CN108169397A - 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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- CN108169397A CN108169397A CN201711237702.1A CN201711237702A CN108169397A CN 108169397 A CN108169397 A CN 108169397A CN 201711237702 A CN201711237702 A CN 201711237702A CN 108169397 A CN108169397 A CN 108169397A
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- gsta2
- broiler chicken
- glutathione
- transferase
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
Abstract
The invention discloses a kind of methods of relative quantitative assay broiler chicken glutathione s-transferase GSTA2, 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, so as to fulfill 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 SEQ ID NO:1、SEQ ID NO:2 and SEQ ID NO:Shown in 3.The method of the present invention eliminates the tedious steps for preparing antibody relative to stronger based on antibody Western Blot method specificity, improves chicken source GSTA2 Protein Detections 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 technology
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 animal growing environment temperature be more than its metabolic stability area upper limit, by body physics adjusting cannot maintain thermal balance when, lead
It causes body that can generate nonspecific response caused by response environment high temperature to react.Numerous studies show broiler chicken under the conditions of heat stress
Feed intake can decline 14%~17%, while can also cause that immunity of meat chickens declines, digestive disease is multiple, digestibility declines, sternly
Ghost image rings broiler chicken survival rate and efficiency of feed utilization, and economic loss is brought to poultry husbandry.It is reported that the U.S. is every year caused by heat stress
Husbandry sector loses about 16.9~23.6 hundred million dollars, and wherein up to 1.25~1.65 hundred million dollars, broiler chicken loses 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 causes cellular damage and Apoptosis by approach such as oxidative stress, inflammatory reactions, to animal machine
Body tissue causes to damage.When cell is in oxidative stress status, a series of activity of intracellular antioxidases improves, to maintain
Cellular oxidation-anti-oxidant balance environment 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 enzymes.
II phase xenobiotic metabolism enzyme alpha type glutathione S-transferases 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 carrying
It takes and is inverted to cDNA in organizer after RNA, designing specific primer according to GSTA2 genes expands cDNA templates, uses
Fluorescence real-time quantitative PCR technology is compared analysis to GSTA2 transcriptional levels 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 outcome albumen.At present, GSTA2 is analyzed on protein level can most reflect the albumen in tissue, cell
With the expression contents in body fluid, it will usually the methods of using enzyme-linked immunization, Western Blot, Tissue in situ hybridization, however
More than protein level detection method is required for the antibody specific recognition GSTA2 albumen of high quality.Due to preparation currently on the market
The antigen protein majority of antibody even derived from people, mouse and rat isotype biology, is gone 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 want 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
Clonal antibody can improve the specificity of antibody, reduce non-specific binding and cross reaction, but the monoclonal control body period is long, takes
With height；If expense can be reduced by preparing GSTA2 polyclonal antibodies, 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 considered, the present invention uses multiple ion
The method of detection establishes a kind of GSTA2 protein quantification methods 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 pairs and collision energy are realized and GSTA2 albumen are carried out on protein level
Relative quantitative assay substantially increases detection sensitivity, accuracy and detection flux.
Invention content
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.It is of the invention mainly to analyze difference using mass spectrum
Peptide fragment quality, and fragmentation, and analysis particular polypeptide production again can be carried out to the specific peptide fragment in complex samples in controlled condition
The quality of raw fragmentation small peptide searches out special primary and secondary ion pair, using special according to principles above by optimizing collision energy
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,
So as to fulfill 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 SEQ ID NO:1、SEQ IDNO:2 and SEQ ID NO:Shown in 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, SEQ ID NO:The parent ion of specific peptide fragment shown in 1 is 504.8m/z,
Daughter ion is 551.3m/z, 711.3m/z, 824.4m/z, and the collision voltage corresponding to daughter ion is 30~35V；SEQ ID NO:
The parent ion of specific peptide fragment shown in 2 be 465.3m/z, daughter ion 631.4m/z, 702.4m/z, 815.5m/z, daughter ion
Corresponding collision voltage is 35~40V；SEQ ID NO:The parent ion of specific peptide fragment shown in 3 be 775.4m/z, son from
Son is 816.5m/z, 963.6m/z, 1034.6m/z, and the collision voltage corresponding to daughter ion is 30~35V.
In some embodiments of the invention, SEQ ID NO:The signal strength of specific peptide fragment shown in 1 is daughter ion
The signal strength summation of 551.3m/z, 711.3m/z, 824.4m/z, SEQ ID NO:The signal of specific peptide fragment shown in 2 is strong
It spends for daughter ion 631.4m/z, the signal strength summation of 702.4m/z, 815.5m/z, SEQ ID NO:Specific peptide shown in 3
The signal strength of section is the signal strength summation of daughter ion 816.5m/z, 963.6m/z, 1034.6m/z, and broiler chicken glutathione S turns
The signal strength for moving enzyme GSTA2 is SEQ ID NO:Specific peptide fragment, SEQ ID NO shown in 1:Specific peptide fragment shown in 2
With SEQ ID NO:The signal strength summation of the specific peptide fragment of specific peptide fragment this three shown in 3.
In some embodiments of the invention, first using the high performance liquid chromatography in LC-MS instrument to 3 specific peptides
Duan Jinhang is detached, and is detected subsequently into mass spectrum；Wherein, the condition of the specific peptide fragments of separation 3 is：Chromatographic column is filled out for C18
The capillary chromatographic column of material, flow velocity are arranged on 200~500nL/min, and mobile phase A is：Water+0.1wt% formic acid+5wt%DMSO,
Mobile phase B is：Acetonitrile+0.1wt% formic acid+5wt%DMSO, when detaching 3 specific peptide fragments using gradient elution, mobile phase
Start gradient is the Mobile phase B of 95% mobile phase A+5%, and popular phase gradient is：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 is：It uses
Broiler chicken glutathione s-transferase GSTA2 under protein extraction buffer extraction different disposal in Broiler Tissues, and to extracting
Gross protein quantify；
Digestion broiler chicken broiler chicken glutathione s-transferase GSTA2 obtains specific peptide fragment step：Take 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 after terminating endonuclease reaction, obtains specific peptide fragment.
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 is：50~100mM Tris-HCl, 0.1~0.5MNaCl, 10~20mM dithiothreitol (DTT)s,
0.5~2wt% Lauryl.beta.-maltosides, 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 in digestion generates specific peptide fragment step, 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, in digestion generates specific peptide fragment step, intracellular protein enzyme is sequentially added
With trypsase carry out digestion the specific steps are：It is first 1 according to the mass ratio of intracellular protein enzyme and total protein:100~200
Ratio adds in intracellular protein enzyme, and digestion total protein was 1 according still further to the mass ratio of trypsase and total protein after 4~6 hours:30
~60 ratio adds in trypsase, digestion 12~16 hours.
In the present invention, a kind of method of relative quantitative assay GSTA2 albumen, includes the following steps：
(1) GSTA2 protein extractions
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 15s~30min in the lower Broiler Tissues of processing, and the gross protein extracted is quantified；
(2) digestion generates the specific peptide fragment represented
The total protein of the accurate different disposal sample for weighing phase homogenous quantities, passes through the sulfydryl inside acetamide closed protein matter
Afterwards, using according to 1：100~200 (intracellular protein enzyme Lys-C：Total protein, mass ratio) ratio, digestion total protein 4~6 hours
Afterwards, according still further to 1：30~60 (trypsase：Total protein, mass ratio) ratio add in trypsase 12~16 hours, addition 1~
The formic acid or trifluoroacetic acid of 5wt% terminates endonuclease reaction, and the buffer solution in volatilizing sample is rotated under the conditions of 40~50 DEG C,
Obtain specific peptide fragment；
Sequence is obtained as AILC [CAM] YLAGK (SEQ ID NO by operating above:Shown in 1, wherein " [] " represents acetyl
Change modification), NIALITER (SEQ ID NO:Shown in 2) and SDILSAFPLLQAFK (SEQ ID NO:Shown in 3) three it is special
Peptide fragment；
(3) separation of GSTA2 specificity peptide fragment
Specific peptide section sequence is detached using LC-MS instrument and signal acquisition, wherein, detach specific peptide fragment
Chromatographic column be C18 fillers capillary chromatographic column, flow velocity is arranged on 200~500nL/min, and mobile phase A is water+0.1wt%
Formic acid+5wt%DMSO, Mobile phase B are acetonitrile+0.1wt% formic acid+5wt%DMSO, and gradient is used in the specific peptide fragment of separation
Elution, popular phase gradient are：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；Ensure 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
Three sub- parent ions of the specific peptide fragment of setting are to being respectively：504.8m/z be AILC [CAM] YLAGK it is female from
Son, the daughter ion generated under 30~35V collision voltages 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 voltages 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 voltages 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 generations
The signal strength of Table A ILC [CAM] YLAGK, the 631.4m/z generated using 465.3m/z, 702.4m/z, 815.5m/z signals 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 the specific peptide fragments of SDILSAFPLLQAFK tri- 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
Lower advantageous effect：
The present invention is realized by multiple ion detection method to GSTA2 protein in different samples using LC-MS instrument
Relative quantification, mainly pass through the digestion twice of intracellular protein enzyme Lys-C and trypsase generate GSTA2 AILC [CAM]
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.It is of the invention opposite
In stronger based on antibody Western Blot method specificity, and the tedious steps for preparing antibody are eliminated, improve Ji Yuan
GSTA2 Protein Detections flux and efficiency.
Description of the drawings
Fig. 1 is the mass spectrogram of AILC [CAM] YLAGK in heat treatment group broiler chicken liver organization in the embodiment of the present invention 1；
Fig. 2 is the mass spectrogram of the NIALITER in heat treatment group broiler chicken liver organization in the embodiment of the present invention 1；
Fig. 3 is the mass spectrogram of the SDILSAFPLLQAFK in heat treatment group broiler chicken liver organization in the embodiment of the present invention 1.
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 1 relative quantitative assay broiler chicken glutathione s-transferase GSTA2 of embodiment a kind of
In the present embodiment, the method for the relative quantitative assay broiler chicken glutathione s-transferase GSTA2 includes following step
Suddenly：
(1) GSTA2 protein extractions
Taking 0.1g control groups and heat treatment group broiler chicken liver organization respectively, (heat treatment group liver specimens are from broiler chicken 33
DEG C processing broiler chicken of 24 hours, control group liver specimens are from the broiler chicken of same period broiler chicken processing 24 hours at 25 DEG C), according to 1：
The ratios of 5 (mass ratioes) add in protein extraction buffer (50mMTris-HCl, 0.5M NaCl, 10mM dithiothreitol (DTT),
1wt% Lauryl.beta.-maltosides, pH 8.0), it is extracted in 90~100 DEG C of water-baths under different disposal in Broiler Tissues
GSTA2 protein 15s~30min, by taking supernatant after 4 DEG C of centrifugation 30min of 20000g after homogenate, using BCA methods 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 restore 1h opened disulfide bonds at 50 DEG C using 10mM dithiothreitol (DTT)s, then
Sulfydryl reaction 30min is closed under dark condition with the iodoacetamide of 20mM；According to 1：The ratio of 200 (mass ratioes) adds in Lys-
C digestion 4h under the conditions of 37 DEG C, according still further to 1：The ratio of 50 (mass ratioes) adds in trypsase digestion 12 under the conditions of 37 DEG C
Hour, reaction is terminated, and all enzyme cutting buffering liquids are vapored away under conditions of 40 DEG C using the formic acid of 1wt%, obtain 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 counterweights of nanoLC-QTRAQ carries out multiple ion monitoring quantitative analysis, the C18 hairs that when analysis uses
Tubule analytical column specification is：75μm×15cm ChromXP C18-CL 3μmMobile phase A for water+0.1wt% formic acid+
5wt%DMSO, Mobile phase B are acetonitrile+0.1wt% formic acid+5wt%DMSO, and popular phase gradient is：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,
Ensure 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 are set as：504.8m/z is AILC [CAM] YLAGK (SEQ ID NO:Shown in 1) parent ion,
The daughter ion generated under 33.5V collision voltages is 551.3m/z, 711.3m/z, 824.4m/z；465.3m/z is NIALITER
(SEQ ID NO:Shown in 2) parent ion, the daughter ion generated under 38.8V collision voltages be 631.4m/z, 702.4m/z,
815.5m/z；775.4m/z is SDILSAFPLLQAFK (SEQ ID NO:Shown in 3) parent ion, produced under 31V collision voltages
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 generations
The signal strength of Table A ILC [CAM] YLAGK, the 631.4m/z generated using 465.3m/z, 702.4m/z, 815.5m/z signals 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 the specific peptide fragments of SDILSAFPLLQAFK tri- 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 2 relative quantitative assay broiler chicken glutathione s-transferase GSTA2 of embodiment a kind of
In the present embodiment, the method for the relative quantitative assay broiler chicken glutathione s-transferase GSTA2 includes following step
Suddenly：
(1) GSTA2 protein extractions
Taking 0.1g control groups and heat treatment group broiler chicken liver organization respectively, (heat treatment group liver specimens are from broiler chicken 33
DEG C processing broiler chicken of 18 hours, control group liver specimens are from the broiler chicken of same period broiler chicken processing 18 hours at 25 DEG C), according to 1：
The ratios of 5 (mass ratioes) add in protein extraction buffer (100mMTris-HCl, 0.1M NaCl, 20mM dithiothreitol (DTT),
2wt% Lauryl.beta.-maltosides, pH 7.2), it is extracted in 90~100 DEG C of water-baths under different disposal in Broiler Tissues
GSTA2 protein 15s~30min, by taking supernatant after 4 DEG C of centrifugation 30min of 20000g after homogenate, using BCA methods 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 restore 1h opened disulfide bonds at 50 DEG C using 10mM dithiothreitol (DTT)s, then
Sulfydryl reaction 30min is closed under dark condition with the iodoacetamide of 20mM；According to 1：The ratio of 100 (mass ratioes) adds in Lys-
C digestion 6h under the conditions of 37 DEG C, according still further to 1：The ratio of 30 (mass ratioes) adds in trypsase digestion 16 under the conditions of 37 DEG C
Hour, reaction is terminated, and all enzyme cutting buffering liquids are vapored away under conditions of 50 DEG C using the trifluoroacetic acid of 1wt%, 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 counterweights of nanoLC-QTRAQ carries out multiple ion monitoring quantitative analysis, the C18 hairs that when analysis uses
Tubule analytical column specification is：75μm×15cm ChromXP C18-CL 3μmMobile phase A for water+0.1wt% formic acid+
5wt%DMSO, Mobile phase B are acetonitrile+0.1wt% formic acid+5wt%DMSO, and popular phase gradient is：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,
Ensure 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 are set as：504.8m/z is AILC [CAM] YLAGK (SEQ ID NO:Shown in 1) parent ion,
The daughter ion generated under 32.5V collision voltages is 551.3m/z, 711.3m/z, 824.4m/z；465.3m/z is NIALITER
(SEQ ID NO:Shown in 2) parent ion, the daughter ion generated under 36.3V collision voltages be 631.4m/z, 702.4m/z,
815.5m/z；775.4m/z is SDILSAFPLLQAFK (SEQ ID NO:Shown in 3) parent ion, under 31.2V collision voltages
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 generations
The signal strength of Table A ILC [CAM] YLAGK, the 631.4m/z generated using 465.3m/z, 702.4m/z, 815.5m/z signals 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 the specific peptide fragments of SDILSAFPLLQAFK tri- 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 3 relative quantitative assay broiler chicken glutathione s-transferase GSTA2 of embodiment a kind of
In the present embodiment, the method for the relative quantitative assay broiler chicken glutathione s-transferase GSTA2 includes following step
Suddenly：
(1) GSTA2 protein extractions
Taking 0.1g control groups and heat treatment group broiler chicken liver organization respectively, (heat treatment group liver specimens are from broiler chicken 33
DEG C processing broiler chicken of 30 hours, control group liver specimens are from the broiler chicken of same period broiler chicken processing 30 hours at 25 DEG C), according to 1：
The ratios of 5 (mass ratioes) add in protein extraction buffer (80mMTris-HCl, 0.3M NaCl, 15mM dithiothreitol (DTT),
1wt% Lauryl.beta.-maltosides, pH 7.5), it is extracted in 90~100 DEG C of water-baths under different disposal in Broiler Tissues
GSTA2 protein 15s~30min, by taking supernatant after 4 DEG C of centrifugation 30min of 20000g after homogenate, using BCA methods 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 restore 1h opened disulfide bonds at 50 DEG C using 10mM dithiothreitol (DTT)s, then
Sulfydryl reaction 30min is closed under dark condition with the iodoacetamide of 20mM；According to 1：The ratio of 150 (mass ratioes) adds in Lys-
C digestion 5h under the conditions of 37 DEG C, according still further to 1：The ratio of 60 (mass ratioes) adds in trypsase digestion 14 under the conditions of 37 DEG C
Hour, reaction is terminated, and all enzyme cutting buffering liquids are vapored away under conditions of 45 DEG C using the trifluoroacetic acid of 5wt%, 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 counterweights of nanoLC-QTRAQ carries out multiple ion monitoring quantitative analysis, the C18 hairs that when analysis uses
Tubule analytical column specification is：75μm×15cm ChromXP C18-CL 3μmMobile phase A for water+0.1wt% formic acid+
5wt%DMSO, Mobile phase B are acetonitrile+0.1wt% formic acid+5wt%DMSO, and popular phase gradient is：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,
Ensure 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 are set as：504.8m/z is AILC [CAM] YLAGK (SEQ ID NO:Shown in 1) parent ion,
The daughter ion generated under 34.2V collision voltages is 551.3m/z, 711.3m/z, 824.4m/z；465.3m/z is NIALITER
(SEQ ID NO:Shown in 2) parent ion, the daughter ion generated under 35.7V collision voltages be 631.4m/z, 702.4m/z,
815.5m/z；775.4m/z is SDILSAFPLLQAFK (SEQ ID NO:Shown in 3) parent ion, under 34.5V collision voltages
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 generations
The signal strength of Table A ILC [CAM] YLAGK, the 631.4m/z generated using 465.3m/z, 702.4m/z, 815.5m/z signals 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 the specific peptide fragments of SDILSAFPLLQAFK tri- 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
It spends for 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 is realized by multiple ion detection method to not similary using LC-MS instrument
The relative quantification of GSTA2 protein in product is mainly generated by the digestion twice of intracellular protein enzyme Lys-C and trypsase
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 eliminated and prepares the cumbersome of antibody relative to stronger based on antibody Western Blot method specificity
Step improves chicken source GSTA2 Protein Detections flux and efficiency.
Those of ordinary skills in the art should understand 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 is limited to these examples (including claim)；Under the thinking of the present invention, above example
Or it can also be combined between the technical characteristic in different embodiments, and there is different aspect 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 for being made, modification, equivalent replacement, improvement 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 (9)
- A kind of 1. 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 transferases 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, so as to fulfill to different disposal The relative quantification of broiler chicken glutathione s-transferase GSTA2 in broiler chicken sample.
- 2. the method for relative quantitative assay broiler chicken glutathione s-transferase GSTA2 according to claim 1, feature exist In 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 SEQ ID NO:1、SEQ ID NO:2 and SEQ ID NO:Shown in 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.
- 3. the method for relative quantitative assay broiler chicken glutathione s-transferase GSTA2 according to claim 2, feature exist In SEQ ID NO:The parent ion of specific peptide fragment shown in 1 be 504.8m/z, daughter ion 551.3m/z, 711.3m/z, 824.4m/z, the collision voltage corresponding to daughter ion are 30~35V；SEQ ID NO:The parent ion of specific peptide fragment shown in 2 For 465.3m/z, daughter ion 631.4m/z, 702.4m/z, 815.5m/z, collision voltage corresponding to daughter ion for 35~ 40V；SEQ ID NO:The parent ion of specific peptide fragment shown in 3 be 775.4m/z, daughter ion 816.5m/z, 963.6m/z, 1034.6m/z, the collision voltage corresponding to daughter ion are 30~35V.
- 4. the method according to claim 3 for relative quantitative assay broiler chicken glutathione s-transferase GSTA2, special Sign is, SEQ ID NO:The signal strength of specific peptide fragment shown in 1 is daughter ion 551.3m/z, 711.3m/z, 824.4m/ The signal strength summation of z, SEQ ID NO:The signal strength of specific peptide fragment shown in 2 is daughter ion 631.4m/z, 702.4m/ The signal strength summation of z, 815.5m/z, SEQ ID NO:The signal strength of specific peptide fragment shown in 3 is 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 is SEQ ID NO:Specific peptide fragment, SEQ ID NO shown in 1:Specific peptide fragment and SEQ ID NO shown in 2:Specific peptide fragment shown in 3 The signal strength summation of this three specific peptide fragments.
- 5. the method for relative quantitative assay broiler chicken glutathione s-transferase GSTA2 according to claim 2, feature exist In first using the high performance liquid chromatography in LC-MS instrument, to 3, specific peptide fragment detaches, and is examined subsequently into mass spectrum It surveys；Wherein, the condition of the specific peptide fragments of separation 3 is：Chromatographic column is the capillary chromatographic column of C18 fillers, and flow velocity is arranged on 200 ~500nL/min, mobile phase A are：Water+0.1wt% formic acid+5wt%DMSO, Mobile phase B are：Acetonitrile+0.1wt% formic acid+ 5wt%DMSO uses gradient elution when detaching 3 specific peptide fragments, and popular phase gradient is：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.
- 6. 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 is：Meat under different disposal is extracted using protein extraction buffer Broiler chicken glutathione s-transferase GSTA2 in chicken tissues, and the gross protein extracted is quantified；Digestion broiler chicken broiler chicken glutathione s-transferase GSTA2 obtains specific peptide fragment step：Take 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.
- 7. the method for relative quantitative assay broiler chicken glutathione s-transferase GSTA2 according to claim 6, feature exist In in the extraction step of broiler chicken glutathione s-transferase GSTA2, the ingredient of the protein extraction buffer is：50~ 100mM Tris-HCl, 0.1~0.5M NaCl, 10~20mM dithiothreitol (DTT)s, 0.5~2wt% Lauryl.beta.-maltosides, PH 7.2~8.0；It is extracted in water-bath under different disposal in Broiler Tissues at 90~100 DEG C using protein extraction buffer 15~30min of broiler chicken glutathione s-transferase GSTA2.
- 8. the method for relative quantitative assay broiler chicken glutathione s-transferase GSTA2 according to claim 6, feature exist In in digestion generates specific peptide fragment step, first using dithiothreitol (DTT) opened disulfide bond, then passing through acetamide closed protein Sulfydryl inside matter, adds formic acid or trifluoroacetic acid terminates endonuclease reaction.
- 9. the method for relative quantitative assay broiler chicken glutathione s-transferase GSTA2 according to claim 6, feature exist In in digestion generates specific peptide fragment step, sequentially adding intracellular protein enzyme and trypsase carry out the specific steps of digestion For：It is first 1 according to the mass ratio of intracellular protein enzyme and total protein:100~200 ratio adds in intracellular protein enzyme, the total egg of digestion It is 1 according still further to the mass ratio of trypsase and total protein after white 4~6 hours:30~60 ratio adds in trypsase, digestion 12~16 hours.
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