CN115197298A - Peptide fragment composition for relatively quantitatively analyzing porcine cytochrome P450 enzyme CYP2E1 and application - Google Patents

Peptide fragment composition for relatively quantitatively analyzing porcine cytochrome P450 enzyme CYP2E1 and application Download PDF

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CN115197298A
CN115197298A CN202210585167.3A CN202210585167A CN115197298A CN 115197298 A CN115197298 A CN 115197298A CN 202210585167 A CN202210585167 A CN 202210585167A CN 115197298 A CN115197298 A CN 115197298A
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peptide fragment
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高杰
朴香淑
郝月
顾先红
张名媛
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Abstract

The application provides a peptide fragment composition, a method and a kit for relatively quantitatively analyzing porcine cytochrome P450 enzyme CYP2E1. The peptide fragment composition for relatively quantitatively analyzing the porcine cytochrome P450 enzyme CYP2E1 comprises 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 segment is shown as SEQ ID NO. 2; the amino acid sequence of the third peptide segment is shown in SEQ ID NO. 3. The porcine CYP2E1 can be detected quickly and efficiently in a quantitative manner on the protein level, and the detection flux and efficiency of the porcine CYP2E1 are improved. Compared with the Western Blot method based on an antibody, the method has stronger specificity.

Description

Peptide fragment composition for relatively quantitatively analyzing porcine cytochrome P450 enzyme CYP2E1 and application
Technical Field
The application relates to the field of relative quantitative analysis of protein, in particular to a peptide fragment composition for relative quantitative analysis of porcine cytochrome P450 enzyme CYP2E1 and application thereof.
Background
CYP2E1 is Cytochrome P450 enzyme (CYP) with the highest content in normal pig liver tissue, consists of 493 amino acids, has the molecular weight of 56.9Kda, is mainly distributed in the liver and is enriched in the central area of hepatic lobule. The main function is to participate in the biotransformation of precancerogen and environmental toxicants (benzene, ethanol, carbon tetrachloride, vinyl chloride and the like). CYP2E1 catalyzes a plurality of oxidation biochemical reactions requiring NADPH, hydroxyethyl, aldehyde, peroxide and other intermediate products are often generated in the process of catalyzing substrate metabolism, incomplete oxidation leads to the generation of oxidation free radicals and active derivatives (ROS), especially, under the catalysis of iron, the ability of CYP2E1 to generate ROS (such as superoxide anion free radicals and hydrogen peroxide) is enhanced, strong oxidants (such as hydroxyl free radicals) are generated, the balance of an in-vivo oxidation resistance system is broken, lipid peroxidation is caused, cell membrane damage, DNA damage and the like are caused, the health level of pigs is influenced, and the loss of the pig industry is caused.
Therefore, there is a strong need to intensively study a relative quantitative analysis method of the cytochrome P450 enzyme CYP2E1 of swine.
Disclosure of Invention
In view of the above, the present application aims to provide a peptide fragment composition for relatively quantitatively analyzing porcine cytochrome P450 enzyme CYP2E1 and an application thereof.
In view of the above objects, the present application provides a peptide fragment composition for relatively quantitative analysis of porcine cytochrome P450 enzyme CYP2E1, comprising a first peptide fragment, a second peptide fragment and a third peptide fragment;
wherein the amino acid sequence of the first peptide segment is shown as SEQ ID NO. 1; the amino acid sequence of the second peptide segment is shown as SEQ ID NO. 2; the amino acid sequence of the third peptide segment is shown as SEQ ID NO. 3.
In some of these embodiments, the parent ion of the first peptide fragment is 861.96m/z, the daughter ions are 1234.65m/z,1121.57m/z and 710.36m/z, and the corresponding collision energy of the daughter ions is 27V; the parent ion of the second peptide fragment is 599.83m/z, the daughter ion is 998.58m/z,861.52m/z and 714.45m/z, and the corresponding collision energy of the daughter ion is 27V; the parent ion of the third peptide fragment is 578.31m/z, the daughter ion is 856.47m/z,658.37m/z and 284.17m/z, and the corresponding collision energy of the daughter ion is 27V.
The embodiment of the application also provides a method for relatively quantitatively analyzing the porcine cytochrome P450 enzyme CYP2E1, wherein the peptide fragment composition is applied to relatively quantitatively analyzing the porcine cytochrome P450 enzyme CYP2E1.
In some of these embodiments, the performing a relative quantification of porcine cytochrome P450 enzyme CYP2E1 comprises:
providing different groups of to-be-detected pig tissue samples, and respectively carrying out protein extraction and proteolysis treatment to obtain different groups of to-be-detected peptide fragments;
respectively detecting the peptide fragment compositions of different groups by a liquid chromatography-mass spectrometry combined method;
and comparing the detection results of the peptide fragment compositions of different groups to relatively quantify the porcine cytochrome P450 enzyme CYP2E1 in the porcine tissue samples to be detected of different groups.
In some of the embodiments, the pig tissue sample to be tested is a pig liver sample to be tested.
In some embodiments, the method of liquid chromatography-mass spectrometry uses a high performance liquid chromatography-tandem mass spectrometer.
In some of these embodiments, the conditions for the combined liquid chromatography-mass spectrometry are as follows:
chromatographic conditions are as follows: and (3) chromatographic column: a C18 column; a mobile phase A:0.1% aqueous formic acid, mobile phase B:0.1% aqueous acetonitrile formate; gradient elution procedure: 0-2min, 5-10% of solution B; 2-45min, 10% -30% of solution B; 45-55min, 30-100% of solution B; 55-60min, 100 percent of solution B; flow rate: 250-450 nl/min;
mass spectrum conditions: collecting data in a positive ion mode; primary mass spectrum scanning range: 300-1800m/z, mass spectral resolution: 60000 (m/z 200), AGC target:3e6, maximum IT:200ms; MS2 scans:20; isolation window:1.6Th, mass spectral resolution: 30000 (m/z 200), AGC target:3e6, maximum IT:120ms, MS2 Activation Type: HCD, collision energy: and 27V.
In some of these embodiments, the detection results include detection results with isotopically labeled peptide fragment compositions; the isotope-labeled internal standard peptide segment is PRTC: saagafgpersr ( 13 C 6 15 N 4 ,+10Da)。
The embodiment of the application also provides a kit for relatively quantitatively analyzing the porcine cytochrome P450 enzyme CYP2E1, wherein the kit contains a reagent for detecting the peptide fragment composition.
In some embodiments, the reagent comprises a first peptide fragment standard, a second peptide fragment standard, a third peptide fragment standard, a dithiothreitol solution, an iodoacetamide solution, and a trypsin solution.
From the above, the peptide fragment composition, the method and the kit for relatively quantitatively analyzing the porcine cytochrome P450 enzyme CYP2E1 provided by the application can specifically detect and quantitatively analyze the CYP2E1 by using a liquid chromatograph-mass spectrometer on the protein level through the peptide fragment composition comprising the first peptide fragment, the second peptide fragment and the third peptide fragment, can rapidly and efficiently quantitatively detect the porcine CYP2E1, and improve the detection flux and efficiency of the porcine CYP2E1. Compared with an antibody-based Western Blot method, the method has stronger specificity, avoids the complex steps, long period and high cost for preparing the CYP2E1 monoclonal antibody, and also avoids the problems of more serious cross reaction, low success rate and the like for preparing the CYP2E1 polyclonal antibody.
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In order to more clearly illustrate the technical solutions in the present application or the related art, the drawings needed to be used in the description of the embodiments or the related art will be briefly introduced below, and it is obvious that the drawings in the following description are only embodiments of the present application, and it is obvious for those skilled in the art that other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a flowchart of a method for relatively quantitatively analyzing a porcine cytochrome P450 enzyme CYP2E1 according to an embodiment of the present application;
FIG. 2 is a secondary mass spectrum of the first peptide fragment FIDLIPSNLPHEATR in example 2;
FIG. 3 is a secondary mass spectrum of the second peptide segment EAHFLLELR of example 2;
FIG. 4 is a second mass spectrum of the third peptide fragment GEIPTFQVHK of example 2;
FIG. 5 is a boxplot of the relative quantification results of CYP2E1 protein for each of the treatment groups and the control group in example 2;
fig. 6 is a boxplot of relative quantification results of CYP2E1 protein in each of the treatment groups and the control group in comparative example 1.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application is further described in detail below with reference to the accompanying drawings in combination with specific embodiments.
It should be noted that technical terms or scientific terms used in the embodiments of the present application should have a general meaning as understood by those having ordinary skill in the art to which the present application belongs, unless otherwise defined. The use of "first," "second," and similar terms in the embodiments of the present application do not denote any order, quantity, or importance, but rather the terms are used to distinguish one element from another. The word "comprising" or "comprises", and the like, means that the element or item preceding the word comprises the element or item listed after the word and its equivalent, but does not exclude other elements or items.
In the pig industry, the ROS is increased sharply, so that the oxidative stress in the pig body is easily caused. The oxidative stress can cause the damage of the pig liver to cause the reduction of the health level of the organism and the like, thereby bringing economic loss to the pig raising industry. The excessive accumulated ROS in the pig body mainly attacks biomacromolecules such as lipid, protein, nucleic acid and the like, so that cell components are damaged, and the structure and the function of the biomacromolecules are changed, and the lipid peroxidation chain reaction can also damage the structure and the function of a biological membrane, so that the oxidative damage and the cell apoptosis are aggravated. Therefore, when CYP2E1 catalyzes a plurality of oxidation biochemical reactions requiring NADPH, incomplete oxidation can cause oxidation stress of a pig body, and the generated ROS can cause inflammatory reaction and liver lipid oxidation, so that fatty infiltration and liver swelling are caused, diseases such as fatty liver and hepatic fibrosis are caused, and the health of the pig is damaged.
Some assays for determining the relative amount of CYP2E1 in vivo have included analyses at the gene level and protein level. The gene level analysis is mainly performed on the mRNA level, for example, cDNA formed by reverse transcription after extracting RNA in a tissue body is amplified according to a primer pair specific to the gene design of CYP2E1, and the transcription level of CYP2E1 in the template is compared and analyzed by using a fluorescent real-time quantitative PCR technology. However, after gene transcription, the protein is usually required to be subjected to regulation such as shearing and splicing to be expressed into the CYP2E1 protein, so that the expression level of the CYP2E1 protein is analyzed on the mRNA level and the expression level of the gene product protein CYP2E1 cannot be truly reflected. CYP2E1 is analyzed on the protein level by methods such as enzyme-linked immunization, western Blot and tissue in situ hybridization, and the detection methods generally need high-quality antibodies to specifically recognize CYP2E1. The CYP2E1 antigen protein for preparing the antibody on the market is usually from model organisms such as human beings, mice, rats and the like, and the expression quantity of the porcine cytochrome P450 enzyme CYP2E1 cannot be quantitatively analyzed on the protein level.
The method for quantitatively analyzing the porcine cytochrome P450 enzyme CYP2E1 at the protein level can be established by preparing antibodies with strong specificity, such as monoclonal antibodies, polyclonal antibodies and the like, for identifying the porcine cytochrome P450 enzyme CYP2E1. However, monoclonal antibodies have problems of long preparation period, high cost and the like; polyclonal antibodies, although less expensive, have a more severe cross-reactivity and a lower success rate.
Therefore, the current analysis of the porcine cytochrome P450 enzyme CYP2E1 has the problems of inaccurate detection, high complexity and cost of antibody preparation and the like.
Based on this, the embodiment of the application provides the peptide fragment composition for relatively quantitatively analyzing the porcine cytochrome P450 enzyme CYP2E1 and the application thereof, and the peptide fragment composition can be used for relatively quantitatively analyzing the porcine cytochrome P450 enzyme CYP2E1 on the protein level, so that the problems that the existing analysis on the porcine-derived CYP2E1 is not accurate enough in detection, the antibody preparation is complex and high in cost and the like are solved to a certain extent.
Table 1 shows peptide fragment compositions for relatively quantitative analysis of porcine cytochrome P450 enzyme CYP2E1 provided in the examples of the present application.
Referring to table 1, the present application provides a peptide composition for relatively quantitative analysis of porcine cytochrome P450 enzyme CYP2E1, including a first peptide, a second peptide and a third peptide. Wherein the amino acid sequence of the first peptide segment is shown as SEQ ID NO. 1; the amino acid sequence of the second peptide segment is shown as SEQ ID NO. 2; the amino acid sequence of the third peptide segment is shown as SEQ ID NO. 3.
TABLE 1 peptide fragment composition for relatively quantitative analysis of porcine cytochrome P450 enzyme CYP2E1
Figure BDA0003663190450000051
The peptide fragment composition for relatively quantitatively analyzing the porcine cytochrome P450 enzyme CYP2E1 comprises a first peptide fragment, a second peptide fragment and a third peptide fragment, and is specifically detected by using a high-efficiency liquid chromatography-mass spectrometer (LC/MS/MS) and quantitatively analyzed for the CYP2E1, so that the peptide fragment composition in the porcine CYP2E1 can be rapidly and efficiently quantitatively detected on the protein level, and the detection flux and efficiency of the porcine CYP2E1 are improved. Compared with an antibody-based Western Blot method, the method has stronger specificity, can avoid the problems of complicated steps, long period, high cost and the like when the porcine CYP2E1 monoclonal antibody is prepared, and can also avoid the problems of serious cross reaction, low success rate and the like when the porcine CYP2E1 polyclonal antibody is prepared.
Table 2 shows mass-to-charge ratio information provided in the examples of the present application for the relative quantitative analysis of the peptide fragment composition of porcine cytochrome P450 enzyme CYP2E1.
TABLE 2 Mass to Charge ratio information for the relative quantitative analysis of peptide fragment composition of porcine cytochrome P450 enzyme CYP2E1
Figure BDA0003663190450000061
Referring to Table 2, in the peptide fragment composition for relatively quantitative analysis of the porcine cytochrome P450 enzyme CYP2E1 provided in the examples of the present application, the parent ion of the first peptide fragment FIDLIPSNLPHEATR is 861.96m/z, the daughter ion is 1234.65m/z,1121.57m/z and 710.36m/z, and the corresponding collision energy of the daughter ion is 27V. The parent ion of the second peptide segment EAHFLLELR is 599.83m/z, the daughter ion is 998.58m/z,861.52m/z and 714.45m/z, and the corresponding collision energy of the daughter ion is 27V; the parent ion of the third peptide segment GEIPTFQVHK is 578.31m/z, the daughter ions are 856.47m/z,658.37m/z and 284.17m/z, and the corresponding collision energy of the daughter ions is 27V. The mass-to-charge ratio, the collision energy and the like of parent ions and daughter ions of the combined peptide fragment composition can be used for separating and collecting signals of peptide fragments when analyzing porcine cytochrome P450 enzyme CYP2E1 by High performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).
Based on the same inventive concept, the present embodiments also provide a kit for relatively quantitative analysis of porcine cytochrome P450 enzyme CYP2E1, the kit comprising reagents for detecting the peptide fragment composition as described in any of the previous items.
In some embodiments, the reagents may include a first peptide fragment (i.e., first peptide fragment fidhlipsnlpheartr) standard, a second peptide fragment (second peptide fragment EAHFLLEALR) standard, a third peptide fragment (i.e., third peptide fragment GEIPTFQVHK) standard, a protein extraction reagent, a proteolytic digestion reagent, and the like.
The protein extraction reagent can be a common protein lysate in the field, such as an SDT protein lysate. The proteolytic reagent can be a conventional proteolytic reagent in the field, such as a dithiothreitol solution, an iodoacetamide solution, a 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 porcine cytochrome P450 enzyme CYP2E1 in any one of the above embodiments, and has the beneficial effects of the corresponding peptide fragment composition embodiment, which are not described again here.
Based on the same inventive concept, the embodiments of the present application further provide a method for relatively quantitatively analyzing the porcine cytochrome P450 enzyme CYP2E1, and the peptide fragment composition according to any of the previous embodiments is applied to relatively quantitatively analyze the porcine cytochrome P450 enzyme CYP2E1.
Fig. 1 shows a flowchart of a method for relatively quantitatively analyzing a porcine cytochrome P450 enzyme CYP2E1 according to an embodiment of the present application.
As shown in fig. 1, the method for relatively quantitatively analyzing the porcine cytochrome P450 enzyme CYP2E1 provided in the embodiment of the present application may include:
s100, providing different groups of to-be-detected pig tissue samples, and respectively performing protein extraction and proteolysis treatment to obtain different groups of to-be-detected peptide fragments;
s200, respectively detecting the peptide fragment compositions of different groups by a liquid chromatography-mass spectrometry combined method;
s300, comparing the detection results of the peptide fragment compositions of different groups to relatively quantify CYP2E1 in the pig tissue samples to be detected of different groups.
In some embodiments, in step S100, the pig tissue sample to be tested may be a pig liver sample to be tested. Each group can be set up with 4 biological sample replicates, respectively.
The protein extraction may include: and extracting the 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 treatment may comprise: and (3) sequentially treating the obtained protein extract by dithiothreitol to open a disulfide bond, treating free sulfydryl in the blocked protein by acetamide, and carrying out enzyme digestion treatment by trypsin to obtain a peptide fragment.
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;
and (3) digesting the second product by using trypsin.
In some embodiments, in step S200, the method may include:
screening peptide fragment compositions for relatively quantitatively analyzing porcine cytochrome P450 enzyme CYP2E 1;
the peptide fragment compositions were identified in a targeted manner and corrected by isotopically labeled internal standards.
In some embodiments, the peptide fragment composition of the porcine cytochrome P450 enzyme CYP2E1 can be screened for relative quantification by a high performance liquid chromatography-mass spectrometry method. Wherein, the conditions of the high performance liquid chromatography-mass spectrometry combined method are as follows:
chromatographic conditions are as follows: and (3) chromatographic column: a C18 column; a mobile phase A:0.1% acetonitrile in water, mobile phase B:0.1% aqueous acetonitrile formate; gradient elution procedure: 0-2min, 5-10% of solution B; 2-45min, 10% -30% of solution B; 45-55min, 30-100% of solution B; 55-60min, 100 percent of solution B; flow rate: 250-450 nl/min;
mass spectrum conditions: collecting data in a positive ion mode; primary mass spectrum scanning range: 300-1800m/z, first-order mass spectrum resolution: 60000 (m/z 200), AGC target:3e6, primary Maximum IT:50ms; secondary mass spectrometry analysis: MS2 scans:20; isolation window:1.6Th, secondary mass spectral resolution: 15000 (m/z 200), AGC target:1e5, secondary Maximum IT:50ms, MS2 Activation Type: HCD, collision energy: and 27V.
In some embodiments, the screening is repeated three or more times by using the method for screening the peptide fragment composition for relatively quantitatively analyzing the porcine cytochrome P450 enzyme CYP2E1, so that the method for relatively quantitatively analyzing the porcine cytochrome P450 enzyme CYP2E1 has reliable reproducibility. And comparing, screening and calculating the peptide chromatographic peak and other information obtained by mass spectrometry to determine the specific peptide information for realizing accurate and relative quantification of the protein.
Through the operation, the peptide sequence of the CYP2E1 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 CYP2E1 can be identified through the established data acquisition mode, and the consistency of signal response among technical repeats is preliminarily evaluated. Three specific peptide fragments with sequences of FIDLIPSNLPHEATR, EAHFLLEALR and GEIPTFQVHK are obtained (namely, the peptide fragment composition for relatively quantitatively analyzing the porcine cytochrome P450 enzyme CYP2E1 shown in the table 1 is obtained). And simultaneously obtaining the mass-to-charge ratio information, collision energy and the like of the parent-child ion pairs of the three specific peptide fragments (namely obtaining the mass-to-charge ratio information of the peptide fragment composition of the porcine cytochrome P450 enzyme CYP2E1 shown in the table 2 for relative quantitative analysis).
In some embodiments, targeted identification of the peptide fragment composition and correction by isotopically labeled internal standards may specifically include:
and (4) respectively taking preset quantities of different groups of peptide fragments to be detected in the step S100, doping the internal standard peptide fragments marked by the heavy isotopes with equal quantity for detection, and separating the peptide fragments by adopting LC/MS/MS and collecting signals. In the step, a target peptide fragment parallel reaction monitoring result can be obtained. The result may include information such as peptide fragment chromatographic peaks, raw peak areas, and a histogram of the raw peak areas.
The conditions of the high performance liquid chromatography-mass spectrometry combined method can be as follows:
chromatographic conditions are as follows: and (3) chromatographic column: a C18 column; mobile phase A:0.1% acetonitrile in water, mobile phase B:0.1% acetonitrile in formic acid; gradient elution procedure: 0 to 2min,5 to 10 percent of solution B; 2-45min, 10% -30% of solution B; 45-55min, 30-100% of solution B; 55-60min, 100 percent of solution B; flow rate: 250-450 nl/min;
mass spectrum conditions: collecting data in a positive ion mode; primary mass spectrum scanning range: 300-1800m/z, mass spectral resolution: 60000 (m/z 200), AGC target:3e6, maximum IT:200ms; MS2 scans:20; isolation window:1.6Th, mass spectral resolution: 30000 (m/z 200), AGC target:3e6, maximum IT:120ms, MS2 Activation Type: HCD, collision energy: 27V.
Three primary and secondary ion mass-to-charge ratio information pairs of the specific polypeptide are respectively set as follows: m/z861.96 is the parent ion of FIDLIPSNLPHEATR, and the daughter ion generated by fragmentation is m/z 1234.65, m/z1121.57 and m/z 710.36; m/z 599.83 is the parent ion of EAHFLLEALR, and the daughter ion generated by fragmentation is m/z998.58, m/z 861.52, m/z 714.45; m/z 578.31 is the parent ion of GEIPTFQVHK, and the daughter ion generated by fragmentation is m/z 856.47, m/z 658.37, m/z 284.17.
In some embodiments, the isotopically labeled internal standard peptide fragment can be PRTC: saagafgpersr ( 13 C 6 15 N 4 ,+10Da)。
In step S300, the CYP2E1 in the pig tissue samples to be detected of different groups can be relatively quantified by analyzing the peptide fragment chromatographic peak obtained by parallel reaction monitoring, the original peak area, the histogram of the original peak area, and the like, and comparing the detection results of the peptide fragment compositions of different groups.
Specifically, data analysis is performed on the original file by using the Skyline software, and 3 sub-ions with high abundance of the peptide segment and continuous as much as possible can be selected to perform quantitative analysis, and first, the peak areas of the sub-ions of the target peptide segment (that is, the peak areas of the sub-ions of the target peptide segment obtained in step S200) are integrated to obtain the original peak area of the peptide segment in the sample; then, the peak area of the heavy isotope labeled internal standard peptide segment (namely, the peak area of the ion of the target peptide segment obtained in the step S200) is used for correction, so that the relative expression amount information of each peptide segment in the samples of different groups is obtained; and finally, calculating the average value of the relative expression quantity of the target peptide in each group of samples, and performing statistical analysis. And analyzing the expression quantity of the target protein, and further calculating to obtain 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 among different sample groups. The specific calculation is the prior art, and may be implemented by, for example, skyline software, so that detailed calculation steps and the like are not described herein again.
The method of the above embodiment is used for realizing the detection of the peptide fragment composition of the corresponding relative quantitative analysis porcine cytochrome P450 enzyme CYP2E1 in any one 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 the above 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 may also be possible or may be advantageous.
The technical solution of the present invention will be further described with reference to the following embodiments.
The experimental procedures in the following examples are conventional unless otherwise specified.
The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
Example 1 screening for relative quantitative analysis of peptide fragment composition of porcine cytochrome P450 enzyme CYP2E1
1. CYP2E1 protein extraction: pig liver tissue samples of a diquat induced oxidative stress treatment group (DQ), an antioxidant lipoic acid treatment group (LA), a diquat + lipoic acid treatment group (DL) and a control group (CK) are respectively taken and added with a proper amount of SDT lysate, and the obtained mixture is transferred into a 2ml centrifuge tube which is filled with a proper amount of quartz sand in advance, and a homogenizer is used for homogenizing and crushing (24 multiplied by 2,6.0M/S,60S and twice). Then, the ultrasonic wave (100W, working time 10s, intermittent time 10s, circulation time 10 times) is carried out, and the water is boiled for 10min.14000g, centrifuging for 10min, taking the supernatant, filtering by a 10kD ultrafiltration membrane, and collecting the filtrate. Protein quantification was performed by BCA method. Samples were aliquoted and stored at-80 ℃. Each group contained 4 biological replicates.
2. And (3) proteolysis: in each treatment group and the control group, about 200ug of protein was sampled from each group, dithiothreitol (DTT) was added to a final concentration of 100mM to open disulfide bonds, followed by 15min in a boiling water bath, cooling to room temperature, adding 200. Mu.L of urea buffer (UA buffer,8M Urea,150mM Tris-HCl, pH 8.0), mixing well, transferring to a 10KD ultrafiltration tube, and centrifuging 14000g 30min. Add 200. Mu.l of UA buffer and centrifuge 14000g 30min, discard the filtrate. Add 100. Mu.L iodoacetamide (IAA, 50mM IAA in UA) to alkylate the free thiol groups inside the blocked protein, shake at 600rpm for 1min, protect from light for 30min at room temperature, and centrifuge for 14000g 20min. Add 100. Mu.L of UA buffer and centrifuge 14000g for 20min for 3 times. Add 100. Mu.L of NH 4 HCO 3 buffer (50 mM), centrifuge 14000g 20min for 2 times. Add 40. Mu.L NH 4 HCO 3 buffer (containing Trypsin in a 1-enzyme ratio), shaking at 600rpm for 1min, and carrying out digestion at 37 ℃ for 16h. The collection tube was replaced with a fresh one and centrifuged for 14000g 15min. Add 40. Mu.L NH 4 HCO 3 The buffer (50 mM) was centrifuged at 14000g 30min and the filtrate collected. The peptide fragment after enzymolysis is desalted and lyophilized, then is redissolved by 0.1 percent Formic Acid (FA), and the concentration of the peptide fragment is determined by OD 280.
3. Screening the specific peptide fragment of the target protein and establishing a data acquisition method: firstly, randomly selecting a sample from a plurality of repetitions of each group in the step 2, respectively taking a proper amount of peptide fragments after enzymolysis, and equivalently mixing the peptide fragments into a sample; taking 1ug of mixed peptide fragment, and performing high performance liquid chromatography separation by using an HPLC system; the buffer solution A is 0.1% formic acid aqueous solution, and the solution B is 0.1% formic acid acetonitrile aqueous solution (acetonitrile is 84%); the chromatographic column is balanced by 95 percent of solution A; samples were run through a Trap Column (100 μm. By 50mm,5 μm-C18, dr. Maisch phases, r25. Aq), and an Analytical Column (180 μm. By 150mm,3 μm-C18, dr. Maisch phases, r23. Aq) at a flow rate of 300nl/min; the liquid phase separation gradient was as follows: from 0min to 2min, linear gradient of liquid B from 5% to 10%, from 2min to 45min, linear gradient of liquid B from 10% to 30%; 45-55 minutes, linear gradient of B liquid from 30% to 100%; the linear gradient of the liquid B is maintained at 100 percent within 55-60 minutes. Then, performing targeted qualitative analysis by using a Q-active HF mass spectrometer through a parallel reaction monitoring method; analysis duration: 60min, detection mode: positive ion, parent ion scan range: 300-1800m/z, first order mass spectral resolution: 60000 (m/z 200), AGC target:3e6, primary Maximum IT:50ms; peptide fragment secondary mass spectrometry was collected as follows: acquisition of 20 secondary mass spectra (MS 2 scan) was triggered after each full scan (full scan), secondary mass resolution: 15000 (m/z 200), AGC target:1e5, secondary Maximum IT:50ms, MS2 Activation Type: HCD, isolation window:1.6Th, normalized collagen energy:27; and performing MS2 scanning on the candidate peptide fragment of the target protein by LC-MS/MS by adopting a targeted shotgun scanning mode.
Through comparison, screening and calculation of information such as peptide chromatographic peaks obtained by mass spectrometry, pig liver tissue samples of a diquat induced oxidative stress treatment group (DQ), an antioxidant lipoic acid treatment group (LA), a diquat + lipoic acid treatment group (DL) and a control group (CK) are determined to obtain sequence information of the peptide composition of the pig cytochrome P450 enzyme CYP2E1 used for relatively quantitative analysis in the table 1, and to obtain mass-to-charge ratio information of the peptide composition of the pig cytochrome P450 enzyme CYP2E1 used for relatively quantitative analysis in the table 2, and to determine specific steps of the method for relatively quantitative analysis of the pig cytochrome P450 enzyme CYP2E1, specific parameter conditions of the steps, and the like.
Example 2 application of liver CYP2E1 protein relativity quantification of aquacide induced oxidative stress fattening pig model
1. CYP2E1 protein extraction: pig liver tissue samples of a diquat induced oxidative stress treatment group (DQ), an antioxidant lipoic acid treatment group (LA), a diquat + lipoic acid treatment group (DL) and a control group (CK) are respectively taken and added with a proper amount of SDT lysate, and the obtained mixture is transferred into a 2ml centrifuge tube which is filled with a proper amount of quartz sand in advance, and a homogenizer is used for homogenizing and crushing (24 multiplied by 2,6.0M/S,60S and twice). Then, the ultrasonic wave (100W, working time 10s, intermittent time 10s, circulation time 10 times) is carried out, and the water is boiled for 10min.14000g, centrifuging for 10min, taking the supernatant, filtering by a 10kD ultrafiltration membrane, and collecting the filtrate. Protein quantification was performed using BCA method. Samples were aliquoted and stored at-80 ℃. Each group contained 4 biological replicates.
2. And (3) proteolysis: in each treatment group and control group, about 200ug of protein was sampled, dithiothreitol (DTT) was added to a final concentration of 100mM to open disulfide bonds, the mixture was boiled in a boiling water bath for 15min, cooled to room temperature, mixed with 200. Mu.L of urea buffer (UA buffer,8M Urea,150mM Tris-HCl, pH 8.0), transferred to a 10KD ultrafiltration tube, and centrifuged at 14000g 30min. 200 mul of UA buffer was added and centrifuged at 14000g 30min, and the filtrate was discarded. Add 100. Mu.L iodoacetamide (IAA, 50mM IAA in UA) to alkylate the free thiol groups inside the blocked protein, shake at 600rpm for 1min, protect from light for 30min at room temperature, and centrifuge for 14000g for 20min. Add 100. Mu.L of UA buffer, centrifuge 14000g for 20min and repeat 3 times. Add 100. Mu.L NH 4 HCO 3 buffer (50 mM), centrifuge 14000g 20min for 2 times. Add 40. Mu.L NH 4 HCO 3 buffer (containing trypsin T)rypsin, enzyme ratio 1:50 ) the cleavage was carried out, shaking at 600rpm for 1min, 16h at 37 ℃. The collection tube was replaced with a fresh one and centrifuged for 14000g 15min. Add 40. Mu.L NH 4 HCO 3 The buffer (50 mM) was centrifuged at 14000g 30min and the filtrate collected. The peptide fragment after enzymolysis is desalted and lyophilized, then is redissolved by 0.1 percent Formic Acid (FA), and the concentration of the peptide fragment is determined by OD 280.
3. Target identification of target protein specific peptide fragments and correction by isotopic internal standard: firstly, taking about 1ug of peptide fragment from each group of multiple repeated samples in the step 2, respectively doping 20fmol heavy isotope labeled internal standard peptide fragment (PRTC: SAAGAFGPELSR) for detection, and performing high performance liquid chromatography separation on the polypeptide by adopting an HPLC system; the buffer solution A is 0.1% formic acid aqueous solution, and the solution B is 0.1% formic acid acetonitrile aqueous solution (acetonitrile is 84%); the chromatographic column is balanced by 95 percent of solution A; injecting a sample into a chromatographic analysis column for gradient separation, wherein the flow rate is 300nl/min; the liquid phase separation gradient was as follows: from 0min to 2min, linear gradient of liquid B from 5% to 10%, from 2min to 45min, linear gradient of liquid B from 10% to 30%; 45-55 minutes, linear gradient of B liquid from 30% to 100%; the linear gradient of the liquid B is maintained at 100 percent within 55-60 minutes. After the high performance liquid chromatography separation, performing parallel reaction monitoring mass spectrometry on five target peptide segments of the identified target protein by using a Q-active HF mass spectrometer, wherein the analysis time is as follows: 60min, detection mode: a positive ion; primary mass spectrum scanning range: 300-1800m/z, mass spectral resolution: 60000 (m/z 200), AGC target:3e6, maximum IT:200ms; after each one-stage MS scan (full MS scan), 20 parallel reaction monitoring scans (MS 2 scans) were collected according to the Inclusion list, isolation window:1.6Th, mass spectral resolution: 30000 (m/z 200), AGC target:3e6, maximum IT:120ms, MS2 Activation Type: HCD, normalized fusion energy:27.
three primary and secondary ion mass-to-charge ratio information pairs for setting specific polypeptide are respectively as follows: m/z861.96 is the parent ion of FIDLIPSNLPHEATR, and the daughter ion generated by fragmentation is m/z 1234.65, m/z1121.57 and m/z 710.36; m/z 599.83 is the parent ion of EAHFLLEALR, and the daughter ion generated by fragmentation is m/z998.58, m/z 861.52, m/z 714.45; m/z 578.31 is the parent ion of GEIPTFQVHK, and the daughter ion generated by fragmentation is m/z 856.47, m/z 658.37, m/z 284.17.
4. Analyzing the monitoring result of the parallel reaction of the target peptide fragment: and analyzing the target protein specific peptide segment data obtained by parallel reaction monitoring, wherein the target protein specific peptide segment data comprises information such as a peptide segment chromatographic peak, an original peak area, a comparison histogram of the original peak area and the like. 3 daughter ions with high abundance of peptide fragments and continuous as much as possible are selected for quantitative analysis. Firstly, integrating the peak area of a daughter ion of a target peptide fragment to obtain the original peak area of the peptide fragment in a sample; then, correcting the peak area of the heavy isotope labeled internal standard peptide segment to obtain the relative expression amount information of each peptide segment in different samples; and finally, calculating the average value of the relative expression quantity of the target peptide in each group of samples, and performing statistical analysis. And analyzing the expression quantity of the target protein, and further calculating to obtain the relative expression quantity difference of the target protein in different sample groups according to the relative expression quantity of the corresponding peptide segment of each target protein among different sample groups.
And (3) test results: see fig. 2-5. In the diquat + lipoic acid (DL) treatment group, the second-order mass spectrum of the first peptide fragment FIDLIPSNLPHEATR is shown in figure 2, the second-order mass spectrum of the second peptide fragment EAHFLLEALR is shown in figure 3, and the second-order mass spectrum of the third peptide fragment GEIPTFQVHK is shown in figure 4.
DQ in FIG. 5 is the relative quantification result of the porcine-derived CYP2E1 protein in the diquat-induced oxidative stress treated group (DQ) relative to the porcine-derived CYP2E1 protein in the control group (CK). LA is a relative quantification result of the swine-derived CYP2E1 protein in the antioxidant lipoic acid treatment group (LA) with respect to the swine-derived CYP2E1 protein in the control group (CK). DL is the relative quantitative result of the porcine-derived CYP2E1 protein in the diquat + lipoic acid treatment group (DL) relative to the porcine-derived CYP2E1 protein in the control group (CK). CK is the relative quantification of the porcine-derived CYP2E1 protein in the control group (CK) compared to the average of the porcine-derived CYP2E1 protein in the three treatment groups, the diquat induced oxidative stress treatment group (DQ), the antioxidant lipoic acid treatment group (LA), and the diquat + lipoic acid treatment group (DL).
Comparative example 1
The difference from the embodiment 2 is that based on the LC-MS/MS method, the peptide fragment after enzymolysis is detected by adopting the TMT (Tandem Mass Tag) protein isotope labeling quantitative technology according to the specific operation instruction of the TMT protein labeling kit of Thermo company.
Specifically, the peptide fragments after enzymolysis are obtained through step 1 and step 2 in example 2.
And (3) test results: see fig. 6.
DQ in FIG. 6 is the relative quantification result of the porcine-derived CYP2E1 protein in the diquat-induced oxidative stress treated group (DQ) relative to the porcine-derived CYP2E1 protein in the control group (CK). LA is the relative quantification of the swine-derived CYP2E1 protein in the antioxidant lipoic acid treatment group (LA) relative to the swine-derived CYP2E1 protein in the control group (CK). DL is the relative quantitative result of the porcine-derived CYP2E1 protein in the diquat + lipoic acid treatment group (DL) relative to the porcine-derived CYP2E1 protein in the control group (CK). CK is the relative quantification of the porcine-derived CYP2E1 protein in the control group (CK) compared to the average of the porcine-derived CYP2E1 protein in the three treatment groups (diquat induced oxidative stress treatment (DQ), antioxidant lipoic acid treatment (LA) and diquat + lipoic acid treatment (DL)).
Comparing the relative quantification results obtained in example 2 and comparative example 1 in the boxplot, it can be seen that the change trend of the swine-origin CYP2E1 protein identified in example 2 between different treatment groups is consistent with the change trend of the swine-origin CYP2E1 protein identified in comparative example 1 between different treatment groups. The peptide fragment composition for the relative quantitative analysis of the porcine cytochrome P450 enzyme CYP2E1, which comprises the first peptide fragment FIDLIPSNLPHEATR, the second peptide fragment EAHFLLELR and the third peptide fragment GEIPTFVHK, has good accuracy, and can be used for identifying the target peptide fragment composition of the porcine cytochrome P450 enzyme CYP2E1 in a targeted manner, so that compared with the existing in vitro labeled detection method, such as the high-throughput identification of all porcine proteins by the TMT (Tandem Mass Tag) protein isotope labeling quantitative technology, the method has higher sensitivity.
Those of ordinary skill in the art will understand that: the discussion of any embodiment above is meant to be exemplary only, and is not intended to intimate that the scope of the disclosure, including the claims, is limited to these examples; within the idea of the present disclosure, also technical features in the above embodiments or in different embodiments may be combined, 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 detail for the sake of brevity.
While the present disclosure has been described in conjunction with specific embodiments thereof, many alternatives, modifications, and variations of these embodiments will be apparent to those of ordinary skill 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. Therefore, any omissions, modifications, equivalents, improvements, and the like that may be made 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 animal husbandry and veterinary institute of Chinese academy of agricultural sciences
<120> peptide fragment composition for relatively quantitative analysis of porcine cytochrome P450 enzyme CYP2E1 and application thereof
<130> FI221034
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 15
<212> PRT
<213> amino acid sequence of first peptide fragment
<400> 1
Phe Ile Asp Leu Ile Pro Ser Asn Leu Pro His Glu Ala Thr Arg
1 5 10 15
<210> 2
<211> 10
<212> PRT
<213> amino acid sequence of second peptide fragment
<400> 2
Glu Ala His Phe Leu Leu Glu Ala Leu Arg
1 5 10
<210> 3
<211> 10
<212> PRT
<213> amino acid sequence of third peptide fragment
<400> 3
Gly Glu Ile Pro Thr Phe Gln Val His Lys
1 5 10

Claims (9)

1. The peptide fragment composition for relatively quantitatively analyzing the porcine cytochrome P450 enzyme CYP2E1 is characterized by comprising a first peptide fragment, a second peptide fragment and a third peptide fragment;
wherein the amino acid sequence of the first peptide segment is shown as SEQ ID NO. 1; the amino acid sequence of the second peptide segment is shown as SEQ ID NO. 2; the amino acid sequence of the third peptide segment is shown as SEQ ID NO. 3.
2. The peptide fragment composition for relatively quantitatively analyzing the porcine cytochrome P450 enzyme CYP2E1 according to claim 1, wherein the parent ion of the first peptide fragment is 861.96m/z, the daughter ion is 1234.65m/z,1121.57m/z and 710.36m/z, and the corresponding collision energy of the daughter ion is 27V; the parent ion of the second peptide fragment is 599.83m/z, the daughter ion is 998.58m/z,861.52m/z and 714.45m/z, and the corresponding collision energy of the daughter ion is 27V; the parent ion of the third peptide fragment is 578.31m/z, the daughter ion is 856.47m/z,658.37m/z and 284.17m/z, and the corresponding collision energy of the daughter ion is 27V.
3.A method for relatively quantitatively analyzing a porcine cytochrome P450 enzyme CYP2E1, wherein the peptide fragment composition according to any one of claims 1 to 2 is applied to relatively quantitatively analyze the porcine cytochrome P450 enzyme CYP2E1.
4. The method of claim 3, wherein the performing a relative quantitative analysis of porcine cytochrome P450 enzyme CYP2E1 comprises:
providing different groups of to-be-detected pig tissue samples, and respectively performing protein extraction and proteolysis treatment to obtain different groups of to-be-detected peptide fragments;
respectively detecting the peptide fragment compositions of different groups by a liquid chromatography-mass spectrometry combined method;
and comparing the detection results of the peptide fragment compositions of different groups to relatively quantify the porcine cytochrome P450 enzyme CYP2E1 in the porcine tissue samples to be detected of different groups.
5. The method of claim 4, wherein the test porcine tissue sample is a test porcine liver sample.
6. The method of claim 4, wherein the LC-MS is performed using HPLC-MS.
7. The method of claim 4, wherein the detection results comprise detection results with isotopically labeled peptide fragment compositions; the isotope-labeled internal standard peptide segment is PRTC: saagafgpersr ( 13 C 6 15 N 4 ,+10Da)。
8. A kit for relatively quantitatively analyzing the porcine cytochrome P450 enzyme CYP2E1, comprising a reagent for detecting the peptide fragment composition of any one of claims 1 to 2.
9. The kit of claim 8, wherein the reagents comprise a first peptide fragment standard, a second peptide fragment standard, a third peptide fragment standard, a dithiothreitol solution, an iodoacetamide solution, and a trypsin solution.
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