CN115043905B - Peptide fragment composition for relatively quantitatively analyzing pig glycine guanyltransferase GATM and application thereof - Google Patents

Peptide fragment composition for relatively quantitatively analyzing pig glycine guanyltransferase GATM and application thereof Download PDF

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CN115043905B
CN115043905B CN202210585161.6A CN202210585161A CN115043905B CN 115043905 B CN115043905 B CN 115043905B CN 202210585161 A CN202210585161 A CN 202210585161A CN 115043905 B CN115043905 B CN 115043905B
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gatm
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郝月
顾宪红
高杰
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Institute of Animal Science of CAAS
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Abstract

The application provides a peptide fragment composition, a method and a kit for relatively quantitatively analyzing pig glycine guanyltransferase GATM. A peptide fragment composition for relatively quantitatively analyzing porcine glycine guanyltransferase GATM, comprising a first peptide fragment and a second peptide fragment; the amino acid sequence of the first peptide is shown as SEQ ID NO. 1; the amino acid sequence of the second peptide fragment is shown as SEQ ID NO. 2. The method can rapidly and efficiently quantitatively detect the porcine GATM at the protein level, and improves the detection flux and efficiency of the porcine GATM. Has stronger specificity than the Western Blot method based on antibody.

Description

Peptide fragment composition for relatively quantitatively analyzing pig glycine guanyltransferase GATM and application thereof
Technical Field
The application relates to the field of pork food detection, in particular to a peptide fragment composition for relatively quantitatively analyzing pig glycine guanyltransferase GATM and application thereof.
Background
With the improvement of the intensive degree of livestock industry in China, particularly modern breeding industry, pigs are affected by more and more stress factors during growth, fattening and slaughter, and the distribution of organism nutrient substances and the change of free radical metabolism are caused. Oxidative stress is a common stress factor in production, and various stress factors can promote oxidation reaction in a body, so that oxidative stress with different degrees is caused.
GATM is a mitochondrial enzyme that is primarily responsible for catalyzing the first rate-limiting step in creatine biosynthesis. In the body, creatine is synthesized mainly in the liver using three different amino acids including arginine, glycine and methionine. The first step is the transfer of the arginine group to glycine by glycine amidino transferase to produce ornithine and guanidinoacetic acid. And in the second step, the N-methyltransferase of the guanylacetic acid is responsible for methylation of the guanylacetic acid to generate creatine, so that the synthesis of endogenous creatine is completed. Creatine is released into the blood circulation after the animal liver is synthesized, and then transported into the muscle fibers and other tissues via specific creatine transport proteins. The phosphorylated form of creatine is creatine phosphate, and when the bond between the creatine molecule and phosphate is broken, a large amount of free energy is generated, which plays an important role in relieving oxidative stress and the like.
Thus, there is a need to investigate the relative quantitative analysis of porcine glycine guanyltransferase GATM.
Disclosure of Invention
Accordingly, the present application is directed to a peptide composition for the relative quantitative analysis of porcine glycine guanyltransferase GATM and uses thereof.
In view of the above, the present application provides a peptide fragment composition for relatively quantitatively analyzing porcine glycine guanyltransferase GATM, comprising a first peptide fragment and a second peptide fragment;
the amino acid sequence of the first peptide segment is shown as SEQ ID NO. 1; the amino acid sequence of the second peptide is shown as SEQ ID NO. 2.
In some embodiments, the parent ion of the first peptide fragment is 606.82m/z, the child ion is 844.46m/z,634.32m/z and 519.29m/z, and the collision energy of the child ion is 27V; the parent ion of the second peptide is 484.73m/z, the child ion is 805.40m/z,748.38m/z and 554.30m/z, and the collision energy corresponding to the child ion is 27V.
Embodiments of the present application also provide a method for the relative quantitative analysis of porcine glycine guanyltransferase GATM using a peptide fragment composition as described in any preceding claim.
In some of these embodiments, the relative quantitative analysis of porcine glycine guanyltransferase GATM comprises:
providing pig tissue samples to be tested in different groups, and respectively carrying out protein extraction and proteolytic treatment to obtain peptide fragments to be tested in different groups;
detecting the peptide fragment compositions of different groups respectively by a liquid chromatography-mass spectrometry method;
the results of the detection of the different groups of peptide fragment compositions are compared to relatively quantify porcine glycine guanyltransferase GATM in the different groups of porcine tissue samples to be tested.
In some embodiments, the test pig tissue sample is a test pig liver sample.
In some embodiments, the method of liquid chromatography-mass spectrometry employs a high performance liquid chromatography-tandem mass spectrometer.
In some of these embodiments, the conditions for the liquid chromatography-mass spectrometry combination are as follows:
chromatographic conditions: chromatographic column: a C18 column; mobile phase a:0.1% formic acid in water, mobile phase B:0.1% acetonitrile in water; gradient elution procedure: 0-2 min, 5-10% B solution; 2-45 min, 10-30% of B solution; 45-55 min, 30-100% of B solution; 55-60 min,100% of B solution; flow rate: 250 to 450nl/min;
mass spectrometry conditions: collecting data in a positive ion mode; primary mass spectrum scan range: 300-1800m/z, mass spectrum resolution: 60000 (m/z 200), AGC target:3e6, maximum IT:200ms; MS2 scan: 20, a step of; isolation window:1.6Th, mass spectral resolution: 30000 (m/z 200), AGC target:3e6, maximum IT:120ms,MS2 Activation Type: HCD, collision energy: 27V.
In some of these embodiments, the detection result comprises a detection result of a peptide fragment composition having an isotopic label; the isotopically labeled internal standard peptide fragment is PRTC: SAAGAFGPELSR% 13 C 6 15 N 4 ,+10Da)。
The embodiment of the application also provides a kit for relatively quantitatively analyzing pig glycine guanyltransferase GATM, wherein the kit contains a reagent for detecting the peptide fragment composition as set forth in any one of the preceding claims.
In some of these embodiments, the reagent comprises a first peptide standard, a second peptide standard, a dithiothreitol solution, an iodoacetamide solution, and a trypsin solution.
From the above, it can be seen that the peptide composition, the method and the kit for relatively quantitatively analyzing pig glycine guanyltransferase GATM provided by the application can be used for specifically detecting and quantitatively analyzing GATM on a protein level by using a liquid chromatograph, can be used for rapidly and efficiently quantitatively detecting pig GATM, and improves the detection flux and efficiency of pig GATM. Compared with the Western Blot method based on the antibody, the method has stronger specificity, avoids the complicated steps, long period and high cost for preparing the GATM monoclonal antibody, and also avoids the problems of serious cross reaction, low success rate and the like for preparing the GATM polyclonal antibody.
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In order to more clearly illustrate the technical solutions of the present application or related art, the drawings that are required to be used in the description of the embodiments or related art will be briefly described below, and it is apparent that the drawings in the following description are only embodiments of the present application, and other drawings may be obtained according to the drawings without inventive effort to those of ordinary skill in the art.
FIG. 1 is a flow chart of a method for relatively quantitatively analyzing porcine glycine guanyltransferase GATM according to an embodiment of the present application;
FIG. 2 is a second mass spectrum of the first peptide RPDPIDWSVK of example 2;
FIG. 3 is a second-order mass spectrum of the second peptide fragment YGHYFPK in example 2;
FIG. 4 is a box plot of the relative quantification of GATM protein for each of the treatment and control groups of example 2;
FIG. 5 is a box plot of the relative quantification of GATM protein for each of the treatment and control groups of comparative example 1.
Detailed Description
The present application will be further described in detail below with reference to specific embodiments and with reference to the accompanying drawings, in order to make the objects, technical solutions and advantages of the present application more apparent.
It should be noted that unless otherwise defined, technical or scientific terms used in the embodiments of the present application should be given the ordinary meaning as understood by one of ordinary skill in the art to which the present application belongs. The terms "first," "second," and the like, as used in embodiments of the present application, do not denote any order, quantity, or importance, but rather are used to distinguish one element from another. The word "comprising" or "comprises", and the like, means that elements or items preceding the word are included in the element or item listed after the word and equivalents thereof, but does not exclude other elements or items.
In pig farming, oxidative stress in pigs is easily caused after ROS are sharply increased. Oxidative stress can cause the reduction of body health level and the like by causing the damage of pork liver, thereby bringing economic loss to the pig industry. The ROS excessively accumulated in the pig body mainly attacks biomacromolecules such as lipid, protein, nucleic acid and the like, so that cell components are damaged, the structure and the function are changed, and the lipid peroxidation linkage reaction can damage the structure and the function of a biomembrane, so that oxidative damage and apoptosis are aggravated. The phosphorylated form of creatine is creatine phosphate, and when the bond between the creatine molecule and phosphate is broken, a large amount of free energy is generated, which plays an important role in relieving oxidative stress and the like.
Some assays for determining the relative in vivo content of GATM primarily involve analysis at both the gene level and the protein level. The analysis at the gene level is mainly at the mRNA level, for example, cDNA formed after reverse transcription after RNA in a tissue is extracted, a cDNA template is amplified according to a gene design specific primer pair of GATM, and the transcription level of GATM in the template is compared and analyzed by using a fluorescent real-time quantitative PCR technology. However, after gene transcription, it is usually necessary to perform regulation such as cleavage and splicing to express the GATM protein, and therefore analysis of the expression level of the GATM protein at the mRNA level does not truly reflect the expression level of the gene product protein GATM. Whereas GATM is analyzed at the protein level, methods such as ELISA, western Blot, and tissue in situ hybridization are generally employed, and these detection methods generally require high quality antibodies to specifically recognize GATM. However, the GATM antigen proteins for preparing antibodies on the market at present are usually derived from model organisms such as human beings, mice and rats, and the expression level of the pig glycine guanyltransferase GATM cannot be quantitatively analyzed at the protein level.
The method for quantitative analysis of porcine glycine guanyltransferase GATM at the protein level can be established by preparing antibodies, such as monoclonal antibodies and polyclonal antibodies, which recognize porcine glycine guanyltransferase GATM and have strong specificity. However, monoclonal antibodies have the problems of long preparation period, high cost and the like; polyclonal antibodies, while relatively inexpensive, have relatively severe cross-reactions and low success rates.
Therefore, the analysis of swine glycine guanyltransferase GATM has the problems of inaccurate detection, complex antibody preparation, high cost and the like.
Based on the above, the embodiment of the application provides a peptide fragment composition for relatively quantitatively analyzing pig glycine guanyltransferase GATM and application thereof, and the relative quantitative analysis of pig glycine guanyltransferase GATM on the protein level can be performed through the peptide fragment composition, so that the problems that the detection is inaccurate, the preparation of antibodies is complex and the cost is high in the existing analysis of pig GATM are solved to a certain extent.
Table 1 shows the peptide fragment compositions provided in the examples of the present application for relative quantitative analysis of porcine glycine guanyltransferase GATM.
Referring to Table 1, embodiments of the present application provide compositions of peptide fragments for relative quantitative analysis of porcine glycine guanyltransferase GATM, comprising a first peptide fragment and a second peptide fragment. The amino acid sequence of the first peptide segment is shown as SEQ ID NO. 1; the amino acid sequence of the second peptide is shown as SEQ ID NO. 2.
Table 1 peptide fragment compositions for relative quantitative analysis of porcine glycine guanyltransferase GATM
Peptide fragment numbering Peptide fragment name Sequence numbering Sequence information
First peptide fragment RPDPIDWSVK SEQ ID NO.1 Arg-Pro-Asp-Pro-Ile-Asp-Trp-Ser-Val-Lys
Second peptide fragment YGGHYFPK SEQ ID NO.2 Tyr-Gly-Gly-His-Tyr-Phe-Pro-Lys
The peptide fragment composition for relatively quantitatively analyzing the pig glycine guanyltransferase GATM provided by the embodiment of the application comprises the first peptide fragment and the second peptide fragment, and can be used for rapidly and efficiently quantitatively detecting the peptide fragment composition in the pig GATM on the protein level by using a high-efficiency liquid chromatography-mass spectrometry (LC/MS/MS) for specific detection and quantitative analysis of the GATM, so that the detection flux and the detection efficiency of the pig GATM are improved. Compared with a Western Blot method based on an antibody, the method has stronger specificity, can avoid the problems of complicated steps, long period, high cost and the like when the swine GATM monoclonal antibody is prepared, and can also avoid the problems of serious cross reaction, low success rate and the like when the swine GATM polyclonal antibody is prepared.
Table 2 shows mass to charge ratio information for relatively quantitative analysis of peptide fragment compositions of porcine glycine guanyltransferase GATM provided by examples of the present application.
TABLE 2 Mass to charge ratio information for relative quantitative analysis of peptide fragment compositions of porcine glycine guanyltransferase GATM
Referring to Table 2, in the peptide composition for relatively quantitatively analyzing porcine glycine guanyltransferase GATM provided by the embodiment of the present application, the parent ion of the first peptide RPDPIDWSVK is 606.82m/z, the child ions are 844.46m/z,634.32m/z and 519.29m/z, and the collision energy corresponding to the child ions is 27V. The parent ion of YGYFPK of the second peptide fragment is 484.73m/z, the child ion is 805.40m/z,748.38m/z and 554.30m/z, and the collision energy corresponding to the child ion is 27V. The combined peptide fragment composition has mass-to-charge ratio, collision energy and the like of parent ions and child ions, and can be used for peptide fragment separation and signal acquisition when high performance liquid chromatography-tandem mass spectrometry (High performance liquid chromatography tandem mass spectrometry, HPLC-MS/MS) is used for analyzing pig glycine guanyltransferase GATM.
Based on the same inventive concept, embodiments of the present application also provide a kit for the relative quantitative analysis of porcine glycine guanyltransferase GATM, the kit comprising reagents for detecting the peptide fragment composition as described in any one of the preceding claims.
In some embodiments, the reagents may include a first peptide (i.e., first peptide RPDPIDWSVK) standard, a second peptide (second peptide YGGHYFPK) standard, a protein extraction reagent, a proteolytic reagent, and the like.
The protein extraction reagent may be a protein lysate commonly used in the art, such as SDT protein lysate. The proteolytic reagent may be a proteolytic reagent commonly used in the art, such as dithiothreitol solution, iodoacetamide solution, trypsin solution, and the like.
The kit of the above embodiment is used for realizing detection of the peptide fragment composition of the corresponding relative quantitative analysis pig glycine guanyltransferase GATM in any of the above embodiments, and has the beneficial effects of the corresponding peptide fragment composition embodiments, and will not be described in detail herein.
Based on the same inventive concept, the embodiment of the application also provides a method for relatively quantitatively analyzing pig glycine guanyltransferase GATM, and the peptide fragment composition described in any embodiment is used for relatively quantitatively analyzing pig glycine guanyltransferase GATM.
FIG. 1 shows a flow chart of a method for relatively quantitatively analyzing porcine glycine guanyltransferase GATM according to an embodiment of the present application.
As shown in FIG. 1, the method for relatively quantitatively analyzing pig glycine guanyltransferase GATM according to the embodiment of the present application may include:
s100, providing pig tissue samples to be tested in different groups, and respectively carrying out protein extraction and proteolytic treatment to obtain peptide fragments to be tested in different groups;
s200, detecting the peptide fragment compositions of different groups respectively by a liquid chromatography-mass spectrometry method;
s300, comparing the detection results of the peptide fragment compositions of different groups to relatively quantify GATM in the pig tissue samples to be detected of different groups.
In some embodiments, in step S100, the porcine tissue sample to be tested may be a porcine liver sample to be tested. Each group may be provided with 4 biological sample replicates, respectively.
The protein extraction may include: and (3) extracting protein in the pig tissue sample to be detected by adopting SDT lysate to obtain a protein extract, and quantifying the protein.
In some embodiments, the proteolytic processing may include: and sequentially adopting dithiothreitol to treat the obtained protein extract to open disulfide bonds, adopting acetamide to treat free sulfhydryl groups in the blocked protein, and adopting trypsin digestion to obtain peptide fragments.
That is, the protease treatment includes:
treating the protein extract with dithiothreitol to open disulfide bonds to obtain a first product;
treating the first product with acetamide to block free sulfhydryl groups in the protein to obtain a second product;
the second product was digested with trypsin.
In some embodiments, in step S200, it may include:
screening the peptide fragment composition for relative quantitative analysis of porcine glycine guanyltransferase GATM;
the peptide fragment compositions were targeted identified and corrected by isotopically labeled internal standards.
In some embodiments, the peptide fragment composition of porcine glycine guanyltransferase GATM may be screened for relative quantification by high performance liquid chromatography-mass spectrometry. The conditions of the high performance liquid chromatography-mass spectrometry method are as follows:
chromatographic conditions: chromatographic column: a C18 column; mobile phase a:0.1% acetonitrile in water, mobile phase B:0.1% acetonitrile in water; gradient elution procedure: 0-2 min, 5-10% B solution; 2-45 min, 10-30% of B solution; 45-55 min, 30-100% of B solution; 55-60 min,100% of B solution; flow rate: 250 to 450nl/min;
mass spectrometry conditions: collecting data in a positive ion mode; primary mass spectrum scan range: 300-1800m/z, primary mass spectrum resolution: 60000 (m/z 200), AGC target:3e6, first order maximumit: 50ms; secondary mass spectrometry: MS2 scan: 20, a step of; isolation window:1.6Th, secondary mass spectrum resolution: 15000 (m/z 200), AGC target:1e5, second order maximumit: 50ms,MS2 Activation Type: HCD, collision energy: 27V.
In some embodiments, repeated screening more than three times using the above-described method of screening a peptide fragment composition of porcine glycine guanyltransferase GATM results in reliable reproducibility of the method of relatively quantitatively analyzing porcine glycine guanyltransferase GATM. And specific peptide fragment information for realizing accurate relative quantification of the protein can be obtained through comparison, screening and calculation of the information such as the chromatographic peak of the peptide fragment obtained by mass spectrum analysis.
Through the above operation, the peptide sequence of the GATM protein in the mixed sample is subjected to targeted monitoring, a data acquisition method can be established and optimized, the specific peptide of the target protein GATM can be identified through the established data acquisition mode, and the consistency of signal response among technical repetitions is primarily evaluated. Two specific peptides of sequences RPDPIDWSVK and YGGHYFPK were obtained (i.e. peptide compositions for relative quantitative analysis of porcine glycine guanyltransferase GATM as shown in table 1 were obtained). Simultaneously obtaining the mass-to-charge ratio information and collision energy of the primary and secondary ion pairs of the two specific peptide fragments (namely obtaining the mass-to-charge ratio information of the peptide fragment composition of the pig glycine guanyltransferase GATM which is relatively quantitatively analyzed as shown in the table 2).
In some embodiments, targeted identification of the peptide fragment composition and correction by isotopically labeled internal standards may specifically include:
and (3) respectively taking the preset amounts of the peptide fragments to be detected in the step S100 in different groups, doping the internal standard peptide fragments marked by the equivalent weight isotopes for detection, and adopting LC/MS/MS separation peptide fragments and signal acquisition. In the step, the parallel reaction monitoring result of the target peptide fragment can be obtained. The results may contain information such as the chromatographic peak of the peptide fragment, the area of the original peak, and a comparison histogram of the area of the original peak.
The conditions of the high performance liquid chromatography-mass spectrometry method can be as follows:
chromatographic conditions: chromatographic column: a C18 column; mobile phase a:0.1% acetonitrile in water, mobile phase B:0.1% acetonitrile in water solution of formic acid acetonitrile in acetonitrile; gradient elution procedure: 0-2 min, 5-10% B solution; 2-45 min, 10-30% of B solution; 45-55 min, 30-100% of B solution; 55-60 min,100% of B solution; flow rate: 250 to 450nl/min;
mass spectrometry conditions: collecting data in a positive ion mode; primary mass spectrum scan range: 300-1800m/z, mass spectrum resolution: 60000 (m/z 200), AGC target:3e6, maximum IT:200ms; MS2 scan: 20, a step of; isolation window:1.6Th, mass spectral resolution: 30000 (m/z 200), AGC target:3e6, maximum IT:120ms,MS2 Activation Type: HCD, collision energy: 27V.
The two primary and secondary ion mass charge ratio information pairs for setting the specific polypeptide are respectively as follows: m/z606.82 is RPDPIDWSVK parent ion, and the daughter ions generated by fragmentation are m/z 844.46, m/z634.32 and m/z519.29; m/z 484.73 is the parent ion of YGHTYFPK, and the daughter ions generated by fragmentation are m/z 805.40, m/z 748.38, and m/z 554.30.
In some embodiments, the isotopically labeled internal standard peptide fragment may be PRTC: SAAGAFGPELSR% 13 C 6 15 N 4 ,+10Da)。
In step S300, the comparison bar graph of the chromatographic peak, the original peak area and the original peak area of the peptide fragment obtained by the parallel reaction monitoring is analyzed, and the detection results of the peptide fragment compositions of different groups are compared, so that the relative quantification of GATM in the pig tissue samples to be detected of different groups can be performed.
Specifically, skyline software is adopted to perform data analysis on an original file, 3 sub-ions with higher abundance of peptide fragments and as continuous as possible can be selected to perform quantitative analysis, and the sub-ion peak areas of target peptide fragments (namely, the sub-ion peak areas of target peptide fragments obtained in the step S200) are integrated to obtain the original peak areas of the peptide fragments in a sample; then correcting the peak area of the heavy isotope labeled internal standard peptide fragment (namely, the sub-ion peak area of the target peptide fragment obtained in the step S200) to obtain the relative expression amount information of each fragment of peptide in different groups of samples; and finally, calculating the average value of the relative expression quantity of the target peptide fragments in each group of samples, and carrying out statistical analysis. And analyzing the expression quantity of the target protein, and further calculating the difference of the relative expression quantity of the target protein in different sample groups according to the relative expression quantity of the corresponding peptide segment of each target protein in different sample groups. The specific calculation is in the prior art, for example, may be implemented by Skyline software, so detailed calculation steps and the like are not repeated here.
The method of the above embodiment is used to realize detection of the peptide fragment composition of pig glycine guanyltransferase GATM according to the corresponding relative quantitative analysis of any of the above embodiments, and has the beneficial effects of the corresponding peptide fragment composition embodiment, which will not be described in detail herein.
It should be noted that the foregoing describes some embodiments of the present application. Other embodiments are within the scope of the following claims. In some cases, the actions or steps recited in the claims may be performed in a different order than in the embodiments described above and still achieve desirable results. In addition, the processes depicted in the accompanying figures do not necessarily require the particular order shown, or sequential order, to achieve desirable results. In some embodiments, multitasking and parallel processing are also possible or may be advantageous.
The technical scheme of the application is further described below with reference to the specific embodiments.
The experimental methods in the following examples are conventional methods unless otherwise specified.
The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.
Example 1 screening of peptide fragment compositions for relative quantitative analysis of porcine glycine guanyltransferase GATM
1. GATM protein extraction: and respectively taking pig liver tissue samples of a diquat induced oxidative stress treatment group (DQ), an antioxidant lipoic acid treatment group (LA), a diquat plus lipoic acid treatment group (DL) and a control group (CK), adding a proper amount of SDT lysate, transferring the pig liver tissue samples into a 2ml centrifuge tube which is pre-filled with a proper amount of quartz sand, and homogenizing and crushing (24X 2,6.0M/S,60S and twice) by using a homogenizer. Then sonicated (100W, 10s on duty, 10s off, 10 cycles) and boiled water bath for 10min. And (3) centrifuging for 10min at 14000g, filtering the supernatant by using a 10kD ultrafiltration membrane, and collecting filtrate. Protein quantification was performed using BCA method. Subpackaging the sample, and preserving at-80 ℃. Each group contained 4 biological replicates.
2. Proteolysis: in each of the treatment group and the control group, about 200ug of protein was taken as a sample, dithiothreitol (DTT) was added to the sample to a final concentration of 100mM, disulfide bonds were opened, the sample was boiled in water for 15min, cooled to room temperature, and then added200. Mu.L of urea buffer (UA buffer,8M Urea,150mM Tris-HCl, pH 8.0) was added and the mixture was transferred to a 10KD ultrafilter tube and centrifuged at 14000g for 30min. 200 μl UA buffer was added and centrifuged for 14000g 30min, and the filtrate was discarded. 100. Mu.L of iodoacetamide (IAA, 50mM IAA in UA) was added to alkylate and block free thiol groups in the protein, the mixture was shaken at 600rpm for 1min, and centrifuged at 14000g for 20min at room temperature in the absence of light. 100. Mu.L of UA buffer was added and centrifugation was repeated 3 times for 14000g of 20min. 100. Mu.L NH was added 4 HCO 3 buffer (50 mM), centrifugation 14000g was repeated 2 times for 20min. 40 mu LNH was added 4 HCO 3 The buffer (containing Trypsin with an enzyme ratio of 1:50) was digested, and 1min was shaken at 600rpm for 16h at 37 ℃. The collection tube was replaced with a new one and centrifuged 14000g for 15min. 40. Mu.L of NH was added 4 HCO 3 buffer (50 mM) was centrifuged at 14000g for 30min and the filtrate was collected. The digested peptide was desalted and lyophilized, then reconstituted with 0.1% Formic Acid (FA) and OD280 was used to determine the peptide concentration.
3. Screening specific peptide fragments of target proteins and establishing a data acquisition method: firstly, randomly selecting one sample from multiple repetitions of each group in the step 2, respectively taking a proper amount of peptide fragments after enzymolysis, and mixing the peptide fragments into one sample in an equivalent manner; taking 1ug of the mixed peptide segment, and adopting an HPLC system to carry out high performance liquid chromatography separation; buffer A is 0.1% formic acid aqueous solution, and buffer B is 0.1% formic acid acetonitrile aqueous solution (84% acetonitrile); the column was equilibrated with 95% solution a; sample passing Trap Column (100 μm. Times.50 mm,5 μm-C18, dr. Maisch phase, r25.aq), analytical Column (180 μm. Times.150 mm,3 μm-C18, dr. Maisch phase, r23.aq) flow rate 300nl/min; the liquid phase separation gradient is as follows: 0 min-2 min, linear gradient of B liquid from 5% to 10%,2 min-45 min, linear gradient of B liquid from 10% to 30%;45 minutes to 55 minutes, the linear gradient of the liquid B is from 30% to 100%;55 minutes-60 minutes, the linear gradient of the B liquid is maintained at 100 percent. Then, carrying out targeted qualitative analysis by using a Q-exact HF mass spectrometer through a parallel reaction monitoring method; analysis duration: 60min, detection mode: positive ion, parent ion scan range: 300-1800m/z, primary mass spectrum resolution: 60000 (m/z 200), AGC target:3e6, first order maximumit: 50ms; peptide fragment secondary mass spectrometry was collected as follows: after each full scan (full scan) 20 secondary mass spectra (MS 2 scan) were triggered to be acquired, secondary mass spectrum resolution: 15000 (m/z 200), AGC target:1e5, second order maximumit: 50ms,MS2 Activation Type: HCD, isolation window:1.6Th,Normalized collision energy:27; LC-MS/MS adopts a targeted shotgun scanning mode to carry out MS2 scanning on candidate peptide fragments of target proteins.
By comparing, screening and calculating the information such as the chromatographic peaks of the peptide fragments obtained by mass spectrometry, determining the porcine liver tissue samples of the diquat induced oxidative stress treatment group (DQ), the antioxidant lipoic acid treatment group (LA), the diquat + lipoic acid treatment group (DL) and the control group (CK), the sequence information of the peptide fragment composition for relatively quantitatively analyzing the porcine glycine amidino transferase GATM in Table 1 can be obtained, the mass-to-charge ratio information of the peptide fragment composition for relatively quantitatively analyzing the porcine glycine amidino transferase GATM in Table 2 can be obtained, and the specific steps and specific parameter conditions of the individual steps of the method for relatively quantitatively analyzing the porcine glycine amidino transferase GATM and the like can be determined.
Example 2 application in quantitative relative liver GATM protein of diquat-induced oxidative stress pig model
1. GATM protein extraction: and respectively taking pig liver tissue samples of a diquat induced oxidative stress treatment group (DQ), an antioxidant lipoic acid treatment group (LA), a diquat plus lipoic acid treatment group (DL) and a control group (CK), adding a proper amount of SDT lysate, transferring the pig liver tissue samples into a 2ml centrifuge tube which is pre-filled with a proper amount of quartz sand, and homogenizing and crushing (24X 2,6.0M/S,60S and twice) by using a homogenizer. Then sonicated (100W, 10s on duty, 10s off, 10 cycles) and boiled water bath for 10min. And (3) centrifuging for 10min at 14000g, filtering the supernatant by using a 10kD ultrafiltration membrane, and collecting filtrate. Protein quantification was performed using BCA method. Subpackaging the sample, and preserving at-80 ℃. Each group contained 4 biological replicates.
2. Proteolysis: in each treatment group and control group, about 200ug protein is taken as a sample of each group, dithiothreitol (DTT) is added to a final concentration of 100mM, disulfide bonds are opened, boiling water is used for 15min, cooling is carried out to room temperature, 200 ul urea buffer (UA buffer,8M Urea,150mM Tris-HCl, pH 8.0) is added to be evenly mixed, the mixture is transferred to a 10KD ultrafilter tube, and the mixture is centrifuged 14000g for 30min. 200 μl UA buffer was added and centrifuged for 14000g 30min, and the filtrate was discarded. 100. Mu.L of iodoacetamide (IAA, 50mM IAA in UA) was added to alkylate and block free thiol groups in the protein, the mixture was shaken at 600rpm for 1min, and centrifuged at 14000g for 20min at room temperature in the absence of light. 100. Mu.L of UA buffer was added and centrifugation was repeated 3 times for 14000g of 20min. 100. Mu.L NH was added 4 HCO 3 buffer (50 mM), centrifugation 14000g was repeated 2 times for 20min. 40. Mu.L of NH was added 4 HCO 3 The buffer (containing Trypsin with an enzyme ratio of 1:50) was digested, and 1min was shaken at 600rpm for 16h at 37 ℃. The collection tube was replaced with a new one and centrifuged 14000g for 15min. 40. Mu.L of NH was added 4 HCO 3 buffer (50 mM) was centrifuged at 14000g for 30min and the filtrate was collected. The digested peptide was desalted and lyophilized, then reconstituted with 0.1% Formic Acid (FA) and OD280 was used to determine the peptide concentration.
3. Targeting identification of the target protein specific peptide fragment and correction by isotopic internal standard: firstly, taking about 1ug peptide fragment from each sample in the multiple repetitions of each group in the step 2, respectively doping 20fmol heavy isotope labeled internal standard peptide fragment (PRTC: SAAGAFGPELSR) for detection, and carrying out high performance liquid chromatography separation on the polypeptide by adopting an HPLC system; buffer A is 0.1% formic acid aqueous solution, and buffer B is 0.1% formic acid acetonitrile aqueous solution (84% acetonitrile); the column was equilibrated with 95% solution a; sample is injected into a chromatographic analysis column for gradient separation, and the flow rate is 300nl/min; the liquid phase separation gradient is as follows: 0 min-2 min, linear gradient of B liquid from 5% to 10%,2 min-45 min, linear gradient of B liquid from 10% to 30%;45 minutes to 55 minutes, the linear gradient of the liquid B is from 30% to 100%;55 minutes-60 minutes, the linear gradient of the B liquid is maintained at 100 percent. After high performance liquid chromatography separation, carrying out parallel reaction monitoring mass spectrometry on five target peptide fragments of the identified target protein by using a Q-exact HF mass spectrometer, wherein the analysis duration is as follows: 60min, detection mode: a positive ion; primary mass spectrum scan range: 300-1800m/z, mass spectrum resolution: 60000 (m/z 200), AGC target:3e6, maximum IT:200ms; 20 parallel reaction monitoring scans (MS 2 scans) were acquired according to the Inclusion list after each level MS scan (full MS scan), isolation window:1.6Th, mass spectral resolution: 30000 (m/z 200), AGC target:3e6, maximum IT:120ms,MS2 Activation Type: HCD, normalized collision energy:27.
the two primary and secondary ion mass charge ratio information pairs for setting the specific polypeptide are respectively as follows: m/z606.82 is RPDPIDWSVK parent ion, and the daughter ions generated by fragmentation are m/z 844.46, m/z634.32 and m/z519.29; m/z 484.73 is the parent ion of YGHTYFPK, and the daughter ions generated by fragmentation are m/z 805.40, m/z 748.38, and m/z 554.30.
4. Analysis of the monitoring results of the parallel reaction of the target peptide fragment: and analyzing the target protein specific peptide fragment data obtained by parallel reaction monitoring, wherein the target protein specific peptide fragment data comprises information such as a peptide fragment chromatographic peak, an original peak area, a comparison histogram of the original peak area and the like. 3 sub-ions with higher abundance and as continuous as possible are selected for quantitative analysis. Firstly, integrating the sub-ion peak areas of target peptide fragments to obtain the original peak areas of the peptide fragments in a sample; correcting the peak area of the internal standard peptide fragment marked by the heavy isotope to obtain the relative expression quantity information of each fragment of peptide in different samples; and finally, calculating the average value of the relative expression quantity of the target peptide fragments in each group of samples, and carrying out statistical analysis. And analyzing the expression quantity of the target protein, and further calculating the difference of the relative expression quantity of the target protein in different sample groups according to the relative expression quantity of the corresponding peptide segment of each target protein in different sample groups.
Test results: see fig. 2-4. In the diquat+lipoic acid (DL) treated group, the second mass spectrum of the first peptide RPDPIDWSVK is shown in fig. 2, and the second mass spectrum of the second peptide YGGHYFPK is shown in fig. 3.
DQ in FIG. 4 is the relative quantification of porcine GATM protein in the diquat-induced oxidative stress treatment group (DQ) versus porcine GATM protein in the control group (CK). LA is the relative quantification of porcine GATM protein in the antioxidant lipoic acid treated group (LA) relative to porcine GATM protein in the control group (CK). DL is the relative quantification of porcine-derived GATM protein in the diquat + lipoic acid treated group (DL) versus porcine-derived GATM protein in the control group (CK). CK is a relative quantification of the average value of porcine GATM protein in the control group (CK) compared to the porcine GATM protein in the three treatment groups (diquat induced oxidative stress (DQ), antioxidant Lipoic Acid (LA) and diquat + lipoic acid (DL)).
Comparative example 1
The difference from example 2 is that the peptide fragment after enzymolysis was detected based on the LC-MS/MS method using TMT (Tandem Mass Tag) protein isotope labeling quantitative technique according to the specific operation instructions of the TMT protein labeling kit of Thermo company.
Specifically, the peptide fragment after enzymolysis is obtained through step 1 and step 2 in example 2.
Test results: see fig. 5.
DQ in FIG. 5 is the relative quantification of porcine GATM protein in the diquat-induced oxidative stress treatment group (DQ) versus porcine GATM protein in the control group (CK). LA is the relative quantification of porcine GATM protein in the antioxidant lipoic acid treated group (LA) relative to porcine GATM protein in the control group (CK). DL is the relative quantification of porcine-derived GATM protein in the diquat + lipoic acid treated group (DL) versus porcine-derived GATM protein in the control group (CK). CK is a relative quantification of the average value of porcine GATM protein in the control group (CK) compared to the porcine GATM protein in the three treatment groups (diquat induced oxidative stress (DQ), antioxidant Lipoic Acid (LA) and diquat + lipoic acid (DL)).
Comparing the box plots of the relative quantitative results obtained in example 2 and comparative example 1, it can be seen that the trend of change in the porcine-derived GATM protein identified in example 2 between the different treatment groups is consistent with the trend of change in the porcine-derived GATM protein identified in comparative example 1 between the different treatment groups. The peptide composition comprising the first peptide RPDPIDWSVK and the second peptide YGGYFPK for relatively quantitatively analyzing pig glycine guanyltransferase GATM according to the embodiment of the application has good accuracy, and the peptide composition can target and identify the target peptide composition of pig glycine guanyltransferase GATM, and has higher sensitivity compared with the existing in-vitro labeling detection method, such as high throughput identification of all pig proteins by TMT (Tandem Mass Tag) protein isotope labeling quantitative technology.
Those of ordinary skill in the art will appreciate that: the discussion of any of the embodiments above is merely exemplary and is not intended to suggest that the scope of the disclosure, including the claims, is limited to these examples; the technical features of the above embodiments or in the different embodiments may also be combined under the idea of the present disclosure, the steps may be implemented in any order, and there are many other variations of the different aspects of the embodiments of the present disclosure as described above, which are not provided in details for the sake of brevity.
While the present disclosure has been described in conjunction with specific embodiments thereof, many alternatives, modifications, and variations of those embodiments will be apparent to those skilled in the art in light of the foregoing description.
The disclosed embodiments are intended to embrace all such alternatives, modifications and variances which fall within the broad scope of the appended claims. Accordingly, any omissions, modifications, equivalents, improvements, and the like, which are within the spirit and principles of the embodiments of the disclosure, are intended to be included within the scope of the disclosure.
Sequence listing
<110> Beijing livestock veterinary research institute of China agricultural sciences
<120> peptide fragment composition for relative quantitative analysis of porcine glycine guanyltransferase GATM and use thereof
<130> FI221398
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 10
<212> PRT
<213> amino acid sequence of first peptide fragment
<400> 1
Arg Pro Asp Pro Ile Asp Trp Ser Val Lys
1 5 10
<210> 2
<211> 8
<212> PRT
<213> amino acid sequence of second peptide fragment
<400> 2
Tyr Gly Gly His Tyr Phe Pro Lys
1 5

Claims (4)

1. A composition of peptide fragments for relative quantitative analysis of porcine glycine guanyltransferase GATM, comprising a first peptide fragment and a second peptide fragment;
the amino acid sequence of the first peptide segment is shown as SEQ ID NO. 1; the amino acid sequence of the second peptide is shown as SEQ ID NO. 2.
2. The composition of matter of claim 1, wherein the parent ion of the first peptide fragment is 606.82m/z, the child ion is 844.46m/z,634.32m/z and 519.29m/z, and the ion has a collision energy of 27V; the parent ion of the second peptide is 484.73m/z, the child ion is 805.40m/z,748.38m/z and 554.30m/z, and the collision energy corresponding to the child ion is 27V.
3.A kit for the relative quantitative analysis of porcine glycine guanyltransferase GATM, comprising a peptide fragment composition according to any one of claims 1 to 2.
4. The kit of claim 3, wherein the kit comprises a first peptide standard, a second peptide standard, a dithiothreitol solution, an iodoacetamide solution, and a trypsin solution.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110286181A (en) * 2019-07-26 2019-09-27 中国肉类食品综合研究中心 A method of based on pork content in LC-MS/MS measurement pork balls

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CN110286181A (en) * 2019-07-26 2019-09-27 中国肉类食品综合研究中心 A method of based on pork content in LC-MS/MS measurement pork balls

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