogen or a C1-C4 short-chain alkane, and R 24 is a hydroxyl group or a methoxy group.

[0032] Correspondingly, a method for labeling a protein with the mass spectrometry tag, the method comprising the following steps:

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

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

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

[0036] Correspondingly, a method for labeling a protein with the mass spectrometry tag, the method comprising the following steps:

[0037] (1) Treat a 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, then add the mass spectrometry signal compound solution, then add acetonitrile and formic acid, and react for 1-4 h.

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

[0042] Correspondingly, a method for quantitatively analyzing a protein, the method comprising the following steps:

[0043] (1) Label the protein to be measured using the method;

[0044] (2) Separate the labeled protein using chromatography-mass spectrometry coupling technology;

[0045] (3) Perform quantitative analysis on the protein to be measured using the mass spectrometry mode of in-source fragmentation combined with multiple reaction monitoring.

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

[0047] In-Source Dissociation (ISD) refers to the fragmentation that occurs during the initial ionization of an analyte within the ion source. For example, when analyzing intact antibodies using the ISD mode of MALDI-TOF, semi-quantification can be achieved by detecting the Fc fragment (m / z = 25 kDa); or by applying a high voltage (>6000 V) within the ESI source to cleave intact proteins into peptide fragments, followed by quantification using QqQ-MS. This method has problems such as uncontrollable cleavage sites and poor reproducibility of fragments, and lacks specific marker ions, making it difficult to distinguish homologous proteins. If direct quantification analysis of intact proteins is based on QqQ-MS, for example, separating intact proteins using reverse-phase chromatography (RP-LC), proteins with a molecular weight >50 kDa exceed the mass-to-charge ratio detection range (m / z < 4000) of QqQ-MS.

[0048] Therefore, the present invention realizes high-sensitivity absolute quantification of intact proteins from top to bottom on a low-cost QqQ-MS through a combined strategy of chemical labeling - in-source dissociation - MRM. Specifically:

[0049] Using bottom-up protein analysis involves complex, cumbersome, and time-consuming digestion operations, which can easily lead to inaccurate quantification results. Therefore, the present invention adopts a top-down quantification method to avoid the digestion step. However, if intact proteins are directly used for mass spectrometry analysis, the molecular weight of the proteins often exceeds the detection range of the mass spectrometer, and the ionization efficiency is relatively low, making it difficult to detect low-concentration proteins. Therefore, the present invention labels the proteins, introducing mass spectrometry tags with higher ionization efficiency onto the proteins. The introduced mass spectrometry tags can easily undergo directional cleavage during in-source dissociation, combined with the mass spectrometry detection method of multiple reaction monitoring (MRM), to achieve absolute quantification analysis of labeled proteins from top to bottom.

[0050] Moreover, the present invention has made a targeted selection of the mass spectrometry tags. By chemically labeling small-molecule mass spectrometry tags covalently to proteins, the high ionization efficiency of the tags is used to enhance the signal. In the prior art, techniques for signal enhancement based on mass spectrometry tags rely on high-resolution mass spectrometry (such as Orbitrap) to analyze complex fragment spectra and cannot be adapted to low-cost QqQ-MS. In the present invention, the synergistic effect of bifunctional groups is utilized: the R1 group contains highly ionizable groups such as quaternary ammonium and pyridyl groups, which can improve the mass spectrometry response; the R2 group uses highly efficient reactive groups such as N-hydroxysuccinimide ester (NHS) and sulfonimidoyl fluoride, which can improve the labeling efficiency; in addition, the special linker chemical chain can easily undergo directional cleavage during in-source dissociation, facilitating subsequent mass spectrometry detection.

[0051] In the method of combining in-source cleavage with MRM, the present invention precisely controls the cleavage parameters. By optimizing the DP voltage and CE, the mass spectrometry tags are directionally cleaved during the ionization process to generate stable fragment ions. A specific MRM channel is used to only monitor the characteristic daughter ion (m / z = 386.2), avoiding complex matrix interference (the signal-to-noise ratio is increased by 20 times compared with the traditional full-scan mode).

[0052] The present invention also optimizes the adaptability of QqQ-MS. It includes: innovation in chromatographic separation: using SEC chromatography to replace traditional reverse-phase chromatography to avoid irreversible adsorption of macromolecular proteins on the reverse-phase column.

[0053] The method for quantitatively analyzing proteins provided by the present invention has strong universality (it can cover secreted proteins (such as BSA), membrane proteins (such as CD20), and antibodies (such as IgG), etc.), good instrument compatibility (it can be adapted to multi-brand QqQ-MS such as Agilent 6495 and Sciex QTRAP 6500+ without hardware modification of the equipment), and supports the quantification of proteins in complex matrices such as serum and tissue lysates, providing reliable data support for subsequent research. Description of the Drawings

[0054] Figure 1 It is a schematic diagram of the binding process of proteins and mass spectrometry tags;

[0055] Figure 2 It is a schematic diagram of the binding process of proteins and mass spectrometry tags in Example 1;

[0056] Figure 3 It is a schematic diagram of the detection result of bovine serum albumin by the two-step method in Example 1;

[0057] Figure 4 It is a schematic diagram of the detection result of the SEC-MS calibration curve working solution in the two-step method of Example 1;

[0058] Figure 5 It is a schematic diagram of the detection result of bovine serum albumin by the one-step method in Example 1;

[0059] Figure 6 It is a schematic diagram of the result of bovine serum albumin in Example 3;

[0060] Figure 7 It is a schematic diagram of the detection result after replacing the mass spectrometry tag in the control example;

[0061] Figure 8 It is a schematic diagram of the detection result after replacing the linker in the control example. Detailed Embodiments

[0062] The present invention provides a method for quantitatively analyzing intact proteins. The principle is as follows: After the amino acids on the protein bind to the linker, a click chemical reaction occurs with the mass spectrometry signal molecule, thereby binding a mass spectrometry tag to the protein. Subsequently, a liquid chromatography-triple quadrupole tandem mass spectrometer is used to separate and quantitatively analyze the analyte. The mass spectrometry acquisition mode is in-source fragmentation combined with multiple reaction monitoring mode (MRM). The mass spectrometry tag on the protein undergoes in-source fragmentation at the ion source, generating a specific fragment ion with m / z = 718.3, and characteristic fragments with m / z = 386.2 generated by the collision-induced fragmentation of this fragment ion, to achieve quantitative analysis. Among them, the method for labeling proteins (binding mass spectrometry tags to proteins) includes a "one-step method" and a "two-step method", and the process is as Figure 1 shown. The "one-step method" means that the mass spectrometry tag is an integral compound, and both the mass spectrometry signal molecule and the linker are groups on the mass spectrometry tag, and the protein reacts with the mass spectrometry signal molecule and the linker simultaneously; the "two-step method" means that the mass spectrometry tag includes two independent compounds, namely a mass spectrometry signal compound and a linker, and the protein first reacts with the linker, 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) Bind the mass spectrometry tag to the protein to be measured by chemical labeling method; specifically: Treat the sample containing the protein to be labeled to obtain a protein solution to be measured. Dissolve the mass spectrometry tag in DMSO solution to obtain a mass spectrometry tag solution. Add the protein solution to be measured to the mass spectrometry tag solution, and control the molar ratio of the protein to be measured to the linker to be 1:5 - 1:100. Subsequently, add acetonitrile and formic acid, and control the acetonitrile concentration in the reaction system to be 10% and the formic acid concentration to be 1%. React at 4°C - 50°C for 1 - 4 h; preferably react at room temperature for 2 h. After the reaction is completed, centrifuge. Add crystal violet as an internal standard solution to the centrifuged solution.

[0065] (2) Separate the labeled protein by size exclusion chromatography-mass spectrometry (SEC-MS);

[0066] (3) Perform quantitative analysis using the mass spectrometry mode of in-source fragmentation combined with multiple reaction monitoring (MRM), specifically achieved through the following ion signals: The specific fragment ion generated by in-source fragmentation (m / z = 718.3); The characteristic ion generated by the collision-induced dissociation of this fragment ion (m / z = 386.2).

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

[0068] Among them, R1 is a mass spectrometry signal molecule, which is a group with mass spectrometry sensitization ability and a molecular weight in the range of 50 - 1000 Da, and is any one of quaternary ammonium group, pyridyl group, quinolinyl group, and alkyl-substituted amino group; R2 and R3 are linker groups, which are groups capable of reacting with amino acids on the protein to be labeled, and are any one of hydrogen, N-hydroxysuccinimide ester, sulfonic acid group-N-hydroxysuccinimide ester, sulfonyl fluoride, sulfimide fluoride, and hydroxylamine.

[0069] Preferably, the structure of R1 is shown in Formula I or Formula II:

[0070] Formula I:

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

[0072] Formula II:

[0073] In Formula II, R7, R8, R9, and R 10 are respectively hydrogen or short-chain alkanes with C1 - C4, R 11 and R 12 represent the presence of one or more substituents, which are hydrogen or short-chain alkanes with C1 - C4, and R 13 is hydroxyl or methoxy.

[0074] Preferably, when R3 is hydrogen, that is, when the click chemical reaction of azide cycloalkyne addition occurs, the structure of R2 is shown in Formula III or Formula IV:

[0075] Formula III: R 14 is a benzene ring, an alkyl-substituted benzene ring, or a halogenated 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 group structure is any one of the following structures:

[0078]

[0079] That is: if the linker structure contains octyne, the SPAAC reaction occurs, and at this time, R2 and R3 form a ring with octyne; if the linker structure is a straight-chain alkyne, the azide reaction occurs, and it is not a ring-forming reaction, then only R2 connects the corresponding group.

[0080] More preferably, the R1 group is selected from any one of the following groups:

[0081]

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

[0083] (1) Binding the mass spectrometry tag to the protein to be measured by chemical labeling method; specifically: treating the sample containing the protein to be labeled to obtain a solution of the protein to be measured. Dissolve the linker in DMSO solution to obtain a linker solution. Dissolve the mass spectrometry signal compound in DMSO solution to obtain a mass spectrometry signal compound solution. Add the protein solution to be measured to the linker solution, and control the molar ratio of the protein to be measured to the linker to be 1:5 - 1:100. Then add acetonitrile and formic acid, and control the acetonitrile concentration in the reaction system to be 10% and the formic acid concentration to be 1%. React at 4°C - 50°C for 1 - 4 h; preferably react at room temperature for 2 h. Then add the mass spectrometry signal compound solution and react at room temperature for 0.5 h. After the reaction, centrifuge. Add crystal violet as an internal standard solution to the centrifuged solution.

[0084] (2) Separating the labeled protein by size exclusion chromatography-mass spectrometry (SEC-MS);

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

[0086] Among them, the general structural formula of the mass spectrometry signal compound is shown as Formula V or Formula VI:

[0087] Formula V:

[0088] In Formula V, x is any integer from 1 to 8, R 15 , R 16 and R 17 are respectively short-chain alkanes with C1 - C4;

[0089] Formula VI:

[0090] In Formula VI, R 18 , R 19 , R 20 and R 21 are respectively hydrogen or short-chain alkanes with C1 - C4, R 22 and R 23 represent the existence of one or more substituents, which are hydrogen or short-chain alkanes with C1 - C4, and R 24 is a hydroxyl group or a methoxy group.

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

[0092]

[0093] Preferably, the linker is selected from the following compounds:

[0094]

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

[0096] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. If not specifically specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. The obtained data are all averages obtained after at least 3 repetitions, and all repetitions obtained are valid data.

[0097] Example 1

[0098] The reaction principle of this example is as Figure 2 shown: After the lysine on the protein reacts with DBCO-CONH-SS-NHS, it then undergoes a strain-promoted alkyne-azide cycloaddition reaction (SPAAC) with 5-carboxytetramethylrhodamine azide with an azide group attached, thereby constructing a mass spectrometry tag on the protein to be analyzed. Bovine serum albumin (BSA) is used for relevant tests in each example. Because the molecular weight of BSA is clear (about 66 kDa) and its structure is stable; moreover, BSA is often added as a standard to serum or tissue lysates, and its experimental data can directly reflect the tolerance of the method to complex biological matrices.

[0099] 1. Two-step method

[0100] (1) Protein labeling

[0101] Weigh 1 mg of bovine serum albumin and dissolve it in 10 mL of ultrapure water to obtain a 0.1 mg / mL bovine serum albumin solution. 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.

[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 make up 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 h. Subsequently, centrifuge with a 10 kDa ultrafiltration centrifugal tube. Add crystal violet as the internal standard solution (the final concentration of crystal violet is 10 ng / mL) to the ultrafiltered solution for machine detection.

[0104] (2) Machine detection

[0105] Liquid phase conditions: The chromatographic column is ACQUITY Protein BEH SEC Column, 1.7 μm, 4.6×150 mm; the flow rate is 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: 5500 V; ion source temperature (TEM): 450 °C; ion source nebulizing gas (GS1): 50 psi; ion source heating auxiliary gas (GS2): 50 psi; curtain gas (CUR): 35 psi; collision gas (CAD): 7 psi; scan mode: MRM; The MRM parameter conditions are set as 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 tag 718.3 386.2 280 55 Internal standard 372.0 356.0 260 45

[0109] Detect the labeled bovine serum albumin standard solution by in-source fragmentation combined with multiple reaction monitoring (MRM). The detection results are as Figure 3 shown. The results show that the mass spectrometry tag on bovine serum albumin was successfully fragmented by in-source fragmentation and detected by multiple reaction monitoring (MRM). The retention time is 3.03 min.

[0110] Use DBCO-CONH-SS-NHS and 5-4TAMRA as labeling reagents and detect the standard curve working solution by SEC-MS. Perform linear fitting with the concentration of BSA as the abscissa and the peak area ratio of BSA to the internal standard as the ordinate. The results are as Figure 4As shown, in the range of bovine serum albumin concentration from 1 μg / mL to 32 μg / mL, the linear correlation coefficient is greater than 0.99, 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 a mass spectrometry tag:

[0113]

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

[0115] The results are as Figure 5 shown. 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, and the two peak areas are close. This indicates that both the one-step method and the two-step method can successfully label proteins.

[0116] Example 2

[0117] Use the two-step method of Example 1 to measure different concentrations of proteins respectively:

[0118] 1. Prepare a BSA solution with a concentration of 2 μg / mL, and use the mass spectrometry tag method described in Example 1 to detect this BSA solution. The measured concentration of the BSA solution is 2.01 μg / mL.

[0119] 2. Prepare a BSA solution with a concentration of 32 μg / mL, and use the mass spectrometry tag method described in Example 1 to detect this BSA solution. The measured concentration of the BSA solution is 31.6 μg / mL.

[0120] The results show that the method of the present invention can accurately quantify proteins with 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), 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), 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 ester sodium solution, then add 10 μL of acetonitrile and 1 μL of formic acid, and make up the solution to 100 μL with ultrapure water. React at room temperature for 2 hours.

[0125] Then add 2.4 μL of 10 mM 5-4TAMRA solution to the above reaction solution, and react at room temperature for 0.5 h. Then centrifuge with a 10 kDa ultrafiltration centrifugal tube. Add crystal violet as an internal standard solution (the final concentration of crystal violet is 10 ng / mL) to the ultrafiltered solution for on-machine detection.

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

[0127] Control example

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

[0129] The on-machine results are as Figure 7 shown. Theoretical fragment ion: m / z = 111.01372, but the corresponding fragment ion peak does not appear in the spectrum. This may be because the tag is relatively small and it is difficult to break at the ion source after reacting with the protein.

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

[0131]

[0132] The theoretical fragments are the same as those in the example, the labeled protein concentration is 1 mg / mL, and the other conditions are exactly the same. The on-machine results are asFigure 8 as shown Figure 8 It shows that the peak area is 7.65E4. This may be due to the relatively short linker chain and the large steric hindrance of the protein, resulting in a sharp decline in the labeling efficiency.

[0133] The embodiments described above are only descriptions of the preferred modes of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations, variations, modifications, and substitutions made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined 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 as follows: Among them, R1 is a mass spectrometry signal molecule, which is a group with mass spectrometry sensitization ability and a molecular weight in the range of 50 - 1000 Da, and is any one of quaternary ammonium, pyridyl, quinoline, and alkyl-substituted amino groups; R2 and R3 are linker groups, which are groups capable of reacting with amino acids on the protein to be labeled, and are any one of hydrogen, N-hydroxysuccinimide ester, sulfonic acid group-N-hydroxysuccinimide ester, sulfonyl fluoride, sulfimide fluoride, and hydroxylamine.

2. The mass spectrometry tag according to claim 1, characterized in that: The structure of R1 is shown as Formula I or Formula II: Formula Ⅰ: In Formula I, x is an integer from 1 to 8, and R4, R5, and R6 are respectively short-chain alkanes with C1 - C4. Formula II: In formula II, R7, R8, R9 and R 10 are each independently hydrogen or a short-chain alkane having 1 to 4 carbon atoms, R 11 and R 12 represent the presence of one or more substituents, which are hydrogen or a short-chain alkane having 1 to 4 carbon atoms, and R 13 is a hydroxyl group or a methoxy group.

3. The mass spectrometry tag according to claim 1, characterized in that: When R3 is hydrogen, the structure of R2 is shown as Formula III or Formula IV: Formula Ⅲ: In formula III, R 14 is any one of a benzene ring, an alkyl-substituted benzene ring, and a halogenated benzene ring; Formula IV: y is an integer from 1 to 15.

4. The mass spectrometry tag according to claim 1, wherein: When R3 is not hydrogen, the structure of R2 is any one of the following structures: Among them, m is any integer from 1 to 15, and n is any integer from 1 to 100.

5. A mass spectrometry tag for labeling proteins, characterized in that: The mass spectrometry tag includes a linker and a mass spectrometry signal compound, and the linker is any one of the following compounds: Among them, y and m are respectively any integers from 1 to 15, and n is any integer from 1 to 100; The structural formula of the mass spectrometry signal compound is shown as Formula V or Formula VI: Formula V: In formula V, x is any integer from 1 to 8, and R 15 , R 16 and R 17 are each a short-chain alkane having 1 to 4 carbon atoms; Formula VI: In formula VI, R 18 , R 19 , R 20 and R 21 are each hydrogen or a short-chain alkane having 1 to 4 carbon atoms, R 22 and R 23 represent the presence of one or more substituents, which are hydrogen or a short-chain alkane having 1 to 4 carbon atoms, and R 24 is a hydroxyl group or a methoxy group.

6. A method for labeling proteins with the mass spectrometry tag according to any one of claims 1 to 4, characterized in that: The method includes the following steps: (1) Treat the sample containing the protein to be labeled to obtain a protein solution; (2) Dissolve the mass spectrometry tag in a solution to obtain a mass spectrometry tag solution; (3) Add the protein solution to the mass spectrometry tag solution, then add acetonitrile and formic acid, and react for 1 - 4 h.

7. A method for labeling proteins using the mass spectrometry tag described in claim 5, characterized in that: The method includes the following steps: (1) Treat the sample containing the protein to be labeled to obtain a protein solution; (2) Dissolve the mass spectrometry signal compound in a solution to obtain a mass spectrometry signal compound solution; (3) Dissolve the linker in a solution to obtain a linker solution; (4) Add the protein solution to be labeled to the linker solution, after the two react, then add the mass spectrometry signal compound solution, and then add acetonitrile and formic acid, and react for 1 - 4 h.

8. The method according to claim 7, characterized in that: The molar ratio of the protein to be labeled to the linker is 1:5 - 1:

100.

9. The method according to claim 7, wherein: The reaction temperature is: 4 °C - 50 °C.

10. A method for quantitatively analyzing proteins, characterized in that: The method includes the following steps: (1) Label the protein to be detected using the method according to any one of claims 6 - 9; (2) Separate the labeled protein using chromatograph-mass spectrometry coupling technology; (3) Perform quantitative analysis on the protein to be detected using the mass spectrometry mode of in-source fragmentation combined with multiple reaction monitoring.

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