CN114740121A - Double-amino-acid stable isotope labeling method based on primary mass spectrometry and application - Google Patents
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Abstract
The invention discloses a double-amino-acid stable isotope labeling method based on primary mass spectrometry, which comprises the following steps: uniformly mixing at least two proteins with different molecular weights to prepare a protein sample, sequentially adding a protein reduction reaction solution and a protein reducing agent into the protein sample, and uniformly mixing and stirring for later use; adding acrylamide to mark cysteine into the reduced protein sample solution, and then adding succinic anhydride to mark lysine; passing the protein solution labeled by the diamino acid through a desalting column to remove unreacted reagents; and (4) carrying out enzymolysis, and carrying out chromatography-mass spectrometry on enzymolysis products. The beneficial effects of the invention are as follows: the double-amino acid labeling method improves the yield and efficiency of protein quantitative analysis by labeling two different amino acids, overcomes the defects of low yield and efficiency of the quantitative analysis of the existing single-amino acid stable isotope labeling method, and can be used for the quantitative analysis of the protein in a complex biological sample.
Description
Technical Field
The invention belongs to the technical field of isotope labeling methods, and particularly relates to a double-amino-acid stable isotope labeling method based on primary mass spectrometry.
Background
The stable isotope labeling technology combined with mass spectrum detection has become a quantitative analysis method for protein in complex biological samples. The existing stable isotope labeling technology mainly includes labeling specific amino acids (e.g., lysine) in proteins or protein enzymatic hydrolysis products (i.e., peptide fragments), and then performing Data Dependent Acquisition (DDA) mass spectrometry on the molecules of the peptide fragments of the protein enzymatic hydrolysis products, that is, selecting parent ion peptide fragment molecules (e.g., m/z 537.827, +2) with a certain mass-to-charge ratio (m/z) in a primary Mass Spectrometry (MS) analysis, and entering a high-energy collision chamber filled with high-purity (> 99.999%) inert gas through an ion isolation window (isolation window) with a certain mass range (e.g., ± 0.6Da) to perform ion fragmentation (fragmentation) so as to obtain a secondary mass spectrometry (MS/MS) analysis map (i.e., an daughter ion map). The aim of quantitative analysis of protein is achieved by detecting ion ions or report ions (reporters) with isotope labels in the secondary mass spectrum. For example, TMT (TMT) developed by Thermo Scientific and iTRAQ (International tags for relative and absolute qualification, iTRAQ) marking technologies developed by AB SCIEX. The two labeling technologies can realize quantitative analysis of proteins in various samples by measuring the peak area of an isotope labeled reporter ion in a secondary mass spectrum.
However, the main problems with these quantitative analysis techniques are:
in addition to the selected parent ion peptide fragment molecule, other peptide fragment parent ions (e.g., + -0.6 Da) in an ion isolation window (isolation window) are simultaneously selected and subjected to secondary mass spectrometry in the primary Mass Spectrum (MS), so that the accurate detection of the secondary mass spectrometry report ions generates irreparable interference.
Secondly, the reported ion peak areas generated by the mixture of two or more peptide fragment parent ions in the ion isolation window (isolation window) selected in the primary mass spectrometry in the secondary mass spectrometry are from the superposition of the reported ion peak areas of the peptide fragment parent ions, the reported ion peak area of the appointed parent ion in the primary mass spectrometry cannot be accurately measured, and the accurate quantitative analysis of the protein represented by the parent ion peptide fragment cannot be carried out.
With the wide application of high-resolution mass spectrometry, a stable isotope labeling technology based on primary mass spectrometry can obtain a relatively accurate quantitative analysis result. However, the prior art has greatly limited the widespread use of this technique due to the limited production of peptide fragment parent ions that can be quantitatively analyzed by labeling only one amino acid (e.g., cysteine) at a time. Therefore, the development of a stable isotope labeling technique that can label more than one amino acid at the same time is significant for improving the efficiency of quantitative analysis.
Disclosure of Invention
The main object of the present application is to provide a method for stably labeling a double amino acid based on primary mass spectrometry, which can improve the yield and efficiency of protein quantitative analysis by stably labeling two different amino acids with isotopes.
In order to achieve the above purpose, the invention provides the following technical scheme:
a double-amino-acid stable isotope labeling method based on primary mass spectrometry comprises the following steps:
(1) uniformly mixing at least two proteins with different molecular weights to prepare a protein sample, sequentially adding a protein reduction reaction solution and a protein reducing agent into the protein sample, and uniformly mixing and stirring for later use;
(2) adding acrylamide to mark cysteine into the reduced protein sample solution obtained in the step (1), and then adding succinic anhydride to mark lysine;
(3) removing unreacted reagents from the protein solution labeled by the diamino acid obtained in the step (2) through a desalting column;
(4) adding Trypsin-Trypsin into the protein sample marked by the diamino acid obtained in the step (3) for enzymolysis; and (3) carrying out chromatographic-mass spectrometric analysis on the enzymolysis products.
In the above method for labeling a stable isotope of a diamino acid based on primary mass spectrometry, as a preferred embodiment, in the step (1), the protein reduction reaction solution is an aqueous solution containing 4M urea, 20mM Tris-HCl and 3% isopropanol; the dosage ratio of the protein sample to the protein reduction reaction solution is 1 mg: 1 ml.
In the above method for labeling a double-amino-acid stable isotope based on primary mass spectrometry, as a preferred embodiment, in step (1), the protein reducing agent is an aqueous solution containing 500mM TCEP, and the ratio of the amount of the protein sample to the amount of the protein reducing agent is 1 mg: 50 ul; the mixing and stirring temperature is 37 deg.C, and the mixing and stirring time is 30 min.
TCEP is tris (2-carboxyethyl) phosphine, is a very effective thiol reducing agent, and is widely used as a quantitative reducing agent for disulfide bonds in protein chemistry and proteomics research. The reagent has good stability and solubility in aqueous solution. The stability in acid and alkaline solution is good. TCEP is not only a highly efficient disulfide bond reducing agent, but also does not require removal in some thiol crosslinking reactions, as compared to other classes of thiol reducing agents such as DTT. TCEP opens the intramolecular or intermolecular disulfide bond formed between cysteines in a protein to form-SH, which can then be subjected to an acrylation labeling reaction with acrylamide.
In the above method for labeling a diamino acid stable isotope based on primary mass spectrometry, as a preferred embodiment, in step (2), acrylamide is added in an amount 5-7 times that of the protein sample, and labeled cysteine is: uniformly mixing the reduced protein sample solution with acrylamide, and reacting for 30min in a dark place at the temperature of 20-25 ℃;
the added succinic anhydride amount is 7-10 times of that of the protein sample, and the labeled lysine is as follows: adding succinic anhydride, mixing, and reacting at 20-25 deg.C under dark condition for 30 min.
Acrylamide (C)3H5ON) and thiol (-SH) groups ON cysteine side chains in the reduced protein peptide chain to form acryloyl groups, i.e. the thiol (-SH) groups ON each cysteine side chain are combined with 71.0366Da acrylamide groups (S-C)3H4ON)。
Succinic anhydride (C)4H4O3) With primary amines (-NH) on lysine side chains in protein peptide chains2) A covalent reaction takes place to form a succinylated group, i.e. a primary amine (-NH) on each lysine side chain2) Incorporating succinamide groups (NH-C) of 100.0160Da4H4O3)。
As a preferred embodiment of the method for labeling a stable isotope of a diamino acid based on primary mass spectrometry, the displacement solution passing through the desalting column in the step (3) is 50mM NH4HCO3. The desalting column (Zeba Spin DesaltingColumns, Thermoscientific) removed the remaining unreacted labeling reagents acrylamide, succinic anhydride and TCEP.
As a preferred embodiment, in the step (4), the enzymolysis is performed at 37 ℃ for 5 hours, and then formic acid is added to terminate the enzymolysis.
In a second aspect of the present application, there is provided an application of a two-amino acid stable isotope labeling method based on primary mass spectrometry in quantitative proteomics analysis of serum.
The invention has the beneficial effects that: the double-amino-acid stable isotope labeling method based on the primary mass spectrometry improves the yield and efficiency of protein quantitative analysis by performing stable isotope labeling on two different amino acids, overcomes the defects of low yield and efficiency of the quantitative analysis of the existing single-amino-acid stable isotope labeling method, and can be used for the quantitative analysis of proteins in complex biological samples (such as serum samples).
The primary mass spectrometry-based double-amino-acid stable isotope labeling method improves the protein identification and quantitative analysis flux (protein number) and coverage (more low-concentration proteins are detected) of complex biological samples (such as blood samples and tissue samples), so that the invention can realize the discovery of protein biomarkers related to diseases from complex biological samples including blood, urine and tissue samples through quantitative white matter omics analysis.
Drawings
FIG. 1 is a flow chart of quantitative analysis of protein by the method of stable isotope labeling with two amino acids based on primary mass spectrometry according to the present invention;
FIG. 2 shows the first-order mass spectrometry-based two-amino acid stable isotope labeled Bovine Serum Albumin (BSA) with different molar ratios12C:13C) Carrying out quantitative analysis;
FIG. 3 shows the two amino acid stable isotope labeled transferrin (transferrin) with different molar ratios based on primary mass spectrometry12C:13C) And (4) carrying out quantitative analysis.
Detailed Description
In order to make the technical solutions in the embodiments of the present application better understood, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to examples, and it is obvious that the described embodiments are only some embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
The efficiency of the amino acid labeling reaction (such as acrylamide labeling of cysteine and succinyl labeling of lysine) is related to the micro-environment spatial structure of the protein polypeptide chain in which the labeled functional group (such as-SH of cysteine side chain and-NH 2 of lysine side chain) is positioned on the amino acid side chain, and a single protein cannot represent the complex micro-environment spatial structure of the protein in the biological sample. Therefore, the invention uses a mixture of several proteins as a sample to be marked so as to better represent the complex microenvironment space structure state of the proteins.
Example 1
The method for labeling the stable isotope of the double amino acids based on the primary mass spectrometry comprises the following steps:
(1) 0.25mg each of transferrin, fetuin, bovine serum albumin and α -acid glycoprotein was weighed and mixed uniformly to obtain 1mg of a protein mixed sample, 1ml of a protein reduction reaction solution (the protein reduction reaction solution was an aqueous solution containing 4M urea, 20mM Tris-HCl and 3% isopropanol) was added to the protein mixed sample to prepare a protein sample solution, 50ul and 500mM of a protein reducing agent (the protein reducing agent was an aqueous solution containing 500mM TCEP) was added to the protein sample solution, and the mixture was mixed and stirred at 37 ℃ for 30 minutes.
(2) Adding 7mg of acrylamide into the reduced protein sample solution, mixing and stirring uniformly, carrying out a light-shielding reaction at the temperature of 20-25 ℃ for 30min, and marking cysteine; then adding 10mg succinic anhydride, mixing uniformly, and reacting for 30min in the dark at 20-25 ℃ to mark lysine.
(3) Passing the solution of the diamino acid-labeled protein obtained in step (2) through a Desalting column (Zeba Spin desaling Columns, Thermoscientific) to remove unreacted reagents (acrylamide, succinic anhydride and TCEP) in a 50mM NH-substituted solution4HCO3。
(4) And (4) adding 20ug (excessive) of Trypsin-Trypsin into the protein sample labeled by the diamino acid obtained in the step (3), uniformly mixing, carrying out enzymolysis for 5 hours at 37 ℃, then adding 20ul of 10% formic acid to stop the enzymolysis reaction, carrying out chromatography-mass spectrometry on the enzymolysis product, and then completing the identification and quantitative analysis of the protein through a data analysis system.
1. Effect of using different masses of labeling reagents on efficiency of double amino acid labeling:
the test method comprises the following steps:
according to the method for labeling the stable isotope of the diamino acid described in example 1, five kinds of labeling reagents with different mass were used to perform labeling reaction with 1mg of protein sample, peak areas of labeled peptide fragments were detected by chromatography-mass spectrometry, and the labeling reaction efficiency was calculated by comparing the peak areas of the labeled peptide fragments with the peak areas of the unlabeled peptide fragments, and the results of the labeling efficiency of the diamino acid are shown in tables 1 and 2, respectively.
TABLE 1 reaction efficiency of acrylamide and cysteine labeling
As can be seen from table 1: when the amount of acrylamide reaches 5mg, the labeling efficiency of the mixed sample of acrylamide and four proteins reaches 100%, and 7mg of acrylamide is used for labeling cysteine in the labeling reaction of the actual biological sample in order to ensure high labeling efficiency.
TABLE 2 succinic anhydride efficiency of reaction with lysine labeling
As can be seen from table 2: when the amount of succinic anhydride used reached 10mg, the labeling efficiency of lysine in the sample mixed with four proteins reached 100%, and thus 10mg of succinic anhydride was used to label lysine in the labeling reaction of the actual biological sample.
2. Quantitative analysis of proteins by the method of stable isotope labeling with diamino acids as described herein
The invention uses non-isotopically labeled acrylamide (A)12C3H5ON，Light/12C3) And isotopically labeled acrylamides (13C3H5ON，Heavy/13C3) Separately labeling cysteine in two protein samples, using non-isotopically labeled succinic anhydride (A)12C4H4O3，Light/12C4) And isotopically labeled succinic anhydride(s) (ii)13C4H4O3，Heavy/13C4) Respective labelThe lysine in the two protein samples is recorded by comparing the non-isotopically labeled peak intensity with the isotopically labeled peak intensity (i.e., Light/Heavy or both) containing the cysteine peptide segment or the lysine peptide segment or both12C/13C) And quantitative analysis of the protein in the two samples is realized, and the technical flow is shown in figure 1.
2.1 test methods
Preparation of protein samples: the protein sample consists of transferrin and bovine serum albumin, the protein sample is divided into 3 groups, and the mol ratio of the transferrin in the 3 groups is respectively 10:1,1: 1,1: 10; the molar ratio of bovine serum albumin in group 3 was 10:1,1: 1,1: 10.
the 3 groups of protein samples with different molar ratios were respectively non-isotopically labeled and isotopically labeled according to the method for stable isotopic labeling of double amino acids described in example 1.
For example: for a molar ratio of 10:1, (molar ratio: sample-1/sample-2: 10:1) using non-isotopically labeled acrylamide (a), (b), (c), (d) and d) 2) and (d) and (d) using non-1) a. a (d) a (d) and (d) a. and (d), (d) a) and (d) a. and (d) a. and (d), (d) a. and (d) a b) a. and (d) a. and (e12C3H5ON，Light/12C) And succinic anhydride (b)12C4H4O3，Light/12C) Respectively marking cysteine and lysine in the sample-1; using isotopically labelled acrylamides (13C3H5ON，Heavy/13C) And succinic anhydride (a)13C4H4O3，Heavy/13C) Cysteine and lysine in sample-2 were labeled separately. Mixing the marked sample-1 and the sample-2, performing Trypsin (Trypsin) enzymolysis, and performing amino acid sequencing and peptide peak intensity calculation on the peptide fragment by a protein enzymolysis product (namely the peptide fragment) through chromatography-mass spectrometry detection and data analysis, wherein the results are shown in a figure 2 and a figure 3.
As can be seen from fig. 2 and 3: the correlation coefficient R2 of the protein quantitative analysis between 3 samples with different molar ratios (namely 10:1, 1:1 and 1:10) and the coefficient of variation CV (CV) of less than or equal to 14 percent prove that the double-amino-acid labeling quantitative analysis technology provided by the invention has good reliability and accuracy.
3. The double-amino-acid stable isotope labeling method disclosed by the invention is applied to serum quantitative proteomics analysis.
3.1 the double-amino-acid stable isotope labeling method is applied to serum quantitative proteomics analysis and is compared and analyzed with a single-amino-acid labeling method. The main comparative analysis experimental procedures were as follows:
first, two 15. mu.L serum samples (1mg protein per serum) were taken and labeled with the diamino acid of the present invention (i.e., acrylamide-labeled cysteine and succinic anhydride-labeled lysine) and the conventional single amino acid (i.e., acrylamide-labeled cysteine), respectively, and the labeling reaction was carried out according to the diamino acid labeling method described in example 1.
② adding 20 μ g Trypsin (Trypsin) into the two marked serum samples respectively for enzymolysis reaction, and reacting for 5 hours at 37 ℃. Then 20. mu.L of 10% formic acid was added to stop the enzymatic reaction, and the enzymatic product was obtained.
Thirdly, performing chromatography-mass spectrometry (LC-MS/MS) detection and data analysis on the enzymolysis products to obtain a protein identification analysis result and a protein quantitative analysis result, as shown in Table 3:
TABLE 3 application of double-amino-acid stable isotope labeling method in serum quantitative proteomics analysis
From table 3, it can be seen that, compared with the conventional single amino acid labeling technique, the double-amino acid stable isotope labeling method provided by the invention is higher than the single amino acid labeling method in both the protein identification number and the protein quantitative number. While 94% of the identified proteins in the two-amino acid labeling method can obtain quantitative information, the single-amino acid labeling technique is only 63%. In addition, 3654 secondary mass spectra are generated in the single amino acid labeling method compared with the double amino acid labeling method, which indicates that the peptide segment generated by the trypsin enzymolysis reaction of the serum protein sample in the double amino acid labeling method is less than that in the single amino acid labeling method. Specifically cutting the C-terminal of lysine (Lys/K) and arginine (Arg/R) in the protein by trypsin, marking cysteine by using acrylamide through single amino acid marking, and not preventing the trypsin specific cutting reaction of the lysine (Lys/K) and arginine (Arg/R) in the protein to generate a peptide segment of which the C-terminal comprises the lysine (Lys/K) or the arginine (Arg/R); on the other hand, lysine (Lys/K) in the diamino acid label cannot be subjected to the enzyme digestion reaction of trypsin due to the labeling by succinic anhydride, and only a peptide fragment containing arginine (Arg/R) at the C-terminal is generated.
The protein identified and quantified from the serum sample is subjected to comparative analysis of the number of second-stage mass spectrums, and the results are shown in table 4:
TABLE 4 comparison of protein quantitation depth by double-amino-acid stable isotope labeling method and single-amino-acid labeling method
Sixthly, as can be seen from table 4: compared with the conventional single amino acid labeling method, the double amino acid stable isotope labeling method provided by the invention has more secondary mass spectrum numbers for identifying and quantitatively analyzing low-concentration protein (for example, 500pg/mL), and the single amino acid labeling method has more secondary mass spectrum numbers for identifying and quantitatively analyzing high-concentration protein (for example, >1.0 mg/mL). Therefore, the two-amino acid marker provided by the invention provides a more effective analysis technology for identifying and quantifying low-concentration protein in serum.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for a person skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be considered as the protection scope of the present invention.
Claims (7)
1. A double-amino-acid stable isotope labeling method based on primary mass spectrometry is characterized by comprising the following steps:
(1) uniformly mixing at least two proteins with different molecular weights to prepare a protein sample, sequentially adding a protein reduction reaction solution and a protein reducing agent into the protein sample, and uniformly mixing and stirring for later use;
(2) adding acrylamide to mark cysteine into the reduced protein sample solution obtained in the step (1), and then adding succinic anhydride to mark lysine;
(3) removing unreacted reagents from the protein solution labeled by the diamino acid obtained in the step (2) through a desalting column;
(4) adding Trypsin-Trypsin into the protein sample marked by the diamino acid obtained in the step (3) for enzymolysis; and (3) carrying out chromatographic-mass spectrometric analysis on the enzymolysis products.
2. The method for labeling a diamino acid stable isotope according to claim 1 wherein in step (1), the protein reduction reaction solution is an aqueous solution containing 4M urea, 20mM Tris-HCl and 3% isopropanol; the dosage ratio of the protein sample to the protein reduction reaction solution is 1 mg: 1 ml.
3. The method for labeling a diamino acid stable isotope according to claim 1 wherein in step (1), the protein reducing agent is an aqueous solution containing 500mM TCEP, and the ratio of the amount of protein sample to the amount of protein reducing agent is 1 mg: 50 ul; the mixing and stirring temperature is 37 deg.C, and the mixing and stirring time is 30 min.
4. The method for labeling diamino acid stable isotopes based on primary mass spectrometry of claim 1 wherein in step (2), the amount of acrylamide added is 5-7 times that of the protein sample, and the labeled cysteine is: uniformly mixing the reduced protein sample solution with acrylamide, and reacting for 30min in a dark place at the temperature of 20-25 ℃;
the added succinic anhydride amount is 7-10 times of that of the protein sample, and the labeled lysine is as follows: adding succinic anhydride, mixing, and reacting at 20-25 deg.C under dark condition for 30 min.
5. The method of claim 1, wherein the displacement solution passing through the desalting column in step (3) is 50mM NH4HCO3。
6. The method for labeling the stable isotope of the diamino acid based on the primary mass spectrometry as claimed in claim 1, wherein in the step (4), the enzymolysis is carried out at 37 ℃ for 5 hours, and then formic acid is added to stop the enzymolysis.
7. Use of the method of stable isotope labeling with two amino acids based on primary mass spectrometry according to any of claims 1 to 6 in quantitative proteomics analysis of serum.
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