CN115166067B - Peptide fragment composition for relatively quantitatively analyzing pig 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 and application - Google Patents

Peptide fragment composition for relatively quantitatively analyzing pig 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 and application Download PDF

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CN115166067B
CN115166067B CN202210587648.8A CN202210587648A CN115166067B CN 115166067 B CN115166067 B CN 115166067B CN 202210587648 A CN202210587648 A CN 202210587648A CN 115166067 B CN115166067 B CN 115166067B
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peptide
peptide fragment
aldh1l1
ion
mass spectrometry
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CN115166067A (en
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高杰
张名媛
顾宪红
朴香淑
郝月
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Institute of Animal Science of CAAS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0026—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5)
    • C12N9/0028—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5) with NAD or NADP as acceptor (1.5.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y105/00—Oxidoreductases acting on the CH-NH group of donors (1.5)
    • C12Y105/01—Oxidoreductases acting on the CH-NH group of donors (1.5) with NAD+ or NADP+ as acceptor (1.5.1)
    • C12Y105/01006—Formyltetrahydrofolate dehydrogenase (1.5.1.6)
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
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    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
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    • G01N30/02—Column chromatography
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    • G01N30/72—Mass spectrometers
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    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid chromatography
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N2030/042—Standards
    • G01N2030/045—Standards internal
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
    • G01N2030/8831—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving peptides or proteins

Abstract

The present application provides a peptide fragment composition, method and kit for relatively quantitatively analyzing pig 10-formyl tetrahydrofolate dehydrogenase ALDH1L1. A composition of peptide fragments of pig 10-formyl tetrahydrofolate dehydrogenase ALDH1L1, comprising a first peptide fragment, a second peptide fragment, and a third 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 and the amino acid sequence of the third peptide fragment is shown as SEQ ID NO. 3. The method can rapidly and efficiently quantitatively detect the swine ALDH1L1 on the protein level, and improves the detection flux and efficiency of the swine ALDH1L1. Has stronger specificity than the Western Blot method based on antibody.

Description

Peptide fragment composition for relatively quantitatively analyzing pig 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 and application
Technical Field
The application relates to the technical field of food detection, in particular to a peptide fragment composition for relatively quantitatively analyzing pig 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 and application thereof.
Background
In the pig industry, oxidative stress can easily cause economic losses. The economic loss caused by oxidative stress is mainly manifested by damage to pig liver, thereby affecting pig health and reducing the quality of pig products (such as pork, etc.). Oxidative stress is often caused by the stimulation of a stressor, disrupting the oxidative-antioxidant balance system in pigs and causing a dramatic increase in oxidative free radicals and their reactive derivatives (Reactive oxygen species, ROS) and the like.
ALDH1L1 is a member of the aldehyde dehydrogenase superfamily and is a regulated enzyme of folate metabolism. ALDH1L1 is also known as 10-formyl tetrahydrofolate dehydrogenase (10-formyltetrahydrofolate dehydrogenase, FDH). The main function is to catalyze the conversion of 10-formyltetrahydrofolate into tetrahydrofolate and CO 2 By NADP + The reaction is relied upon to catalyze the biosynthesis of DNA nucleotides necessary for the synthesis of Tetrahydrofolate (THF) from 10-formyltetrahydrofolate. Has regulation and control effects and higher research value in the physiological and pathological processes related to oxidative stress.
Therefore, it is of great importance to study the relative quantitative analysis of swine 10-formyl tetrahydrofolate dehydrogenase ALDH1L1.
Disclosure of Invention
In view of the above, the present application aims to provide a peptide fragment composition for relatively quantitatively analyzing pig 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 and application thereof.
In view of the above, the present application provides a peptide fragment composition for relatively quantitatively analyzing porcine 10-formyl-tetrahydrofolate dehydrogenase ALDH1L1, comprising a first peptide fragment, a second peptide fragment, and a third 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; the amino acid sequence of the third peptide is shown as SEQ ID NO. 3.
In some embodiments, the parent ion of the first peptide fragment is 770.89m/z, the child ion is 1054.57m/z,907.50m/z and 737.39m/z, and the collision energy of the child ion is 27V; the parent ion of the second peptide is 682.35m/z, the child ion is 1135.57m/z,949.51m/z and 878.47m/z, and the collision energy corresponding to the child ion is 27V; the parent ion of the third peptide is 812.89m/z, the child ion is 1294.65m/z,1157.59m/z and 1100.57m/z, and the collision energy corresponding to the child ion is 27V.
The embodiment of the application also provides a method for relatively quantitatively analyzing the pig 10-formyl tetrahydrofolate dehydrogenase ALDH1L1, wherein the peptide fragment composition is applied to relatively quantitatively analyze the pig liver tissue 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 by adopting a liquid chromatography-mass spectrometry combined method.
In some embodiments, the relative quantitative analysis of 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 of porcine liver tissue using a liquid chromatography-mass spectrometry combination method comprises:
providing different groups of pig liver samples to be tested, and respectively carrying out protein extraction and proteolytic treatment to obtain different groups of peptide segments to be tested;
detecting the peptide fragment compositions of different groups respectively by a liquid chromatography-mass spectrometry method;
the results of the detection of the peptide fragment compositions of the different groups are compared to relatively quantify 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 in the swine tissue samples of the different groups.
In some embodiments, the detecting the peptide fragment compositions of different groups by a method of liquid chromatography-mass spectrometry comprises:
respectively incorporating isotopically labeled internal standard peptide fragments into the peptide fragment compositions of different groups; the weight of the internal standard peptide fragment is the same as the weight of the peptide fragment composition;
and (3) performing liquid chromatography-mass spectrometry detection on the obtained peptide fragment composition.
In some embodiments, the liquid chromatography-mass spectrometry detection is high performance liquid chromatography-tandem mass spectrometry.
In some of these embodiments, each of the different groups contains 4 biological duplicate samples, respectively.
The present embodiments also provide a kit for the relative quantitative analysis of porcine 10-formyl tetrahydrofolate dehydrogenase ALDH1L1, comprising a reagent for the detection of the peptide fragment composition of any one of claims 1-2.
In some of these embodiments, the reagent comprises a standard for the first peptide fragment, a standard for the second peptide fragment, a standard for the third peptide fragment, 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 the pig 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 provided by the application can be used for specifically detecting and quantitatively analyzing the ALDH1L1 on the protein level by using a liquid chromatograph through the peptide composition comprising the first peptide, the second peptide and the third peptide, and can be used for rapidly and efficiently quantitatively detecting the pig ALDH1L1, so that the detection flux and the detection efficiency of the pig ALDH1L1 are improved. Compared with a Western Blot method based on an antibody, the method has stronger specificity, avoids complicated steps, long period and high cost for preparing the ALDH1L1 monoclonal antibody, and also avoids the problems of serious cross reaction, low success rate and the like for preparing the ALDH1L1 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 these drawings without inventive effort to those of ordinary skill in the art.
FIG. 1 is a flow chart of a method for the relative quantitative analysis of porcine 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 in an embodiment of the present application;
FIG. 2 is a second mass spectrum of the first peptide ANATEFGLASGVFTR of example 2;
FIG. 3 is a second mass spectrum of the second peptide DLGEAALNEYLR of example 2;
FIG. 4 is a second-order mass spectrum of the third peptide DTNHGPQNHQAHLR in example 2;
FIG. 5 is a box plot of the relative quantification of ALDH1L1 protein in each of the treatment and control groups of example 2;
FIG. 6 is a box plot of the relative quantification of ALDH1L1 protein in each of the treatment group and the control group of comparative example 1.
Detailed Description
For the purposes of making the objects, technical solutions and advantages of the present application more apparent, the present application will be further described in detail below with reference to the accompanying drawings.
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.
10-formyl tetrahydrofolate dehydrogenase ALDH1L1 is a tetramer composed of identical subunits, each consisting of three functional domains derived from unrelated genes. Increased expression of ALDH1L1 tends to result in increased NADPH levels, increased NADPH oxidase activity, and increased ROS levels. In addition, ALDH1L1 appears to be a major regulator of cellular metabolism, and under certain physiological and pathological conditions the concentration of ALDH1L1 is strongly down-regulated, in which case up-regulation of ALDH1L1 concentration may result in a strong antiproliferative effect. Therefore, the 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 is a candidate tumor suppressor and a potential marker of invasive cancers, and has a regulatory effect and a higher research value in the physiological and pathological processes related to oxidative stress.
In the pig industry, increased expression of ALDH1L1 correlates with increased ROS levels, and excessive accumulation of ROS in pigs due to oxidative stress often results in ROS attack on biological macromolecules such as lipids, proteins, and nucleic acids, thereby damaging cellular components, altering cellular structure, and changing functions accordingly. On the other hand, the chain reaction of lipid peroxidation can cause the damage of the biomembrane structure and the related functions, thereby exacerbating the oxidative damage and apoptosis in pigs. As two major sites of folate metabolism, levels of ALDH1L1 are particularly high in the liver and kidneys of pigs. Therefore, the increased expression of ALDH1L1 is closely related to oxidative stress caused by ROS accumulation, and the oxidative stress further causes inflammatory reaction in pigs, oxidation of liver lipid of the pigs and the like, fat infiltration and liver swelling, occurrence of diseases such as fatty liver, liver fibrosis and the like, and harm to the health of the pigs.
Some assays for determining the relative in vivo levels of ALDH1L1 primarily involve analysis at both the gene level and the protein level. Analysis at the gene level is mainly performed at the mRNA level, for example, cDNA is formed after reverse transcription after RNA is extracted from a tissue, a reverse transcribed cDNA template is amplified according to a gene design specific primer pair of ALDH1L1, and the transcription level of the ALDH1L1 in the template is compared and analyzed by using a fluorescent real-time quantitative PCR technology. However, for comparison at the mRNA level, since the gene is often subjected to regulation such as shearing and splicing after transcription, the expression of the ALDH1L1 protein is not actually reflected by analysis of the expression level at the transcription level. While the analysis of ALDH1L1 at the protein level usually uses methods such as ELISA, western Blot, and tissue in situ hybridization, these detection methods all require high quality antibodies to specifically recognize ALDH1L1. In contrast, most antigen proteins for preparing antibodies in the market today are derived from model organisms such as human, mouse and rat, and the expression level of porcine 10-formyltetrahydrofolate dehydrogenase ALDH1L1 cannot be quantitatively analyzed at the protein level.
The method for quantitative analysis of porcine ALDH1L1 at the protein level can be established by preparing antibodies, such as monoclonal antibodies and polyclonal antibodies, which recognize porcine ALDH1L1 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 existing analysis of swine ALDH1L1 has the problems of inaccurate detection, complex preparation of antibodies, high cost and the like.
Based on the above, the embodiment of the application provides a peptide fragment composition for relatively quantitatively analyzing pig 10-formyl-tetrahydrofolate dehydrogenase ALDH1L1 and application, and the peptide fragment composition is used for relatively quantitatively analyzing pig 10-formyl-tetrahydrofolate dehydrogenase ALDH1L1 on protein level, so that antibodies are not required to be prepared, the specificity is good, and the problems that the detection is inaccurate, the preparation cost of the antibodies is high and the like in the existing analysis of pig ALDH1L1 can be solved to a certain extent.
Table 1 shows the relative quantitative analysis of the peptide compositions of porcine 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 provided in the examples of the present application.
Referring to Table 1, the examples herein provide compositions of peptide fragments comprising a first peptide fragment, a second peptide fragment and a third peptide fragment for relative quantitative analysis of porcine 10-formyl-tetrahydrofolate dehydrogenase ALDH1L1. 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; the amino acid sequence of the third peptide is shown as SEQ ID NO. 3.
TABLE 1 peptide fragment composition for relative quantitative analysis of porcine 10-formyl tetrahydrofolate dehydrogenase ALDH1L1
The first peptide fragment, the second peptide fragment and the third peptide fragment in the peptide fragment composition for relatively quantitatively analyzing the pig 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 provided by the embodiment of the application can be specifically detected by using a liquid chromatograph, so that the ALDH1L1 can be quantitatively analyzed on a protein level, the pig ALDH1L1 can be rapidly and efficiently relatively quantitatively detected, and the detection flux and the detection efficiency of the pig ALDH1L1 can be improved. Compared with a Western Blot method based on antibodies, the method for detecting the swine ALDH1L1 has stronger specificity, can avoid complicated steps for preparing the swine ALDH1L1 monoclonal antibody, has long period and high cost, and also can avoid the problems of serious cross reaction, low success rate and the like for preparing the swine ALDH1L1 polyclonal antibody.
Table 2 shows mass to charge ratio information for the relative quantitative analysis of the peptide fragment composition of porcine 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 provided in the examples of the present application.
TABLE 2 Mass to charge ratio information for relative quantitative analysis of peptide fragment compositions of porcine 10-formyl tetrahydrofolate dehydrogenase ALDH1L1
Referring to Table 2, in the peptide fragment composition for quantitative analysis of pig 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 provided in the example of the present application, parent ion of the first peptide fragment was 770.89m/z, child ion was 1054.57m/z,907.50m/z and 737.39m/z, and collision energy corresponding to the child ion was 27V. The parent ion of the second peptide is 682.35m/z, the child ion is 1135.57m/z,949.51m/z and 878.47m/z, and the collision energy corresponding to the child ion is 27V; the parent ion of the third peptide is 812.89m/z, the child ion is 1294.65m/z,1157.59m/z and 1100.57m/z, and the collision energy corresponding to the child ion is 27V. The mass-to-charge ratio, collision energy and the like of parent ions and child ions of the peptide fragment composition can be used for peptide fragment separation and signal acquisition in a high performance liquid chromatography-tandem mass spectrometry (High performance liquid chromatography tandem mass spectrometry, HPLC-MS/MS) analysis method.
Based on the same inventive concept, the present embodiments also provide a kit for the relative quantitative analysis of porcine 10-formyl-tetrahydrofolate dehydrogenase ALDH1L1, comprising reagents for detecting the peptide fragment composition of any one of the preceding claims.
In some embodiments, the reagents may include a first peptide standard, a second peptide standard, a third peptide standard, a protein extraction reagent, and a proteolytic reagent.
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 the detection of the peptide fragment composition of the corresponding relative quantitative analysis of pig 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 in any of the previous embodiments, and has the beneficial effects of the corresponding peptide fragment composition embodiment, and is not described in detail herein.
Based on the same inventive concept, the present application also provides a method for relatively quantitatively analyzing pig 10-formyl-tetrahydrofolate dehydrogenase ALDH1L1, and the peptide fragment composition described in any of the previous examples is used for relatively quantitatively analyzing pig 10-formyl-tetrahydrofolate dehydrogenase ALDH1L1.
FIG. 1 shows a flow chart of a method for the relative quantitative analysis of porcine 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 in an embodiment of the present application.
As shown in FIG. 1, the method for relatively quantitatively analyzing pig 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 provided in the examples of the present application may comprise:
s100, providing pig liver samples to be tested in different groups, and respectively carrying out protein extraction and proteolytic treatment to obtain peptide segments 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 the 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 in the pig tissue samples to be detected of different groups.
In some embodiments, in step S100, the protein extraction may include: and (3) extracting protein in the pig liver 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 a peptide fragment composition for relatively quantitative analysis of porcine 10-formyl tetrahydrofolate dehydrogenase ALDH1L 1;
the peptide fragment compositions were targeted identified and corrected by isotopically labeled internal standards.
In some embodiments, the peptide fragment composition of porcine 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 can 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 10-formyl-tetrahydrofolate dehydrogenase ALDH1L1 results in reliable reproducibility of the method of relatively quantitatively analyzing porcine 10-formyl-tetrahydrofolate dehydrogenase ALDH1L1. And specific peptide fragment information for realizing accurate relative quantification of protein is obtained through comparison, screening and calculation of information such as peptide fragment chromatographic peaks obtained through mass spectrum analysis.
Through the above operation, the peptide sequence of ALDH1L1 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 ALDH1L1 can be identified through an established data acquisition mode, and the consistency of signal response among technical repetitions is primarily evaluated. And confirmed that ANATEFGLASGVFTR peptide having the amino acid sequence No.1, DLGEAALNEYLR peptide having the amino acid sequence No.2 and DTNHGPQNHQAHLR peptide having the amino acid sequence No.3 were obtained (i.e., a peptide composition for relative quantitative analysis of pig 10-formyltetrahydrofolate dehydrogenase ALDH1L1 as shown in Table 1). And simultaneously determining the mass-to-charge ratio information, collision energy and the like of the primary and secondary ion pairs capable of obtaining three specific peptide fragments (namely, obtaining the mass-to-charge ratio information of the peptide fragment composition of the pig 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 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 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 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 three primary and secondary ion pair mass to charge ratio information of the specific polypeptide are respectively: m/z 770.89 is ANATEFGLASGVFTR parent ion, m/z 1054.57, m/z907.50, m/z 737.39 for the daughter ion produced by fragmentation; m/z 682.35 is DLGEAALNEYLR parent ion, and the daughter ions generated by fragmentation are m/z 1135.57, m/z 949.51 and m/z 878.47; m/z 812.89 is DTNHGPQNHQAHLR parent ion, and the daughter ions generated by fragmentation are m/z 1294.65, m/z 1157.59, and m/z 1100.57.
In some embodiments, the isotopically labeled internal standard peptide fragment may be PRTC: SAAGAFGPELSR% 13 C 6 15 N 4 ,+10Da)。
In step S300, analysis is performed on the chromatographic peak, the original peak area, and the comparison histogram of the original peak area of the peptide fragment obtained by the parallel reaction monitoring, and the detection results of the peptide fragment compositions of different groups are compared, so that the ALDH1L1 in the pig tissue samples to be detected of different groups can be relatively quantified.
Specifically, data analysis is performed on the original file by using Skyline software, 3 sub-ions with higher abundance and as continuous as possible can be selected for quantitative analysis, and the sub-ion peak areas of the target peptide (i.e., the sub-ion peak areas of the first peptide ANATEFGLASGVFTR, the second peptide DLGEAALNEYLR and the third peptide DTNHGPQNHQAHLR obtained in step S200) are integrated to obtain the original peak areas of the peptide in the sample; then correcting the peak areas of the heavy isotope labeled internal standard peptide fragments (namely, the peak areas of the ion peaks of the first peptide fragment ANATEFGLASGVFTR, the second peptide fragment DLGEAALNEYLR and the third peptide fragment DTNHGPQNHQAHLR with heavy isotope labels) to respectively obtain the relative expression amount information of the first peptide fragment ANATEFGLASGVFTR, the second peptide fragment DLGEAALNEYLR and the third peptide fragment DTNHGPQNHQAHLR 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 (3) analyzing the expression quantity of the target protein ALDH1L1, and further calculating the relative expression quantity difference of the target protein in different sample groups according to the relative expression quantity of the corresponding peptide fragment 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 achieve the detection of the peptide fragment composition of pig 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 in any of the above embodiments, and has the beneficial effects of the corresponding peptide fragment composition embodiment, which are not described in detail herein.
It should be noted that some embodiments of the present application are described above. 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 invention 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 10-formyl tetrahydrofolate dehydrogenase ALDH1L1
1. ALDH1L1 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 of protein was taken as the sample of each group, dithiothreitol (DTT) was added to the sample to a final concentration of 100mM, disulfide bonds were opened, the mixture was boiled in a water bath for 15 minutes, cooled to room temperature, 200. Mu.L of urea buffer (UA buffer,8M Urea,150mM Tris-HCl, pH 8.0) was added to the mixture, the mixture was transferred to a 10KD ultrafilter tube, and the mixture was centrifuged for 14000g for 30 minutes. 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. 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 comparison screening and calculation of the information such as the chromatographic peaks of the peptide fragments obtained by the mass spectrometry, it was confirmed that the sequence information of the peptide fragment composition for the relative quantitative analysis of porcine 10-formyl-tetrahydrofolate dehydrogenase ALDH1L1 in Table 1 could be obtained in all the treatment groups, and that the mass-to-charge ratio information of the peptide fragment composition for the relative quantitative analysis of porcine 10-formyl-tetrahydrofolate dehydrogenase ALDH1L1 in Table 2 could be obtained, and that the specific steps and specific parameter conditions of the individual steps of the method for the relative quantitative analysis of porcine 10-formyl-tetrahydrofolate dehydrogenase ALDH1L1, and the like, were determined.
Example 2 application to quantitative relative liver ALDH1L1 protein of diquat-induced oxidative stress pig model
1. ALDH1L1 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 of protein was taken as the sample of each group, dithiothreitol (DTT) was added to the sample to a final concentration of 100mM, disulfide bonds were opened, the mixture was boiled in a water bath for 15 minutes, cooled to room temperature, 200. Mu.L of urea buffer (UA buffer,8M Urea,150mM Tris-HCl, pH 8.0) was added to the mixture, the mixture was transferred to a 10KD ultrafilter tube, and the mixture was centrifuged for 14000g for 30 minutes. 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 three primary and secondary ion pair mass to charge ratio information of the specific polypeptide are respectively: m/z 770.89 is ANATEFGLASGVFTR parent ion, m/z 1054.57, m/z907.50, m/z 737.39 for the daughter ion produced by fragmentation; m/z 682.35 is DLGEAALNEYLR parent ion, and the daughter ions generated by fragmentation are m/z 1135.57, m/z 949.51 and m/z 878.47; m/z 812.89 is DTNHGPQNHQAHLR parent ion, and the daughter ions generated by fragmentation are m/z 1294.65, m/z 1157.59, and m/z 1100.57.
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: as shown in fig. 2-5. In the diquat and lipoic acid treatment group (DL), the second mass spectrum of the first peptide ANATEFGLASGVFTR is shown in fig. 2, the second mass spectrum of the second peptide DLGEAALNEYLR is shown in fig. 3, and the second mass spectrum of the third peptide DTNHGPQNHQAHLR is shown in fig. 4.
FIG. 5 is a graph showing the relative quantification of the average value of ALDH1L1 protein in the control group (CK) compared to the ALDH1L1 protein in the three treatment groups (diquat-induced oxidative stress treatment (DQ), antioxidant lipoic acid treatment (LA) and diquat + lipoic acid treatment (DL)). DL is the relative quantification of ALDH1L1 protein in the diquat + lipoic acid treated group (DL) relative to ALDH1L1 protein in the control group (CK). DQ is the relative quantification of the ALDH1L1 protein in the diquat-induced oxidative stress treated group (DQ) relative to the ALDH1L1 protein in the control group (CK). LA is the relative quantification of ALDH1L1 protein in the antioxidant lipoic acid treated group (LA) relative to ALDH1L1 protein in the control group (CK).
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 1.
Test results: see fig. 6.
In FIG. 6, CK is a relative quantification of the average value of ALDH1L1 in the control group (CK) compared to ALDH1L1 in the three treatment groups (diquat-induced oxidative stress treatment group (DQ), antioxidant lipoic acid treatment group (LA) and diquat + lipoic acid treatment group (DL)). DL is the relative quantification of ALDH1L1 in the diquat + lipoic acid treated group (DL) versus ALDH1L1 in the control group (CK). DQ is the relative quantification of ALDH1L1 in the diquat-induced oxidative stress treated group (DQ) relative to ALDH1L1 in the control group (CK). LA is the relative quantitative result of ALDH1L1 in the antioxidant lipoic acid treated group (LA) versus ALDH1L1 in the control group (CK).
Comparing the relative quantitative result box plots of FIG. 5 obtained in example 2 and FIG. 6 obtained in comparative example 1, it can be seen that the trend of variation of ALDH1L1 identified in example 2 between the different treatment groups was consistent with the trend of variation of ALDH1L1 identified in comparative example 1 between the different treatment groups. The embodiment of the application comprises a relatively quantitative analysis pig for relatively quantitatively analyzing pig source 10-formyltetrahydrofolate dehydrogenase A, has good accuracy, and the peptide fragment composition can target and identify pig 10-formyltetrahydrofolate dehydrogenase ALDH1L1, and has higher sensitivity compared with the existing in-vitro labeling detection method, such as the high-throughput identification pig total protein of 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 pig 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 and use thereof
<130> FI221035
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 15
<212> PRT
<213> amino acid sequence of first peptide fragment
<400> 1
Ala Asn Ala Thr Glu Phe Gly Leu Ala Ser Gly Val Phe Thr Arg
1 5 10 15
<210> 2
<211> 12
<212> PRT
<213> amino acid sequence of second peptide fragment
<400> 2
Asp Leu Gly Glu Ala Ala Leu Asn Glu Tyr Leu Arg
1 5 10
<210> 3
<211> 14
<212> PRT
<213> amino acid sequence of third peptide fragment
<400> 3
Asp Thr Asn His Gly Pro Gln Asn His Gln Ala His Leu Arg
1 5 10

Claims (5)

1. A method for the relative quantitative analysis of porcine 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 for non-disease diagnostic purposes, characterized in that the relative quantitative analysis of porcine liver tissue 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 is performed by liquid chromatography-mass spectrometry using a peptide composition comprising a first peptide fragment, a second peptide fragment and a third 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; the amino acid sequence of the third peptide is shown as SEQ ID NO. 3;
the method for carrying out relative quantitative analysis on the 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 of the pig liver tissue by adopting the liquid chromatography-mass spectrometry comprises the following steps:
providing different groups of pig liver samples to be tested, and respectively carrying out protein extraction and proteolytic treatment to obtain different groups of peptide segments to be tested;
detecting the peptide fragment compositions of different groups respectively by a liquid chromatography-mass spectrometry method; wherein, 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;
the detection results of the peptide fragment compositions of different groups are compared to relatively quantify the 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 in the liver tissue samples of the pigs to be detected of different groups.
2. The method of claim 1, wherein the parent ion of the first peptide fragment is 770.89m/z, the child ion is 1054.57m/z,907.50m/z and 737.39m/z, and the collision energy of the ion is 27V; the parent ion of the second peptide is 682.35m/z, the child ion is 1135.57m/z,949.51m/z and 878.47m/z, and the collision energy corresponding to the child ion is 27V; the parent ion of the third peptide is 812.89m/z, the child ion is 1294.65m/z,1157.59m/z and 1100.57m/z, and the collision energy corresponding to the child ion is 27V.
3. The method according to claim 1, wherein the detection of the different groups of peptide fragment compositions by liquid chromatography-mass spectrometry, respectively, comprises: respectively incorporating isotopically labeled internal standard peptide fragments into the peptide fragment compositions of different groups; the weight of the internal standard peptide fragment is the same as the weight of the peptide fragment composition;
and (3) performing liquid chromatography-mass spectrometry detection on the obtained peptide fragment composition.
4. The method of claim 1, wherein the liquid chromatography-mass spectrometry detection is high performance liquid chromatography-tandem mass spectrometry.
5. The method of claim 1, wherein each of the different groups contains 4 biological duplicate samples.
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WO2000074711A2 (en) * 1999-06-03 2000-12-14 Pharmaproducts Uk Limited 10-formyltetrahydrofolate dehydrogenase as therapeutical agent
WO2015160470A2 (en) * 2014-03-20 2015-10-22 The Trustees Of Princeton University Nadph production by the 10-formyl-thf pathway, and its use in the diagnosis and treatment of disease

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