CN110850102A - Preparation method of specific peptide fragment mass spectrometry sample - Google Patents
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Abstract
A preparation method of a specific peptide fragment mass spectrometry sample comprises the following steps: a: taking sample cells to be analyzed, collecting the number of more than 200 ten thousand cells in a biotin culture environment, dissolving the sample cells in a cell lysis buffer solution, carrying out ultrasonic lysis, and carrying out trypsin digestion on a peptide fragment after quantifying the concentration of a protein solution; b: digesting the peptide fragment by trypsin method, and freeze-drying at-80 deg.C; c: preparing a biotin antibody bound to Protein G beads; d: dissolving the obtained freeze-dried peptide fragment in 1 ml of capture buffer solution; e: combining the antibody and the peptide fragment in C, D, incubating for 2 hours at 4 ℃, and eluting for 4 times by using an elution buffer solution; f: and (4) eluting and desalting the total supernatant through a microfiltration column filled with 200 mul of gun heads and prepared by a 3M C18 solid-phase extraction membrane, dissolving the peptide segments, and performing mass spectrum detection. The invention can accurately check the natural biotinylated protein in the cells.
Description
Technical Field
The invention relates to protein analysis research, in particular to a preparation method of a specific peptide fragment mass spectrometry sample, belonging to the technical field of biochemistry.
Background
The recognition of biotinylated proteins is a major problem in the biological industry, and concerns the search for proteins that function post-translationally and bind to each other. The conventional biotinylated Protein avidin method, generally through avidin binding of biotinylated proteins, Protein G beads on the pancreatic digestion of Protein. But has an affinity constant (K) of 10 due to the covalent bonding force between avidin and biotin15The mol/L is at least 1 ten thousand times higher than the affinity of the antibody for binding antigen, and when the biotin peptide fragment is eluted subsequently, the bound biotinylated lysine residue site is difficult to elute and release, so that the identified peptide fragment lacks a biotinylation mark, and the peptide fragment cannot be distinguished from a protein from which the avidin is specifically bound or a protein which is not specifically bound in the operation, and finally most of the identified protein is easy to be false signals. How to more efficiently and accurately discover the biotinylated protein becomes an important issue concerned by researchers in the field of mass spectrometry.
The main reports of protein labeling using biotinylation followed by protein search are as follows,
rhee et al identified 495 protein Science 2013, 339(6125), 1328-1331 in the human mitochondrial matrix by the biotinylation property of peroxidase using a spatio-temporally resolved proteomic map of the endogenous protein of living cells;
bar et al antibodies directed against the antigen of interest directed biotin deposition onto adjacent proteins in fixed cells and primary tissues, high throughput capture and identification of the interacting protein Nature methods 2018 Feb by mass spectrometry 15(2) 127-.
The above reports both focus on the elaborate discovery of proteins and on how to biotinylate the protein of interest, but often neglect the limitations of mass spectrometric detection and analysis.
Disclosure of Invention
The invention aims to overcome the problems existing in the prior protein research by mass spectrometry of peptide fragments and provides a preparation method of a specific peptide fragment mass spectrometry sample.
In order to realize the purpose of the invention, the following technical scheme is adopted: a preparation method of a specific peptide fragment mass spectrometry sample comprises the following steps:
a: taking sample cells to be analyzed, collecting more than 200 ten thousand cells of a biotin culture environment, dissolving the sample cells in 1-2 ml of cell lysis buffer solution, wherein the cell lysis buffer solution consists of 50mM triethylammonium bicarbonate, 8M urea and 10nM protease inhibitor, performing circulating ultrasonic lysis in ice bath, and performing trypsin digestion on a peptide fragment after quantifying the concentration of a protein solution;
b: digesting the peptide fragment by a trypsin method, adding 5 mM dithiothreitol into 1 mg of protein cracking solution, incubating for 1 hour at room temperature, adding 10 mM iodoacetamide, incubating for 20 minutes at room temperature in a dark place, finally adding 50mM triethylammonium bicarbonate with the total volume being 3 times that of the solution and trypsin with the total amount being 1% of the protein, and incubating and oscillating for more than 12 hours at 37 ℃; carrying out protein gel electrophoresis and Coomassie blue staining on the digested peptide fragment, desalting by using a C18 filter column, carrying out freeze-drying at-80 ℃, taking a dried peptide fragment sample for later use, and carrying out subsequent operations at 4 ℃ when no special description exists;
c: preparing a biotin antibody bound to Protein G beads, adding 100 μ G of biotin-specific antibody into 150 μ l of Protein G beads washed by precooled PBS, incubating overnight at 4 ℃, centrifuging for 1 minute at 800G, discarding supernatant, washing for 2 times by precooled PBS, and washing for 2 times by precooled capture buffer solution for later use, wherein the capture buffer solution consists of 50mM of trihydroxymethyl aminomethane, 150 mM of NaCl and 0.5% of polyethylene glycol octyl phenyl ether by mass concentration, and the pH value of the capture buffer solution is 7.4;
d: dissolving about 15 mg of the freeze-dried peptide fragment obtained in the step B by 1 ml of capture buffer solution, adjusting the pH value to 7.0-7.5 by 3M trihydroxymethyl aminomethane, centrifuging for 5 minutes at 5000g, taking the supernatant, and taking 10mg for later use after the concentration of the peptide fragment is measured by a fluorescence method;
e: combining the antibody and the peptide fragment in C, D, incubating for 2 hours at 4 ℃, centrifuging for 1 minute at 800 g, discarding the supernatant, washing for 2 times with a precooled capture buffer solution, washing for 2 times with 50mM trihydroxymethyl aminomethane with precooled pH of 7.4, washing for 2 times with precooled deionized water, completely absorbing and removing water by a 200 mu l long gun head, and eluting for 4 times with 200 mu l elution buffer solution, wherein the elution buffer solution comprises trifluoroacetic acid with the volume percentage of 0.2%, anhydrous acetonitrile with the volume percentage of 80%, and the rest is deionized water;
f: and (3) eluting and desalting the total supernatant through a microfiltration column which is prepared by filling a 3M C18 solid phase extraction membrane with 200 mul of gun heads, dissolving the peptide sections by 100 mul of trifluoroacetic acid with volume percentage of 0.1%, and performing high-resolution mass spectrum detection.
Further, the method comprises the following steps of; in the step E, the corresponding ratio of the peptide fragment to the biotin antibody is 100 μ g of biotin antibody per 10mg of the purified peptide fragment.
The invention has the following advantages: the biotin antibody and the biotinylated peptide segment have low affinity, and after the non-specific binding peptide segment is removed through a cleaning step, the biotinylated peptide segment can be eluted from a lysine residue position, but biotin imprinting information with small mass number is still reserved at the lysine residue position, so that a true signal can be easily identified and obtained in mass spectrometry; the biotinylation site and avidin are tightly combined, and the conventional method is difficult to elute and separate. The peptide segment detected and analyzed by the method contains biotinylation site information, and subsequent screening can be carried out by taking the biotinylation site information as an option; the peptide fragments obtained by the conventional method do not contain biotin labels, and the peptide fragments are difficult to distinguish from antibody specific binding or non-specific adsorption introduced in the operation. The natural biotinylated protein in the cells can be accurately checked, and the background basic level of biotin of each cell can be researched; the traditional method obtains too many peptide fragments, and cannot distinguish and identify the background natural biotin protein.
Drawings
FIG. 1 is a schematic diagram of a process for identifying biotinylated peptide fragments.
FIG. 2 is a map of the Myc-biotinylase-KRAS fragment inserted into pBABE plasmid.
FIG. 3 shows the expression of Myc-biotinidase-KRAS-containing HEK293T cell clones by Western blotting.
FIG. 4 is a graph of the distribution of the number of biotinylated amino acid residues on the protein identified for HEK293T cells expressing the Myc-biotinylase-KRAS fusion protein.
FIG. 5 shows the result of biotinylated protein analysis in HEK293T cells expressing Myc-biotinylase-KRAS fusion protein.
Detailed Description
In order to more fully explain the implementation of the present invention, the implementation examples of the present invention are provided, which are merely illustrative of the present invention and do not limit the scope of the present invention.
Example 1: the preparation method of the sample for the mass spectrometric analysis of the HEK293T cell specific peptide fragment comprises the following steps:
a: collecting HEK293T cells to be analyzed, collecting 500 ten thousand cells of a biotin culture environment, dissolving in 2 ml of cell lysis buffer solution, wherein the cell lysis buffer solution consists of 50mM triethylammonium bicarbonate, 8M urea and 10nm protease inhibitor, performing circulating ultrasonic lysis in ice bath, and performing trypsin digestion on a peptide fragment after quantifying the concentration of a protein solution;
b: digesting the peptide fragment by a trypsin method, adding dithiothreitol with a final concentration of 5 mM into 15 mg of protein cracking solution, incubating for 1 hour at room temperature, adding iodoacetamide with a final concentration of 10 mM, incubating for 20 minutes at room temperature in a dark place, finally adding 50mM triethylammonium bicarbonate with a total volume of 3 times and 150 mug of trypsin, and incubating and oscillating for more than 12 hours at 37 ℃; carrying out protein gel electrophoresis and Coomassie blue staining on the digested peptide fragment, desalting by using a C18 filter column, carrying out freeze-drying at-80 ℃, taking a dried peptide fragment sample for later use, and carrying out subsequent operations at 4 ℃ when no special description exists;
c: preparing a biotin antibody bound to Protein G beads, adding 100 μ G of biotin-specific antibody to 150 μ l of Protein G beads washed with precooled PBS, wherein the PBS solution is a phosphate buffer solution; incubating at 4 ℃ overnight, centrifuging for 1 minute at 800 g, then discarding the supernatant, washing for 2 times with precooled PBS, and washing for 2 times with precooled capture buffer solution for later use, wherein the capture buffer solution consists of 50mM of trihydroxymethyl aminomethane, 150 mM of NaCl and 0.5% of polyethylene glycol octyl phenyl ether by mass concentration;
d: dissolving the freeze-dried peptide fragment obtained in the step B by 1 ml of capture buffer solution, adjusting the pH value to 7.0-7.5 by 3M trihydroxymethyl aminomethane, centrifuging for 5 minutes at 5000g, taking the supernatant, and taking 10mg of peptide fragment for later use after measuring the concentration of the peptide fragment by a fluorescence method;
e: mixing C, D prepared antibody and 10mg peptide fragment, incubating for 2 hours at 4 ℃, centrifuging for 1 minute at 800 g, discarding the supernatant, washing for 2 times by using precooled capture buffer solution, washing for 2 times by using 50mM trihydroxymethyl aminomethane with precooled pH of 7.4, washing for 2 times by using precooled deionized water, completely absorbing water by using 200 mu l long gun head, and eluting for 4 times by using 200 mu l elution buffer solution, wherein the elution buffer solution comprises trifluoroacetic acid with volume percentage of 0.2%, anhydrous acetonitrile with volume percentage of 80%, and the rest is deionized water;
f: and (3) eluting and desalting the total supernatant through a microfiltration column which is prepared by filling a 3M C18 solid phase extraction membrane with 200 mul of gun heads, dissolving the peptide sections by 100 mul of trifluoroacetic acid with volume percentage of 0.1%, and performing high-resolution mass spectrum detection.
The mass spectrum data is analyzed by protein software PD2.2 of Thermo company, a lysine residue biotinylation peptide fragment is screened out, and the corresponding biotinylation target protein is obtained by splicing and matching, as shown in Table 1, the 22 proteins with biotinylation modification naturally exist in HEK293T cells and can be used as the basic background of a tool cell HEK293T for reference of related research
TABLE 1
Example 2: HEK293T cell example of Myc-biotinylase-KRAS fusion protein
(1) Preparation of HEK293T cells expressing Myc-biotinidase-KRAS fusion protein
The fusion gene fragment containing Myc-tagged biotinidase (5 'end) and KRAS (3' end) synthesized by the gene was inserted into pBABE plasmid behind SV40 promoter by homologous recombination (FIG. 2). Fusion gene fragment plasmid using lipofectamine3000 transient transfection HEK293T cells, packaging into retrovirus. After 24 hours, the HEK293T cells were infected with viruses filtered by a 0.22 μ M filter to remove cell debris, and were subjected to pressure screening for 1 week using 1 μ M puromycin, and the selected monoclones were transferred to a 96-well plate and cultured for 2 weeks. FIG. 3 shows that the specific Myc monoclonal antibody is used in Western blotting to identify the band (55 kDa in size) of Myc-biotinylase-KRAS, and cell clone highly expressing the fusion protein is selected, amplified and stored. Based on the cells, the method of the present invention is exemplified for the high specificity recognition of biotinylated binding proteins.
(2) The preparation method of the HEK293T cell specific peptide fragment mass spectrum analysis sample of the Myc-biotinylase-KRAS fusion protein comprises the following steps:
a: taking HEK293T cells expressing Myc-biotinylase-KRAS fusion protein to be analyzed, collecting 500 ten thousand cells of a biotin culture environment, dissolving in 2 ml of cell lysis buffer solution, wherein the cell lysis buffer solution consists of 50mM triethylammonium bicarbonate, 8M urea and 10nm protease inhibitor, performing circulating ultrasonic lysis in ice bath, and performing trypsin digestion on a peptide segment after quantifying the concentration of a protein solution;
b: digesting the peptide fragment by a trypsin method, adding dithiothreitol with a final concentration of 5 mM into 15 mg of protein cracking solution in total, incubating for 1 hour at room temperature, adding iodoacetamide with a final concentration of 10 mM, incubating for 20 minutes in a dark place at room temperature, finally adding 50mM triethylammonium bicarbonate and 150 mug of trypsin with the total volume of 3 times, and incubating and oscillating for more than 12 hours at 37 ℃; carrying out protein gel electrophoresis and Coomassie blue staining on the digested peptide fragment, desalting by using a C18 filter column, carrying out freeze-drying at-80 ℃, taking a dried peptide fragment sample for later use, and carrying out subsequent operations at 4 ℃ when no special description exists;
c: preparing a biotin antibody bound to Protein G beads, adding 100 μ G of biotin-specific antibody into 150 μ l of Protein G beads washed by precooled PBS, incubating overnight at 4 ℃, centrifuging for 1 minute at 800G, discarding supernatant, washing for 2 times by precooled PBS, and washing for 2 times by precooled capture buffer solution for later use, wherein the capture buffer solution consists of 50mM of trihydroxymethyl aminomethane, 150 mM of NaCl and 0.5% of polyethylene glycol octyl phenyl ether by mass concentration, and the pH value of the capture buffer solution is 7.4;
d: dissolving the freeze-dried peptide fragment in 1 ml of capture buffer solution, adjusting the pH value to 7.0-7.5 by using 3M trihydroxymethyl aminomethane, centrifuging 5000g for 5 minutes, taking the supernatant, and taking 10mg of peptide fragment for later use after measuring the concentration of the peptide fragment by using a fluorescence method;
e: mixing the prepared antibody in C, D with 10mg of peptide fragments, incubating for 2 hours at 4 ℃, centrifuging for 1 minute at 800 g, then discarding the supernatant, washing for 2 times with precooled capture buffer solution, washing for 2 times with 50mM trihydroxymethyl aminomethane with precooled pH of 7.4, washing for 2 times with precooled deionized water, completely absorbing water by a 200 mu l long gun head, and eluting for 4 times with 200 mu l elution buffer solution, wherein the elution buffer solution comprises trifluoroacetic acid with volume percentage of 0.2%, anhydrous acetonitrile with volume percentage of 80%, and the rest is deionized water;
f: after the total supernatant is eluted and desalted by a microfiltration column which is prepared by filling a 3M C18 solid phase extraction membrane with a 200 mul gun head, dissolving a peptide segment by 100 mul trifluoroacetic acid with volume percentage of 0.1%, and performing high-resolution mass spectrum detection;
and (3) analyzing mass spectrum data, screening out lysine residue biotinylation peptide fragments, and splicing and matching to obtain corresponding biotinylation target protein. According to the cell, 5879 pieces of biotinylated peptide fragments are recognized by the method, total 1886 biotinylated proteins are obtained, and the distribution of the number of the biotin-labeled amino acid residue positions in the proteins is shown in figure 4. By removing the background protein compared with KRAS binding protein analyzed by avidin method, the method of the present invention can specifically and accurately identify 1872 protein labeled by biotinases, whereas avidin method has no candidate biotinylated protein considering biotinylation screening conditions (Table 2). Among these, 179 biotinylated binding proteins were detected in parallel assays using the method of the invention, and were classified into 7 different protein functions such as membrane composition (FIG. 5). Aiming at the biotinylated protein identified by the method, more than 50% of lysine residues in the amino acid sequence of 29 proteins are marked by biotin, which is probably potential KRAS unknown binding protein, reduces the verification range for subsequent biological verification and greatly improves the working efficiency (Table 3)
TABLE 2
TABLE 3
Gene sequences used to prepare HEK293T cells expressing Myc-biotinylase-KRAS fusion protein: during preparation, the Myc label, the biotinylase and the KRAS are spliced in sequence, wherein no deoxynucleotide interval exists;
(1) myc tag 30bp
gagcagaaac tcatctctga agaggatctg
(2) Biotinidase GenBank AF201721.1, 768bp, prepared by removing the promoter (5 ') and terminator (3') of the intermediate gene, i.e., the first att and the last tga in the sequence
attcttttga ccggcggacg aagcggcggc cggagttcgc ctgaagattt ccggcacgtc
gcgctcgccg aaacggtctc gaccaacagc gagtgccttc tgcgggcgcg cgagggtgat
cccggcaatc tctggatcac cgcggtgcgc cagaccggag gcaggggcag gcgtggccgc
gcctggttct cggagccggg gaacctctac gcatccctgc tgctcatcga tccggcgccg
gtggacaggc tgcatgcgct gccgcttgcg gcagcggttg cggttcatcg ggccatccgc
cgggtgatgc cgccgggcgg agccgaggcc gcgatcaaat ggccgaacga cattcttatc
gacgggcgga agacctgcgg cattcttctc gaaggcgagg ttctggccga cgggcggcgg
gccctggtga tcggctgcgg catcaacgtc gccgtcatgc ctgaagacgc actttacccc
gtcacatcgc ttcggcgcga gggcgcgaca atctctcccg aggagctgtt cgcgcatctt
ttcgtaacca tggccgagac gctcgccgtt tgggaccgtg gcgccggcgt cgccgcaatc
atcgatcagt ggcgcgccgc ggccaagggc atcggcgagg cgatcacggt caacctgccg
gaccgctcgc tttccggccg ctttgcaggt atcgatcggg acggccggct tctactcgac
acgggttccg gcccgccgca aaccattgcg gcgggcgacg tattttttgg atga
(3) KRAS NM-004985.5, 564bp, was prepared by removing the promoter (5') of the middle gene, i.e., the first atg in this sequence.
atgactgaat ataaacttgt ggtagttgga gctggtggcg taggcaagag
tgccttgacg atacagctaa ttcagaatca ttttgtggac gaatatgatc caacaataga
ggattcctac aggaagcaag tagtaattga tggagaaacc tgtctcttgg atattctcga
cacagcaggt caagaggagt acagtgcaat gagggaccag tacatgagga ctggggaggg
ctttctttgt gtatttgcca taaataatac taaatcatttgaagatattc accattatag
agaacaaatt aaaagagtta aggactctga agatgtacct atggtcctag taggaaataa
atgtgatttg ccttctagaa cagtagacac aaaacaggct caggacttag caagaagtta
tggaattcct tttattgaaa catcagcaaa gacaagacag ggtgttgatg atgccttcta
tacattagtt cgagaaattc gaaaacataa agaaaagatg agcaaagatg gtaaaaagaa
gaaaaagaag tcaaagacaa agtgtgtaat tatgtaa
After the embodiments of the present invention have been described in detail, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention, and it is intended that all simple modifications, equivalent changes and modifications made to the above embodiments based on the technical spirit of the present invention shall fall within the technical scope of the present invention, and the present invention shall not be limited to the embodiments illustrated in the description.
Sequence listing
<110> Anyang college of teachers and schools
<120> preparation method of specific peptide fragment mass spectrometry sample
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<170>SIPOSequenceListing 1.0
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<211>30
<212>DNA
<213>2 Ambystoma laterale x Ambystoma jeffersonianum
<400>1
gagcagaaac tcatctctga agaggatctg 30
<210>2
<211>774
<212>DNA
<213>2 Ambystoma laterale x Ambystoma jeffersonianum
<400>2
attcttttga ccggcggacg aagcggcggc cggagttcgc ctgaagattt ccggcacgtc 60
gcgctcgccg aaacggtctc gaccaacagc gagtgccttc tgcgggcgcg cgagggtgat 120
cccggcaatc tctggatcac cgcggtgcgc cagaccggag gcaggggcag gcgtggccgc 180
gcctggttct cggagccggg gaacctctac gcatccctgc tgctcatcga tccggcgccg 240
gtggacaggc tgcatgcgct gccgcttgcg gcagcggttg cggttcatcg ggccatccgc 300
cgggtgatgc cgccgggcgg agccgaggcc gcgatcaaat ggccgaacga cattcttatc 360
gacgggcgga agacctgcgg cattcttctc gaaggcgagg ttctggccga cgggcggcgg 420
gccctggtga tcggctgcgg catcaacgtc gccgtcatgc ctgaagacgc actttacccc 480
gtcacatcgc ttcggcgcga gggcgcgaca atctctcccg aggagctgtt cgcgcatctt 540
ttcgtaacca tggccgagac gctcgccgtt tgggaccgtg gcgccggcgt cgccgcaatc 600
atcgatcagt ggcgcgccgc ggccaagggc atcggcgagg cgatcacggt caacctgccg 660
gaccgctcgc tttccggccg ctttgcaggt atcgatcggg acggccggct tctactcgac 720
acgggttccg gcccgccgca aaccattgcg gcgggcgacg tattttttgg atga 774
<210>3
<211>567
<212>DNA
<213>2 Ambystoma laterale x Ambystoma jeffersonianum
<400>3
atgactgaat ataaacttgt ggtagttgga gctggtggcg taggcaagag tgccttgacg 60
atacagctaa ttcagaatca ttttgtggac gaatatgatc caacaataga ggattcctac120
aggaagcaag tagtaattga tggagaaacc tgtctcttgg atattctcga cacagcaggt 180
caagaggagt acagtgcaat gagggaccag tacatgagga ctggggaggg ctttctttgt 240
gtatttgcca taaataatac taaatcattt gaagatattc accattatag agaacaaatt 300
aaaagagtta aggactctga agatgtacct atggtcctag taggaaataa atgtgatttg 360
ccttctagaa cagtagacac aaaacaggct caggacttag caagaagtta tggaattcct 420
tttattgaaa catcagcaaa gacaagacag ggtgttgatg atgccttcta tacattagtt 480
cgagaaattc gaaaacataa agaaaagatg agcaaagatg gtaaaaagaa gaaaaagaag 540
tcaaagacaa agtgtgtaat tatgtaa 567
Claims (2)
1. A preparation method of a specific peptide fragment mass spectrometry sample is characterized by comprising the following steps:
a: taking sample cells to be analyzed, collecting more than 200 ten thousand cells of a biotin culture environment, dissolving the sample cells in 1-2 ml of cell lysis buffer solution, wherein the cell lysis buffer solution consists of 50mM triethylammonium bicarbonate, 8M urea and 1X protease inhibitor, and performing trypsin digestion on a peptide fragment after quantifying the concentration of a protein solution by circulating ultrasonic lysis in an ice bath;
b: digesting the peptide fragment by a trypsin method, adding 5 mM dithiothreitol into 1 mg of protein cracking solution, incubating for 1 hour at room temperature, adding 10 mM iodoacetamide, incubating for 20 minutes at room temperature in a dark place, finally adding 50mM triethylammonium bicarbonate with the total volume being 3 times that of the solution and trypsin with the total amount being 1% of the protein, and incubating and oscillating for more than 12 hours at 37 ℃; carrying out protein gel electrophoresis and Coomassie blue staining on the digested peptide fragment, if a protein band which is not digested is not seen, desalting by using a C18 filter column, carrying out freeze-drying at-80 ℃, taking a dried peptide fragment sample for later use, and carrying out subsequent operations at 4 ℃ when no special instruction exists;
c: preparing a biotin antibody bound to Protein G beads, adding 100 μ G of biotin-specific antibody into 150 μ l of Protein G beads washed by precooled PBS, incubating overnight at 4 ℃, centrifuging for 1 minute at 800G, discarding supernatant, washing for 2 times by precooled PBS, and washing for 2 times by precooled capture buffer solution for later use, wherein the capture buffer solution consists of 50mM of trihydroxymethyl aminomethane, 150 mM of NaCl and 0.5% of polyethylene glycol octyl phenyl ether by mass concentration, and the pH value of the capture buffer solution is 7.4;
d: dissolving about 15 mg of the freeze-dried peptide fragment obtained in the step B by 1 ml of capture buffer solution, adjusting the pH value to 7.0-7.5 by 3M trihydroxymethyl aminomethane, centrifuging for 5 minutes at 5000g, taking the supernatant, and taking 10mg for later use after the concentration of the peptide fragment is measured by a fluorescence method;
e: combining the antibody and the peptide fragment in C, D, incubating for 2 hours at 4 ℃, centrifuging for 1 minute at 800 g, discarding the supernatant, washing for 2 times with a precooled capture buffer solution, washing for 2 times with 50mM trihydroxymethyl aminomethane with precooled pH of 7.4, washing for 2 times with precooled deionized water, completely absorbing and removing water by a 20 mu l long gun head, and eluting for 4 times with 200 mu l elution buffer solution, wherein the elution buffer solution comprises trifluoroacetic acid with the volume percentage of 0.2%, anhydrous acetonitrile with the volume percentage of 80%, and the rest is deionized water;
f: and (3) eluting and desalting the total supernatant through a microfiltration column which is filled with 200 mul of gun heads and prepared by 3M C18 solid phase extraction membranes, dissolving the peptide sections by 100 mul of trifluoroacetic acid aqueous solution with volume percentage of 0.1%, and performing high-resolution mass spectrum detection.
2. The method for preparing a sample for mass spectrometry of specific peptide fragments according to claim 1, wherein the method comprises the following steps: in the step D, the corresponding ratio of the peptide fragment to the biotin antibody is 100 μ g of biotin antibody per 10mg of the purified peptide fragment.
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