CN108169397B - A kind of method of relative quantitative assay broiler chicken glutathione s-transferase GSTA2 - Google Patents
A kind of method of relative quantitative assay broiler chicken glutathione s-transferase GSTA2 Download PDFInfo
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- CN108169397B CN108169397B CN201711237702.1A CN201711237702A CN108169397B CN 108169397 B CN108169397 B CN 108169397B CN 201711237702 A CN201711237702 A CN 201711237702A CN 108169397 B CN108169397 B CN 108169397B
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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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Abstract
The invention discloses the methods of relative quantitative assay broiler chicken glutathione s-transferase GSTA2 a kind of, first carry out the extraction of broiler chicken glutathione s-transferase GSTA2, digestion is carried out to broiler chicken glutathione s-transferase GSTA2 again and obtains specific peptide fragment, then the primary and secondary ion pair of specific peptide fragment is detected using LC-MS instrument, obtain the daughter ion signal strength summation of specific peptide fragment, the signal strength of GSTA2 albumen is obtained according to the daughter ion signal strength summation of special peptide fragment, by comparing the signal strength of GSTA2 albumen in different disposal broiler chicken sample, to realize the relative quantification to GSTA2 albumen in different disposal broiler chicken sample；The specific peptide fragment of broiler chicken glutathione s-transferase GSTA2 is 3, and amino acid sequence is respectively shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.Method of the invention is stronger relative to antibody Western Blot method specificity is based on, and eliminates the tedious steps for preparing antibody, improves chicken source GSTA2 Protein Detection flux and efficiency.
Description
Technical field
The present invention relates to biochemical analysis technical fields, particularly relate to a kind of relative quantitative assay broiler chicken glutathione S transfer
The method of enzyme GSTA2.
Background technique
Heat stress be the problem of being widely noticed in broiler production be during growth of animal it is common it is a kind of in vitro stress,
When the growing environment temperature of animal is more than its metabolic stability area upper limit, cannot maintain thermal balance by the adjusting of body physics, lead
Cause body that can generate the reaction of nonspecific response caused by response environment high temperature.A large number of studies show that broiler chicken under the conditions of heat stress
Feed intake can decline 14%~17%, while also will cause that immunity of meat chickens decline, digestive disease is multiple, digestibility decline, sternly
Ghost image rings broiler chicken survival rate and efficiency of feed utilization, brings economic loss to poultry husbandry.It is reported that the U.S. every year because of heat stress caused by
Husbandry sector loses about 16.9~23.6 hundred million dollars, and wherein up to 1.25~1.65 hundred million dollars, broiler chicken is lost about for poultry production loss
52000000 dollars.Compared to the U.S., China is also broiler breeding big country, and production occupies the second in the world, but Special Interest Group with consumption
Change, industrialization level deficiency, Aquaculture Environmental Control ability is weak, and heat stress loses even more serious caused by China's broiler production.
Heat stress mainly passes through the approach such as oxidative stress, inflammatory reaction and causes cellular damage and Apoptosis, to animal machine
Body tissue causes to damage.When cell is in oxidative stress status, a series of activity of intracellular antioxidases is improved, to maintain
The anti-oxidant balance environment of cellular oxidation-improves cell survival rate.Glutathione S-transferase (Glutathione S-
Transferase, GST) it is the important isodynamic enzyme with removing toxic substances antioxidation, it constitutes main with important physiological function
Dimer cytoplasm isodynamic enzyme family, work in the phase il of antitoxin Antioxidation Mechanism, thus also become II phase metabolic enzyme.
II phase xenobiotic metabolism enzyme alpha type glutathione S-transferase GSTA2 can block lipid when oxidative stress occurs for cell
Peroxidating chain reaction reduces the expression of inflammatory factor related gene, reduces oxidativestress damage, improves cell survival rate.It is existing
Judging that the analysis method of relative amount is mainly to be compared in mRNA level in-site, protein level to GSTA2 in vivo, by mentioning
It takes and is inverted to cDNA in organizer after RNA, specific primer is designed according to GSTA2 gene, cDNA template is expanded, used
Fluorescence real-time quantitative PCR technology is compared analysis to GSTA2 transcriptional level in template.After genetic transcription, also need to undergo
The post-transcriptional controls such as shearing and splicing can just be expressed as albumen, therefore expression quantity can not actual response on transcriptional level for gene
The expression quantity of its gene product albumen.Currently, the albumen can most be reflected in tissue, cell by analyzing GSTA2 on protein level
With the expression contents in body fluid, it will usually the methods of enzyme-linked immunization, Western Blot, Tissue in situ hybridization are used, however
The above protein level detection method requires the antibody specific recognition GSTA2 albumen of high quality.Due to preparation currently on the market
The antigen protein majority of antibody is even derived from people, mouse and rat isotype biology, goes back on the market for the antibody of chicken source GSTA2
Do not occur, cause currently not yet can on protein level quantitative analysis broiler chicken GSTA2 expression quantity method.If wanting to establish
The method of protein level quantitative analysis broiler chicken GSTA2, need prepare can specific recognition broiler chicken GSTA2 antibody.It is mono- to prepare GSTA2
The specificity of antibody can be improved in clonal antibody, reduces non-specific binding and cross reaction, but the monoclonal control body period is long, takes
With height；If preparation GSTA2 polyclonal antibody can reduce expense, but nonspecific cross reaction is more serious, success rate
It is very low.
According to the exposition need of quantitative GSTA2 albumen, existing analysis method is comprehensively considered, the present invention uses multiple ion
The method of detection establishes a kind of GSTA2 protein quantification method based on mass-spectrometric technique, and this method does not need to prepare antibody, according to
GSTA2 specificity peptide fragment sequence design primary and secondary ion pair and collision energy are realized and are carried out on protein level to GSTA2 albumen
Relative quantitative assay substantially increases detection sensitivity, accuracy and detection flux.
Summary of the invention
In view of this, it is an object of the invention to propose a kind of relative quantitative assay broiler chicken glutathione s-transferase GSTA2
Method.The present invention primarily to realize relative quantitative assay Broiler Chicken in GSTA2 in different tissues expression contents change,
A kind of high-flux detection method without antibody is established using mass-spectrometric technique.The present invention mainly can analyze difference using mass spectrum
Peptide fragment quality, and fragmentation can be carried out to the specific peptide fragment in complex samples in controlled condition, and analysis particular polypeptide produces again
The quality of raw fragmentation small peptide searches out special primary and secondary ion pair by optimizing collision energy according to principles above, using special
Primary and secondary ion pair realizes the relative quantitative assay to GSTA2.
Based on above-mentioned purpose, a kind of side of relative quantitative assay broiler chicken glutathione s-transferase GSTA2 provided by the invention
Method first carries out the extraction of broiler chicken glutathione s-transferase GSTA2, then carries out digestion to broiler chicken glutathione s-transferase GSTA2
Specific peptide fragment is obtained, the primary and secondary ion pair of specific peptide fragment is then detected using LC-MS instrument, obtains specific peptide fragment
Daughter ion signal strength summation obtains broiler chicken glutathione s-transferase according to the daughter ion signal strength summation of special peptide fragment
The signal strength of GSTA2, by comparing the signal strength of broiler chicken glutathione s-transferase GSTA2 in different disposal broiler chicken sample,
To realize the relative quantification to broiler chicken glutathione s-transferase GSTA2 in different disposal broiler chicken sample.
In some embodiments of the invention, the specific peptide fragment of the broiler chicken glutathione s-transferase GSTA2 is 3,
The amino acid sequence of 3 specific peptide fragments is respectively shown in SEQ ID NO:1, SEQ IDNO:2 and SEQ ID NO:3；
Wherein, SEQ ID NO:1:Ala-Ile-Leu-Cys- [Cys-Ala-Met]-Tyr-Leu-Ala-Gly-Lys；
SEQ ID NO:2:Asn-Ile-Ala-Leu-Ile-Thr-Glu-Arg；
SEQ ID NO:3:Ser-Asp-Ile-Leu-Ser-Ala-Phe-Pro-Leu-Leu-Gln-Ala-Phe-Lys.
In some embodiments of the invention, the parent ion of specificity peptide fragment shown in SEQ ID NO:1 is 504.8m/z,
Daughter ion is 551.3m/z, 711.3m/z, 824.4m/z, and collision voltage corresponding to daughter ion is 30~35V；SEQ ID NO:
The parent ion of specificity peptide fragment shown in 2 is 465.3m/z, daughter ion 631.4m/z, 702.4m/z, 815.5m/z, daughter ion
Corresponding collision voltage is 35~40V；The parent ion of specificity peptide fragment shown in SEQ ID NO:3 be 775.4m/z, son from
Son is 816.5m/z, 963.6m/z, 1034.6m/z, and collision voltage corresponding to daughter ion is 30~35V.
In some embodiments of the invention, the signal strength of specificity peptide fragment shown in SEQ ID NO:1 is daughter ion
The signal of the signal strength summation of 551.3m/z, 711.3m/z, 824.4m/z, specificity peptide fragment shown in SEQ ID NO:2 is strong
Spending is daughter ion 631.4m/z, the signal strength summation of 702.4m/z, 815.5m/z, specific peptide shown in SEQ ID NO:3
The signal strength of section is daughter ion 816.5m/z, the signal strength summation of 963.6m/z, 1034.6m/z, broiler chicken glutathione S turn
The signal strength for moving enzyme GSTA2 is specificity peptide fragment shown in SEQ ID NO:1, specificity peptide fragment shown in SEQ ID NO:2
With the signal strength summation of this three specific peptide fragments of specificity peptide fragment shown in SEQ ID NO:3.
In some embodiments of the invention, first using the high performance liquid chromatography in LC-MS instrument to 3 specific peptides
Duan Jinhang separation, is detected subsequently into mass spectrum；Wherein, the condition of 3 specific peptide fragments is separated are as follows: chromatographic column is filled out for C18
The capillary chromatographic column of material, flow velocity are arranged in 200~500nL/min, mobile phase A are as follows: water+0.1wt% formic acid+5wt%DMSO,
Mobile phase B are as follows: acetonitrile+0.1wt% formic acid+5wt%DMSO uses gradient elution, mobile phase when separating 3 specific peptide fragments
Start gradient is the Mobile phase B of 95% mobile phase A+5%, popular phase gradient are as follows: 0~1min, 95%A；1~1.1min, 90%
A；1.1~60min, 80%A；60~70min, 76%A；70~75min, 50%A；75~75.5min, 20%A；75.5~
80.5min 20%A；80.5~81min, 95%A；81~90min, 95%A.
In some embodiments of the invention, the extraction step of broiler chicken broiler chicken glutathione s-transferase GSTA2 are as follows: use
Protein extraction buffer extracts the broiler chicken glutathione s-transferase GSTA2 under different disposal in Broiler Tissues, and to extracting
Gross protein it is quantitative；
Digestion broiler chicken broiler chicken glutathione s-transferase GSTA2 obtains specific peptide fragment step are as follows: takes different disposal sample
Gross protein, the first re-closed protein interior sulfydryl of opened disulfide bond, then sequentially add intracellular protein enzyme and trypsase into
Row digestion obtains specific peptide fragment after terminating endonuclease reaction.
In some embodiments of the invention, in the extraction step of broiler chicken glutathione s-transferase GSTA2, the egg
The ingredient of white matter Extraction buffer are as follows: 50~100mM Tris-HCl, 0.1~0.5MNaCl, 10~20mM dithiothreitol (DTT),
0.5~2wt% Lauryl.beta.-maltoside, pH 7.2~8.0；Use protein extraction buffer water-bath at 90~100 DEG C
15~30min of broiler chicken glutathione s-transferase GSTA2 under middle extraction different disposal in Broiler Tissues.
In some embodiments of the invention, it generates in specific peptide fragment step in digestion, is first beaten using dithiothreitol (DTT)
Disulfide bond is opened, then by sulfydryl inside acetamide closed protein matter, adds formic acid or trifluoroacetic acid terminates endonuclease reaction.
In some embodiments of the invention, it is generated in specific peptide fragment step in digestion, sequentially adds intracellular protein enzyme
The specific steps of digestion are carried out with trypsase are as follows: be first 1:100~200 according to the mass ratio of intracellular protein enzyme and total protein
Intracellular protein enzyme is added in ratio, is 1:30 according still further to the mass ratio of trypsase and total protein after digestion total protein 4~6 hours
~60 ratio addition trypsase, digestion 12~16 hours.
In the present invention, a kind of method of relative quantitative assay GSTA2 albumen, comprising the following steps:
(1) GSTA2 protein extraction
Using protein extraction buffer, (bis- sulphur of 50~100mM Tris-HCl, 0.1~0.5M NaCl, 10~20mM is revived
Sugar alcohol (DTT), 0.5~2% Lauryl.beta.-maltoside (DDM), pH 7.2~8.0) difference is extracted in 90~100 DEG C of water-baths
GSTA2 protein 15~30min in lower Broiler Tissues is handled, and the gross protein extracted is quantified；
(2) digestion generates the specific peptide fragment represented
The total protein for accurately weighing the different disposal sample of phase homogenous quantities, passes through the sulfydryl inside acetamide closed protein matter
Afterwards, using the ratio according to 1:100~200 (intracellular protein enzyme Lys-C: total protein, mass ratio), digestion total protein 4~6 hours
Afterwards, it is added trypsase 12~16 hours according still further to the ratio of 1:30~60 (trypsase: total protein, mass ratio), addition 1~
The formic acid or trifluoroacetic acid of 5wt% terminates endonuclease reaction, and rotation volatilizes the buffer in sample under the conditions of 40~50 DEG C,
Obtain specific peptide fragment；
It is AILC [CAM] YLAGK (shown in SEQ ID NO:1, wherein " [] " indicates acetyl by operating acquisition sequence above
Change modification), three of NIALITER (shown in SEQ ID NO:2) and SDILSAFPLLQAFK (shown in SEQ ID NO:3) are specifically
Peptide fragment；
(3) separation of GSTA2 specificity peptide fragment
Separation and signal acquisition are carried out to specific peptide section sequence using LC-MS instrument, wherein separate specific peptide fragment
Chromatographic column be C18 filler capillary chromatographic column, in 200~500nL/min, mobile phase A is water+0.1wt% for flow velocity setting
Formic acid+5wt%DMSO, Mobile phase B are acetonitrile+0.1wt% formic acid+5wt%DMSO, and gradient is used when separating specific peptide fragment
Elution, popular phase gradient are as follows: 0~1min, 95%A；1~1.1min, 90%A；1.1~60min, 80%A；60~70min,
76%A；70~75min, 50%A；75~75.5min, 20%A；75.5~80.5min, 20%A；80.5~81min, 95%
A；81~90min, 95%A；Guarantee that three specific peptide fragments sequentially enter mass spectrum and analyzed；
(4) multiple ion monitoring method analyzes the signal strength of specific peptide fragment in sample
Be arranged three sub- parent ions of specific peptide fragment to be respectively as follows: 504.8m/z be AILC [CAM] YLAGK it is female from
Son, the daughter ion generated under 30~35V collision voltage are that daughter ion is 551.3m/z, 711.3m/z, 824.4m/z；
465.3m/z is the parent ion of NIALITER, and the daughter ion generated under 35~40V collision voltage is 631.4m/z, 702.4m/
Z, 815.5m/z；775.4m/z is the parent ion of SDILSAFPLLQAFK, and the daughter ion generated under 30~35V collision voltage is
816.5m/z, 963.6m/z, 1034.6m/z；
In this step, the 551.3m/z generated using 504.8m/z, 711.3m/z, 824.4m/z signal strength summation generation
The signal strength of Table A ILC [CAM] YLAGK, the 631.4m/z generated using 465.3m/z, 702.4m/z, 815.5m/z signal are strong
Degree summation represents the signal strength of NIALITER, the 816.5m/z generated using 775.4m/z, 963.6m/z, 1034.6m/z letter
Number intensity summation represents the signal strength of SDILSAFPLLQAFK.Using AILC [CAM] YLAGK, NIALITER and
The signal summation of SDILSAFPLLQAFK tri- specific peptide fragments represents the signal strength of GSTA2 albumen.Finally, by comparing not
With the signal strength of GSTA2 in sample, the relative quantification of GSTA2 albumen in different samples is realized.
Compared with prior art, the method for relative quantitative assay broiler chicken glutathione s-transferase GSTA2 of the present invention have with
It is lower the utility model has the advantages that
The present invention uses LC-MS instrument, is realized by multiple ion detection method to GSTA2 protein in different samples
Relative quantification, the AILC [CAM] of GSTA2 is mainly generated by intracellular protein enzyme Lys-C and trypsase digestion twice
YLAGK, NIALITER and SDILSAFPLLQAFK totally 3 specific peptide fragments, use specific detection AILC of LC-MS instrument
[CAM] YLAGK, NIALITER and SDILSAFPLLQAFK primary and secondary ion pair realizes the quantitative analysis to GSTA2.The present invention is opposite
In being based on, antibody Western Blot method specificity is stronger, and eliminates the tedious steps for preparing antibody, improves Ji Yuan
GSTA2 Protein Detection flux and efficiency.
Detailed description of the invention
Fig. 1 is the mass spectrogram of AILC [CAM] YLAGK in the embodiment of the present invention 1 in heat treatment group broiler chicken liver organization；
Fig. 2 is the mass spectrogram of the NIALITER in the embodiment of the present invention 1 in heat treatment group broiler chicken liver organization；
Fig. 3 is the mass spectrogram of the SDILSAFPLLQAFK in the embodiment of the present invention 1 in heat treatment group broiler chicken liver organization.
Specific embodiment
To make the objectives, technical solutions, and advantages of the present invention clearer, below in conjunction with specific embodiment, to this hair
Bright further description.
The method of the relative quantitative assay broiler chicken glutathione s-transferase GSTA2 of embodiment 1 a kind of
In the present embodiment, the method for the relative quantitative assay broiler chicken glutathione s-transferase GSTA2 includes following step
It is rapid:
(1) GSTA2 protein extraction
Taking 0.1g control group and heat treatment group broiler chicken liver organization respectively, (heat treatment group liver specimens are from broiler chicken 33
DEG C 24 hours broiler chicken of processing, control group liver specimens handle 24 hours broiler chicken from same period broiler chicken at 25 DEG C), according to 1:
The ratios of 5 (mass ratioes) be added protein extraction buffer (50mMTris-HCl, 0.5M NaCl, 10mM dithiothreitol (DTT),
1wt% Lauryl.beta.-maltoside, pH 8.0), it is extracted in 90~100 DEG C of water-baths under different disposal in Broiler Tissues
GSTA2 protein 15~30min, by taking supernatant after 4 DEG C of centrifugation 30min of 20000g after homogenate, using BCA method to supernatant
In total protein carry out protein quantification.
(2) digestion generates the specific peptide fragment represented
Different group sample 50ug are taken respectively, first using 10mM dithiothreitol (DTT) in 50 DEG C of reduction 1h opened disulfide bonds, then
Sulfydryl reaction 30min is closed under dark condition with the iodoacetamide of 20mM；Lys- is added according to the ratio of 1:200 (mass ratio)
Trypsase is added digestion 12 under the conditions of 37 DEG C according still further to the ratio of 1:50 (mass ratio) in C digestion 4h under the conditions of 37 DEG C
Hour, reaction is terminated using the formic acid of 1wt%, and vapor away all enzyme cutting buffering liquids under conditions of 40 DEG C, obtains drying
Good specific peptide fragment.
(3) separation of GSTA2 specificity peptide fragment
Dried specific peptide fragment is re-dissolved using the 95wt% water+5wt%+0.1wt% formic acid of 50ul, is used
The molten specific peptide fragment of 6500 counterweight of nanoLC-QTRAQ carries out multiple ion and monitors quantitative analysis, the C18 hair that when analysis uses
Tubule analytical column specification are as follows: 75 3 μm of C18-CL of μ m 15cm ChromXPMobile phase A is water+0.1wt% formic acid
+ 5wt%DMSO, Mobile phase B are acetonitrile+0.1wt% formic acid+5wt%DMSO, popular phase gradient are as follows: 0~1min, 95%A；1~
1.1min, 90%A；1.1~60min, 80%A；60~70min, 76%A；70~75min, 50%A；75~75.5min,
20%A；75.5~80.5min, 20%A；80.5~81min, 95%A；81~90min, 95%A；Flow velocity is 300nl/min,
Guarantee that three specific peptide fragments sequentially enter mass spectrum and analyzed.
(4) multiple ion monitoring method analyzes the signal strength of specific peptide fragment in sample
Mass spectrometry parameters setting are as follows: 504.8m/z is the parent ion of AILC [CAM] YLAGK (shown in SEQ ID NO:1), In
The daughter ion generated under 33.5V collision voltage is 551.3m/z, 711.3m/z, 824.4m/z；465.3m/z is NIALITER
The parent ion of (shown in SEQ ID NO:2), the daughter ion generated under 38.8V collision voltage be 631.4m/z, 702.4m/z,
815.5m/z；775.4m/z is the parent ion of SDILSAFPLLQAFK (shown in SEQ ID NO:3), is produced under 31V collision voltage
Raw daughter ion is 816.5m/z, 963.6m/z, 1034.6m/z.
In this step, the 551.3m/z generated using 504.8m/z, 711.3m/z, 824.4m/z signal strength summation generation
The signal strength of Table A ILC [CAM] YLAGK, the 631.4m/z generated using 465.3m/z, 702.4m/z, 815.5m/z signal are strong
Degree summation represents the signal strength of NIALITER, the 816.5m/z generated using 775.4m/z, 963.6m/z, 1034.6m/z letter
Number intensity summation represents the signal strength of SDILSAFPLLQAFK.Using AILC [CAM] YLAGK, NIALITER and
The signal summation of SDILSAFPLLQAFK tri- specific peptide fragments represents the signal strength of GSTA2 albumen, by comparing control group
With the signal strength of GSTA2 albumen in heat treatment group broiler chicken liver organization, control group and heat treatment group broiler chicken liver organization are realized
The relative quantification of middle GSTA2 albumen.
The method of the relative quantitative assay broiler chicken glutathione s-transferase GSTA2 of embodiment 2 a kind of
In the present embodiment, the method for the relative quantitative assay broiler chicken glutathione s-transferase GSTA2 includes following step
It is rapid:
(1) GSTA2 protein extraction
Taking 0.1g control group and heat treatment group broiler chicken liver organization respectively, (heat treatment group liver specimens are from broiler chicken 33
DEG C 18 hours broiler chicken of processing, control group liver specimens handle 18 hours broiler chicken from same period broiler chicken at 25 DEG C), according to 1:
The ratios of 5 (mass ratioes) be added protein extraction buffer (100mMTris-HCl, 0.1M NaCl, 20mM dithiothreitol (DTT),
2wt% Lauryl.beta.-maltoside, pH 7.2), it is extracted in 90~100 DEG C of water-baths under different disposal in Broiler Tissues
GSTA2 protein 15~30min, by taking supernatant after 4 DEG C of centrifugation 30min of 20000g after homogenate, using BCA method to supernatant
In total protein carry out protein quantification.
(2) digestion generates the specific peptide fragment represented
Different group sample 50ug are taken respectively, first using 10mM dithiothreitol (DTT) in 50 DEG C of reduction 1h opened disulfide bonds, then
Sulfydryl reaction 30min is closed under dark condition with the iodoacetamide of 20mM；Lys- is added according to the ratio of 1:100 (mass ratio)
Trypsase is added digestion 16 under the conditions of 37 DEG C according still further to the ratio of 1:30 (mass ratio) in C digestion 6h under the conditions of 37 DEG C
Hour, reaction is terminated using the trifluoroacetic acid of 1wt%, and vapor away all enzyme cutting buffering liquids under conditions of 50 DEG C, obtained
Dried specific peptide fragment.
(3) separation of GSTA2 specificity peptide fragment
Dried specific peptide fragment is re-dissolved using the 95wt% water+5wt%+0.1wt% formic acid of 50ul, is used
The molten specific peptide fragment of 6500 counterweight of nanoLC-QTRAQ carries out multiple ion and monitors quantitative analysis, the C18 hair that when analysis uses
Tubule analytical column specification are as follows: 75 3 μm of C18-CL of μ m 15cm ChromXPMobile phase A is water+0.1wt% formic acid
+ 5wt%DMSO, Mobile phase B are acetonitrile+0.1wt% formic acid+5wt%DMSO, popular phase gradient are as follows: 0~1min, 95%A；1~
1.1min, 90%A；1.1~60min, 80%A；60~70min, 76%A；70~75min, 50%A；75~75.5min,
20%A；75.5~80.5min, 20%A；80.5~81min, 95%A；81~90min, 95%A；Flow velocity is 500nl/min,
Guarantee that three specific peptide fragments sequentially enter mass spectrum and analyzed.
(4) multiple ion monitoring method analyzes the signal strength of specific peptide fragment in sample
Mass spectrometry parameters setting are as follows: 504.8m/z is the parent ion of AILC [CAM] YLAGK (shown in SEQ ID NO:1), In
The daughter ion generated under 32.5V collision voltage is 551.3m/z, 711.3m/z, 824.4m/z；465.3m/z is NIALITER
The parent ion of (shown in SEQ ID NO:2), the daughter ion generated under 36.3V collision voltage be 631.4m/z, 702.4m/z,
815.5m/z；775.4m/z is the parent ion of SDILSAFPLLQAFK (shown in SEQ ID NO:3), under 31.2V collision voltage
The daughter ion of generation is 816.5m/z, 963.6m/z, 1034.6m/z.
In this step, the 551.3m/z generated using 504.8m/z, 711.3m/z, 824.4m/z signal strength summation generation
The signal strength of Table A ILC [CAM] YLAGK, the 631.4m/z generated using 465.3m/z, 702.4m/z, 815.5m/z signal are strong
Degree summation represents the signal strength of NIALITER, the 816.5m/z generated using 775.4m/z, 963.6m/z, 1034.6m/z letter
Number intensity summation represents the signal strength of SDILSAFPLLQAFK.Using AILC [CAM] YLAGK, NIALITER and
The signal summation of SDILSAFPLLQAFK tri- specific peptide fragments represents the signal strength of GSTA2 albumen, by comparing control group
With the signal strength of GSTA2 albumen in heat treatment group broiler chicken liver organization, control group and heat treatment group broiler chicken liver organization are realized
The relative quantification of middle GSTA2 albumen.
The method of the relative quantitative assay broiler chicken glutathione s-transferase GSTA2 of embodiment 3 a kind of
In the present embodiment, the method for the relative quantitative assay broiler chicken glutathione s-transferase GSTA2 includes following step
It is rapid:
(1) GSTA2 protein extraction
Taking 0.1g control group and heat treatment group broiler chicken liver organization respectively, (heat treatment group liver specimens are from broiler chicken 33
DEG C 30 hours broiler chicken of processing, control group liver specimens handle 30 hours broiler chicken from same period broiler chicken at 25 DEG C), according to 1:
The ratios of 5 (mass ratioes) be added protein extraction buffer (80mMTris-HCl, 0.3M NaCl, 15mM dithiothreitol (DTT),
1wt% Lauryl.beta.-maltoside, pH 7.5), it is extracted in 90~100 DEG C of water-baths under different disposal in Broiler Tissues
GSTA2 protein 15~30min, by taking supernatant after 4 DEG C of centrifugation 30min of 20000g after homogenate, using BCA method to supernatant
In total protein carry out protein quantification.
(2) digestion generates the specific peptide fragment represented
Different group sample 50ug are taken respectively, first using 10mM dithiothreitol (DTT) in 50 DEG C of reduction 1h opened disulfide bonds, then
Sulfydryl reaction 30min is closed under dark condition with the iodoacetamide of 20mM；Lys- is added according to the ratio of 1:150 (mass ratio)
Trypsase is added digestion 14 under the conditions of 37 DEG C according still further to the ratio of 1:60 (mass ratio) in C digestion 5h under the conditions of 37 DEG C
Hour, reaction is terminated using the trifluoroacetic acid of 5wt%, and vapor away all enzyme cutting buffering liquids under conditions of 45 DEG C, obtained
Dried specific peptide fragment.
(3) separation of GSTA2 specificity peptide fragment
Dried specific peptide fragment is re-dissolved using the 95wt% water+5wt%+0.1wt% formic acid of 50ul, is used
The molten specific peptide fragment of 6500 counterweight of nanoLC-QTRAQ carries out multiple ion and monitors quantitative analysis, the C18 hair that when analysis uses
Tubule analytical column specification are as follows: 75 3 μm of C18-CL of μ m 15cm ChromXPMobile phase A be water+0.1wt% formic acid+
5wt%DMSO, Mobile phase B are acetonitrile+0.1wt% formic acid+5wt%DMSO, popular phase gradient are as follows: 0~1min, 95%A；1~
1.1min, 90%A；1.1~60min, 80%A；60~70min, 76%A；70~75min, 50%A；75~75.5min,
20%A；75.5~80.5min, 20%A；80.5~81min, 95%A；81~90min, 95%A；Flow velocity is 300nl/min,
Guarantee that three specific peptide fragments sequentially enter mass spectrum and analyzed.
(4) multiple ion monitoring method analyzes the signal strength of specific peptide fragment in sample
Mass spectrometry parameters setting are as follows: 504.8m/z is the parent ion of AILC [CAM] YLAGK (shown in SEQ ID NO:1), In
The daughter ion generated under 34.2V collision voltage is 551.3m/z, 711.3m/z, 824.4m/z；465.3m/z is NIALITER
The parent ion of (shown in SEQ ID NO:2), the daughter ion generated under 35.7V collision voltage be 631.4m/z, 702.4m/z,
815.5m/z；775.4m/z is the parent ion of SDILSAFPLLQAFK (shown in SEQ ID NO:3), under 34.5V collision voltage
The daughter ion of generation is 816.5m/z, 963.6m/z, 1034.6m/z.
In this step, the 551.3m/z generated using 504.8m/z, 711.3m/z, 824.4m/z signal strength summation generation
The signal strength of Table A ILC [CAM] YLAGK, the 631.4m/z generated using 465.3m/z, 702.4m/z, 815.5m/z signal are strong
Degree summation represents the signal strength of NIALITER, the 816.5m/z generated using 775.4m/z, 963.6m/z, 1034.6m/z letter
Number intensity summation represents the signal strength of SDILSAFPLLQAFK.Using AILC [CAM] YLAGK, NIALITER and
The signal summation of SDILSAFPLLQAFK tri- specific peptide fragments represents the signal strength of GSTA2 albumen, by comparing control group
With the signal strength of GSTA2 albumen in heat treatment group broiler chicken liver organization, control group and heat treatment group broiler chicken liver organization are realized
The relative quantification of middle GSTA2 albumen.
The mass spectrogram of AILC [CAM] YLAGK in embodiment 1 in heat treatment group broiler chicken liver organization is as shown in Figure 1, m/z
The signal strength that the signal strength that 551.3 signal strength is 33, m/z 711.3 is 71, m/z 824.4 is 139, AILC
The signal strength of [CAM] YLAGK is 33+71+139=243；In embodiment 1 in heat treatment group broiler chicken liver organization
The mass spectrogram of NIALITER as shown in Fig. 2, the signal strength that the signal strength of m/z 631.4 is 1.9, m/z 702.4 is 9.3,
The signal strength that the signal strength of m/z 815.5 is 0.79, NIALITER is 1.9+9.3+0.79=11.99, heat in embodiment 1
The mass spectrogram of SDILSAFPLLQAFK in processing group broiler chicken liver organization is as shown in figure 3, the signal strength of m/z 816.5 is
The signal that the signal strength that 209, m/z 963.6 signal strength is 152, m/z 1034.6 is 95, SDILSAFPLLQAFK is strong
Degree is 209+152+95=456, i.e., the signal strength of GSTA2 albumen is 243+11.99+ in heat treatment group broiler chicken liver organization
456=710.99.According to identical method, the signal strength for calculating GSTA2 albumen in control group broiler chicken liver organization is
781.31, therefore, in the present embodiment in heat treatment group broiler chicken liver organization in GSTA2 albumen and control group broiler chicken liver organization
The ratio of GSTA2 albumen is 710.99/781.31=0.91.
As shown in the above, the present invention uses LC-MS instrument, is realized by multiple ion detection method to not same
The relative quantification of GSTA2 protein in product is mainly generated by intracellular protein enzyme Lys-C and trypsase digestion twice
AILC [CAM] YLAGK, NIALITER and SDILSAFPLLQAFK of GSTA2 totally 3 specific peptide fragments, use LC-MS instrument
GSTA2 is determined in specific detection AILC [CAM] YLAGK, a NIALITER and SDILSAFPLLQAFK primary and secondary ion pair realization
Amount analysis.The present invention is stronger relative to antibody Western Blot method specificity is based on, and eliminates and prepare the cumbersome of antibody
Step improves chicken source GSTA2 Protein Detection flux and efficiency.
It should be understood by those ordinary skilled in the art that: the discussion of any of the above embodiment is exemplary only, not
It is intended to imply that the scope of the present disclosure (including claim) is limited to these examples；Under thinking of the invention, above embodiments
Or it can also be combined between the technical characteristic in different embodiments, and there are different aspects present invention as described above
Many other variations, in order to it is concise they do not provided in details.Therefore, all within the spirits and principles of the present invention,
Any omission, modification, equivalent replacement, improvement for being made etc., should all be included in the protection scope of the present invention.
Sequence table
<110>Institute of Animal Sciences, Chinese Academy of Agricultural Sciences
<120>a kind of method of relative quantitative assay broiler chicken glutathione s-transferase GSTA2
<130> FI170435
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 12
<212> PRT
<400> 1
Ala-Ile-Leu-Cys-[Cys-Ala-Met]-Tyr-Leu-Ala-Gly-Lys
1 5 12
<210> 2
<211> 8
<212> PRT
<400> 2
Asn-Ile-Ala-Leu-Ile-Thr-Glu-Arg
1 5 8
<210> 3
<211> 14
<212> PRT
<400> 3
Ser-Asp-Ile-Leu-Ser-Ala-Phe-Pro-Leu-Leu-Gln-Ala-Phe-Lys
1 5 10 14
Claims (8)
1. a kind of method of relative quantitative assay broiler chicken glutathione s-transferase GSTA2, which is characterized in that first carry out broiler chicken paddy
The extraction of the sweet peptide S transferase GSTA2 of Guang, then digestion is carried out to broiler chicken glutathione s-transferase GSTA2 and obtains specific peptide fragment,
Then the primary and secondary ion pair of specific peptide fragment is detected using LC-MS instrument, the daughter ion signal strength for obtaining specific peptide fragment is total
With, the signal strength of broiler chicken glutathione s-transferase GSTA2 is obtained according to the daughter ion signal strength summation of special peptide fragment, it is logical
The signal strength for comparing broiler chicken glutathione s-transferase GSTA2 in different disposal broiler chicken sample is crossed, to realize to different disposal
The relative quantification of broiler chicken glutathione s-transferase GSTA2 in broiler chicken sample；
The specific peptide fragment of the broiler chicken glutathione s-transferase GSTA2 is 3, the amino acid sequence point of 3 specific peptide fragments
It Wei not be shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3；
Wherein, SEQ ID NO:1:Ala-Ile-Leu-Cys- [Cys-Ala-Met]-Tyr-Leu-Ala-Gly-Lys；
SEQ ID NO:2:Asn-Ile-Ala-Leu-Ile-Thr-Glu-Arg；
SEQ ID NO:3:Ser-Asp-Ile-Leu-Ser-Ala-Phe-Pro-Leu-Leu-Gln-Ala-Phe-Lys.
2. the method for relative quantitative assay broiler chicken glutathione s-transferase GSTA2 according to claim 1, feature exist
In the parent ion of, specificity peptide fragment shown in SEQ ID NO:1 be 504.8m/z, daughter ion 551.3m/z, 711.3m/z,
824.4m/z, collision voltage corresponding to daughter ion are 30~35V；The parent ion of specificity peptide fragment shown in SEQ ID NO:2
For 465.3m/z, daughter ion 631.4m/z, 702.4m/z, 815.5m/z, collision voltage corresponding to daughter ion is 35~
40V；The parent ion of specificity peptide fragment shown in SEQ ID NO:3 be 775.4m/z, daughter ion 816.5m/z, 963.6m/z,
1034.6m/z, collision voltage corresponding to daughter ion are 30~35V.
3. the method according to claim 2 for relative quantitative assay broiler chicken glutathione s-transferase GSTA2, special
Sign is that the signal strength of specificity peptide fragment shown in SEQ ID NO:1 is daughter ion 551.3m/z, 711.3m/z, 824.4m/
The signal strength summation of z, the signal strength of specificity peptide fragment shown in SEQ ID NO:2 are daughter ion 631.4m/z, 702.4m/
The signal strength summation of z, 815.5m/z, the signal strength of specificity peptide fragment shown in SEQ ID NO:3 are daughter ion 816.5m/
The signal strength summation of z, 963.6m/z, 1034.6m/z, the signal strength of broiler chicken glutathione s-transferase GSTA2 are SEQ ID
Specificity peptide fragment shown in NO:1, specificity peptide fragment shown in specificity peptide fragment and SEQ ID NO:3 shown in SEQ ID NO:2
The signal strength summation of this three specific peptide fragments.
4. the method for relative quantitative assay broiler chicken glutathione s-transferase GSTA2 according to claim 1, feature exist
In first being separated using the high performance liquid chromatography in LC-MS instrument to 3 specific peptide fragments, examined subsequently into mass spectrum
It surveys；Wherein, the condition of 3 specific peptide fragments is separated are as follows: chromatographic column is the capillary chromatographic column of C18 filler, and flow velocity is arranged 200
~500nL/min, mobile phase A are as follows: water+0.1wt% formic acid+5wt%DMSO, Mobile phase B are as follows: acetonitrile+0.1wt% formic acid+
5wt%DMSO uses gradient elution, popular phase gradient are as follows: 0~1min, 95%A when separating 3 specific peptide fragments；1~
1.1min, 90%A；1.1~60min, 80%A；60~70min, 76%A；70~75min, 50%A；75~75.5min,
20%A；75.5~80.5min, 20%A；80.5~81min, 95%A；81~90min, 95%A.
5. the method for relative quantitative assay broiler chicken glutathione s-transferase GSTA2 according to claim 1, feature exist
In the extraction step of broiler chicken glutathione s-transferase GSTA2 are as follows: extract meat under different disposal using protein extraction buffer
Broiler chicken glutathione s-transferase GSTA2 in chicken tissues, and it is quantitative to the gross protein extracted；
Digestion broiler chicken broiler chicken glutathione s-transferase GSTA2 obtains specific peptide fragment step are as follows: takes total egg of different disposal sample
White matter, the first re-closed protein interior sulfydryl of opened disulfide bond, then sequentially adds intracellular protein enzyme and trypsase carries out enzyme
It cuts, after terminating endonuclease reaction, obtains specific peptide fragment.
6. the method for relative quantitative assay broiler chicken glutathione s-transferase GSTA2 according to claim 5, feature exist
In, in the extraction step of broiler chicken glutathione s-transferase GSTA2, the ingredient of the protein extraction buffer are as follows: 50~
100mM Tris-HCl, 0.1~0.5M NaCl, 10~20mM dithiothreitol (DTT), 0.5~2wt% Lauryl.beta.-maltoside,
PH 7.2~8.0；It is extracted in water-bath at 90~100 DEG C using protein extraction buffer under different disposal in Broiler Tissues
15~30min of broiler chicken glutathione s-transferase GSTA2.
7. the method for relative quantitative assay broiler chicken glutathione s-transferase GSTA2 according to claim 5, feature exist
In being generated in specific peptide fragment step in digestion, first use dithiothreitol (DTT) opened disulfide bond, then pass through acetamide closed protein
Sulfydryl inside matter, adds formic acid or trifluoroacetic acid terminates endonuclease reaction.
8. the method for relative quantitative assay broiler chicken glutathione s-transferase GSTA2 according to claim 5, feature exist
In being generated in specific peptide fragment step in digestion, sequentially add intracellular protein enzyme and trypsase carries out the specific steps of digestion
Are as follows: intracellular protein enzyme, the total egg of digestion first is added according to the ratio that the mass ratio of intracellular protein enzyme and total protein is 1:100~200
After white 4~6 hours, trypsase, digestion is added according still further to the ratio that the mass ratio of trypsase and total protein is 1:30~60
12~16 hours.
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| CN107290461A (en) * | 2017-07-14 | 2017-10-24 | 浙江工商大学 | A kind of method for the LC-MS analysis for setting up royal jelly allergic protein |
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| CN102687020A (en) * | 2009-10-09 | 2012-09-19 | 西福根有限公司 | Multiplex quantitation of individual recombinant proteins in a mixture by signature peptides and mass spectrometry |
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