CN107064441B - The method of labelled protein evaluation quality of mutton based on phosphated peptide section screening - Google Patents
The method of labelled protein evaluation quality of mutton based on phosphated peptide section screening Download PDFInfo
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/02—Food
- G01N33/12—Meat; Fish
Landscapes
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The method for the labelled protein evaluation quality of mutton based on phosphated peptide section screening that the invention discloses a kind of, this method is by tree species for bio-energy source to height, the phosphorylation level of peptide fragment carries out qualitatively and quantitatively in low-quality mutton, and GO and KEGG enrichment analysis is carried out to the phosphorylated protein after qualitative, pass through the potential relationship of protein bio information analysis phosphorylated protein and quality of mutton, by protein phosphorylation level in height, phosphorylated protein in low-quality mutton with notable difference is as labelled protein, to judge the superiority and inferiority of quality of mutton according to the content of mutton marker protein.Phosphorylated protein in first identified of the present invention mutton, labelled protein for screening reflection quality of mutton provides foundation, and it can be according to the horizontal result judgement quality of mutton of protein phosphorylation, provide a kind of new method for evaluating quality, be conducive to create more profits for meat enterprise, it realizes high quality and favourable price, has a extensive future.
Description
Technical field
The invention belongs to food quality detection technical fields, and in particular to one kind is based on phosphated peptide section screening reflection meat
The labelled protein of matter, the method that different quality of mutton are evaluated using the labelled protein.
Background technique
In quality of mutton evaluation, the content of main component, current new fresh mutton main component mainly in detection mutton
In the detection technique of content, many most common methods of country are still traditional chemical analysis, and this method has detection speed
Degree is slower, needs the disadvantages of instrument and equipment is more and complicated, and the cost is higher, and detection process can destroy sample, is difficult to accomplish
To the on line real-time monitoring of new fresh mutton.In order to overcome the shortcomings of in conventional method, meat is detected using near infrared spectroscopic method
Quality and degree of metamorphism are a selections well, have detection speed it is fast, without doing excessive pretreatment to sample, can be achieved
The advantages that nondestructive measurement, the meat quality detection based on near-infrared spectral analysis technology were also once becoming one in meat evaluation
A hot topic, and continue till now.
However, when near infrared technology non-destructive testing quality of mutton, it is necessary to detection model is established, a large amount of sample is needed,
And the requirement to sample is high.It is all to study meat in terms of the apparent and chemical substance in view of conventional method and near-infrared method
The variation of matter, the identification of meat fails to go deep into explanation mechanism, and the factor for influencing quality of mutton is relatively more, and postmortem aging,
The structure of protein and functional characteristic are more significant to the qualitative effects of meat and meat products in carcass cutting position and muscle.After government official
Muscle cell metabolism in maturation largely decides the quality characteristic of meat, such as pH, tenderness, color and retentiveness
Equal meat qualities.And the most of myosinogen, fribrillin in muscle are by protein phosphorylation and dephosphorylation mistake
Journey influences, therefore phosphorylated protein can be used as the protein labeling of reflection quality of mutton.Meat quality evaluation index mainly has pH
Value, shearing force, yellowish pink, retentiveness etc..These indexs are all the macroscopic view reflections of micro-variations in muscle cell, and protein is as thin
The row envoy of born of the same parents' vital movement function, phosphorylation modification can lead to a series of physiological acoustic signals in muscle cell, thus shadow
Ring meat after killing.Therefore key protein phosphorylation modification and its phosphorylation level variation can be used as reflection meat quality in muscle
Label.
Summary of the invention
It is difficult to go deep into the deficiency of meat change mechanism and existing quality of mutton point etc. for current mutton evaluation method,
The purpose of the present invention is to provide it is a kind of based on phosphated peptide section quantitative and qualitative screen reflection quality of mutton labelled protein and with
The method of labelled protein evaluation quality of mutton.
Technical solution used by solving the above problems is made of following step:
1,12 hours high-quality mutton and low-quality meat samples after acquisition is killed.
2, extraction step 1 acquires the phosphorylated protein in sample, and the phosphorylated protein of acquisition is digested, and obtains phosphorylation
Peptide fragment.
3, the phosphated peptide section obtained using biological mass spectrometry to step 2 carries out qualitative and quantitative detection, with the phosphorus after qualitative
Peptide fragment is acidified in the logarithm conduct of the relative quantification value in high-quality mutton and the ratio between the relative quantification value in low-quality mutton
Judge that the foundation of peptide fragment phosphorylation level variation, the i.e. logarithm are greater than 1 phosphorylation level up-regulation, which is less than -1
Phosphorylation level lower, the logarithm be between 1 and -1 then the phosphated peptide section in high-quality mutton and low-quality mutton sample
Phosphorylation level in product is without significant changes.
4, GO and KEGG enrichment analysis is carried out to the corresponding phosphorylated protein of phosphated peptide section of the step 3 after qualitative.
5, according to the analysis of step 4 as a result, content difference in high-quality meat samples and low-quality meat samples is significant
Phosphorylated protein as labelled protein.
6, the content of the different meat samples marker proteins to be measured of detection, judges different mutton according to the content of labelled protein
Between quality height: if labelled protein is high-quality mutton labelled protein, the higher explanation of labelled protein content sheep to be measured
Meat sample quality is better；If labelled protein is low-quality mutton labelled protein, the higher explanation sheep to be measured of labelled protein content
Meat sample quality is poorer.
In above-mentioned steps 1, the high-quality mutton is the longissimus dorsi muscle meat of sheep, and low-quality mutton is the abdominal muscles of sheep.
In above-mentioned steps 3, the detection includes the detection of peptide fragment phosphorylation site and phosphorylation level.
In above-mentioned steps 5, the relative quantification value of phosphated peptide section and low high-quality mutton in the high-quality meat samples
The logarithm of the ratio between relative quantification value of phosphated peptide section is greater than 1 in sample or less than -1, then the phosphated peptide section is in high-quality
Content difference in meat samples and low high-quality mutton is significant.
The labelled protein of above-mentioned high-quality mutton is phosphofructokinase, triphosphoric acid isomerase 1, pyruvate kinase, phosphorus
Acid glycerol acid kinase 1, fructosediphosphate aldolase A and glyceraldehyde-3-phosphate dehydrogenase, the labelled protein of low-quality mutton are original
Myosin 1, myoglobulin heavy chain 1, myoglobulin heavy chain 2, myoglobulin heavy chain 6 and titin.
The present invention by low-quality mutton high after government official (using longissimus dorsi muscle as the high-quality mutton of representative and with abdominal muscles
For the low-quality mutton of representative) in phosphorylated protein extract and digest, then using biological mass spectrometry to the phosphorus after enzymatic hydrolysis
Be acidified peptide fragment carry out qualitative and quantitative analysis, and to mutton macroscopic view quality changes after government official (mutton pH value, shearing force, color,
Protein solubility, retentiveness) measurement, then will be qualitative after phosphorylating protein carry out GO and KEGG enrichment analysis, root
According to the potential relationship of protein bio information analysis phosphorylating protein and quality of mutton, by protein phosphorylation level (with egg
Judgment basis of the mass spectrum response signal value of the corresponding phosphated peptide section of white matter as protein phosphorylation level) high low-quality
Then phosphorylated protein in mutton with notable difference integrates the GO of meat samples marker protein to be measured as labelled protein
Analysis is enriched with KEGG as a result, judging the specific quality of mutton that the albumen is reflected.With existing quality of mutton identification method phase
Than, the invention has the following beneficial effects:
1, phosphorylating protein in first identified of the present invention mutton reflects that the protein labeling of the quality of mutton mentions for screening
Foundation is supplied.
2, the present invention is classified for quality of mutton after killing, can be according to the horizontal result judgement mutton product of protein phosphorylation
Matter.
3, the present invention has a extensive future, and has great importance for quality of mutton control and mutton classification point etc.,
Even there is directive significance to the height quality evaluation of beef.
4, the present invention creates more profits by being conducive to after high and low quality grading for meat enterprise, realizes high-quality excellent
Valence.
5, the present invention can be deep into molecule and gene level, embody meat change mechanism, to understand meat in depth
Quality comparison.
Detailed description of the invention
Fig. 1 is phosphated peptide section quantitative result distribution map.
Fig. 2 is phosphorylated protein GO classification results figure.
Fig. 3 is phosphorylated protein KEGG enrichment result figure.
Fig. 4 is Protein interaction mapping in high-quality mutton, and wherein APOBEC2 is that non-carrier B messenger rna editing enzymes are urged
Change subunit 2, PKM is pyruvate kinase, and TPI1 is triphosphoric acid isomerase 1, and PFKM is phosphofructokinase, and TWF2 is mariages albumen
Binding protein 2, ZC3HC1 are the zinc finger protein of type containing C3HC 1, and GAPDH is glyceraldehyde-3-phosphate dehydrogenase, and PGK1 is that phosphoric acid is sweet
Oily kinases 1, ALDOA are fructosediphosphate aldolase A, and HERC1 is HECT structural domain+E3 ubiquitin ligase family protein 1,
EIF4EBP1 is eukaryotic translation initiation factor 4E Binding Protein 1, and EIF4G1 is 4 gamma 1 of eukaryotic translation startup factor, and HSPA1A is
Heat-shock protein family A, HSP90AA1 are Heat shock protein72 family A1 subunit, and PPP1R3A is that protein phosphatase 1 regulation is sub-
Base 3A, CAMK2A are 2 α of cam kinase, and MUSTN1 is embryonic nucleus albumen 1, and ACY1 is 1 antibody of amino-acylase, ALDH1A2
It is phosphoprotein abundant in astroglia for 1 family A2 of aldehyde dehydrogenase, PEA15, POLR2M is rna plymerase ii subunit M,
PDLIM3 is the domain PDZ and LIM 3, and MYH7 is myoglobulin heavy chain 7, and VCL is vinculin, and TPM1 is tropomyosin 1, and TCAP is
Titin.
Fig. 5 is Protein interaction mapping in low-quality mutton, and wherein HSPB6 is heat-shock protein family B6, and ATP5B is
ATP synzyme, H+ transhipment, mitochondria F1 complex, β peptide, MUSTN1 are embryonic nucleus albumen 1, and MYH1 is myoglobulin heavy chain 1,
TPM1 is tropomyosin 1, and TTN is titin, and MYOZ1 is myogen regulatory protein 1, and MYOT is flesh contractile protein, and BAG3 is
Bcl-2 combination anti-apoptotic proteins 3, HSP90AA1 are Heat shock protein72 family A1 subunit, and MYH2 is myoglobulin heavy chain 2,
MYH6 is myoglobulin heavy chain 6, and ANXA2 is Annexin A2, and CLIP1 is cytoplasmic linker proteins 1, and NCBP1 is the combination of core cap
Protein protomer 1, ATP2A2 are myoplasm net type calcium ion ATP enzyme 2, and KIF1C is driving albumen 1C type, and MYOM3 is myosinogen 3,
ARPP19 is that recombined human cAMP adjusts phosphoprotein 19, and XRN1 is 5'-3'RNA excision enzyme 1, and STIM1 is stromal interaction molecule 1,
PSMB5 is proteasome subunit β 5, and PGM1 is that phosphoric acid reconciles enzyme 1, and TWF2 is mariages protein-binding protein 2.
Specific embodiment
The present invention is described in more detail with reference to the accompanying drawings and examples, but protection scope of the present invention is not limited only to
These embodiments.
Embodiment 1
1, choose weight is close, the identical Northern Shaanxi White Cashmere Goat of feeding manner 10 (ram, it is not dead, weight 23.12 ±
1.62kg), stop eating before government official 20h, cut off the water 2h, after government official by trunk hanging be placed in 4 DEG C of acid discharges.12h takes it to carry on the back longest after government official
Muscle as high-quality meat samples, take its abdominal muscles as low-quality meat samples.
2, the high-quality meat samples and each 1g of low-quality meat samples for weighing step 1, are respectively placed in 10mL centrifuge tube,
It is added 6mL buffer solution (including 7M urea, 2M thiocarbamide, 0.1%CHAPS, inhibitors of phosphatases, protease inhibitors), uses whirlpool
It revolves oscillator vortex to mix, is then crushed and is homogenized 3 times, each 60s, amplitude 22% using ultrasonic cell disruption instrument, be homogenized
After be placed at room temperature for 30min and under the conditions of 4 DEG C 15000r/min be centrifuged 20min, collect supernatant；Supernatant is placed in centrifuge tube
In, 100 μ L10mM DTT aqueous solutions are added in centrifuge tube, and place it in 60 DEG C reaction 1 hour in constant-temperature incubation device, then
The 40mM IAA aqueous solution that 40 μ L are newly prepared is added, reacts at room temperature 10 minutes.It is above-mentioned after the reaction was completed, transfer the solution into 10K
Super filter tube in, in 12000r/min be centrifuged 20 minutes, discard solution in collecting pipe, into super filter tube be added trypsase (pancreas
Protease and Proteins In Aqueous Solutions mass ratio are 1:50), and super filter tube is placed into 37 DEG C of enzymatic hydrolysis 12h in couveuse.It will enzymatic hydrolysis
Solution 12000r/min afterwards is centrifuged 20 minutes, obtains phosphated peptide section, freeze-drying.
3, the 0.2mg 100 μ L buffer solutions of phosphated peptide section being lyophilized (are contained into 5%TFA, 1M hydroxyacetic acid and 20%
The aqueous solution of ACN) dissolution after, in the solution be added 1.2mg TiO2(0.6mg TiO is added in every 100 μ g phosphated peptide section2) simultaneously
It is placed in 600r/min on shaking table and vibrates 10min, then 3000r/min is centrifuged 10min, collects supernatant and 1mg TiO is added2Again
It is secondary be placed in shake bed reaction 10min after be centrifuged, abandon supernatant.Merge centrifugation gained TiO twice2Precipitating.The TiO that will be collected into2It is heavy
After shallow lake mixes 1min with 100 μ L buffer solutions (aqueous solution containing 5%TFA, 1M hydroxyacetic acid and 20%ACN) first,
3000r/min be centrifuged 10min, gained precipitating cleaned with 100 μ L buffer solutions (aqueous solution containing 80% and 1%TFA) after again from
The heart, gained precipitating are centrifuged again after being cleaned with 100 μ L buffer solutions (aqueous solution containing 20% and 0.2%TFA), are collected precipitating, are used
50 μ L eluents (contain 28%NH3·H2The aqueous solution of O) TiO will be adsorbed in precipitating2On phosphated peptide section elute, receive
Collect eluent, freeze-drying obtains the phosphated peptide section of enriching and purifying.
After the mixed liquor dissolution for being 20:1 by 20 μ L methanol of the phosphated peptide section of above-mentioned enriching and purifying and formic acid volume ratio
12000r/min is centrifuged 10 minutes, Aspirate supernatant, combines Easy nLC1000 efficient liquid phase using Q-Exactive mass spectrograph
Series LC-the MSn (EASY-Spray column, 12cm × 75m, C18,318) of composition analyzes supernatant, chromatostrip
Part: mobile phase A: 100% ultrapure water, 0.1% formic acid, Mobile phase B: 100% acetonitrile, 0.1% formic acid load flow rate pump:
350nL/min, 15 minutes, separate flow velocity: 350nL/min, loading volume: 10 μ L, it is as shown in table 1 below that mobile phase separates gradient.
1 mobile phase of table separates gradient
The mass spectrum original document of generation is handled using MaxQuant1.5 software, database used in protein retrieval be from
The Ovis aries database obtained in NCBI is 1% filtering false positive with FDR.Respectively to high-quality mutton and low-quality sheep
The relative quantification value for all phosphated peptide sections identified in meat sample product does homogenization processing, asks phosphated peptide section in height later
The logarithm of the ratio between the relative quantification value in relative quantification value and low-quality meat samples in quality meat samples is as judgement
The foundation of peptide fragment phosphorylation level variation, the i.e. logarithm are greater than 1 phosphorylation level up-regulation, which is less than -1 phosphoric acid
Change horizontal down-regulation, the logarithm be between 1 and -1 then the phosphated peptide section in high-quality mutton and low-quality meat samples
Phosphorylation level without significant changes.Analyze the result is shown in Figure 1.
As shown in Figure 1, identification obtains 2125 phosphated peptide sections altogether in high-quality mutton and low-quality meat samples, these
Peptide fragment belongs to 750 phosphorylated proteins.Wherein phosphorylation level raises peptide fragment 387, and phosphorylation level lowers peptide fragment 581,
Peptide fragment 1157 without significant changes of phosphorylation level, they account for about 18%, 27% and the 55% of total peptide segment number respectively.Phosphoric acid
Change level extremely significant albumen of difference in two groups of samples mainly has: phosphofructokinase (PFKM), pyruvate kinase (PKM),
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), triphosphoric acid isomerase 1 (TPI1), fructose diphosphate aldehyde lyase A (ALDOA), flesh ball
Albumen (MYH1, MYH2 and MYH6), tropomyosin 1 (TPM1), titin (TCAP), myosin light chain (MYL2), flesh
Calcium protein I (troponin I), myoplasm net type calcium ion ATP enzyme 2 (ATP2A2), heat shock protein (HSP27 and HSP70-1A/
B) etc..
4, GO enrichment is carried out to the phosphorylated protein that step 3 detects using blast2GO2.8 software, as a result sees Fig. 2, and
Phosphorylated protein KEGG access is analyzed using online DAVID software, as a result sees Fig. 3.
As shown in Fig. 2, learnt by the bioprocess analysis participated in phosphorylated protein, what phosphorylated protein was primarily involved in
Bioprocess includes that bioprocess adjusts (Active Regulation of such as enzyme, GO:0050790), intracellular bioprocess (such as phosphorylation
Reaction, GO:0016310), metabolic process (GO:0008152) and signal transmit (such as cynapse signal transduction, GO:0007268)；It is logical
It crosses and phosphorylated protein, which is primarily present in cytoplasm (GO:0005737), is located at, to be learnt to the cellular localization analysis of phosphorylated protein
In the sarcoplasmic reticulum (GO:0033017) and organelle of cell membrane (such as mitochondria, GO:0005739)；By to phosphorylated protein
Functional analysis learns that most of phosphorylated proteins have binding function (such as in conjunction with actin；GO:0003779), catalytic activity
(such as protein kinase activity；GO:0016301), transport activity (such as oxygen transportation, GO:0005344) and the function point analysis factor
(such as atriphos zymoexciter, GO:0001671).
As can be seen from Figure 3,750 phosphorylated proteins take part in 23 accesses, wherein carbohydrate metabolism, amino acid generation
It thanks, signal transduction and lipid-metabolism etc. are that KEGG is enriched with significant metabolic pathway.
STRING database protein-protein interaction network is constructed by online database, because database lacks correlation
The Protein Information of Ovis aries species, therefore using the Bos taurus albumen database for belonging to same section with it.
As shown in figure 4, participating in enzyme such as phosphofructokinase (PFKM), the triphosphoric acid isomerase of glycolysis in high-quality mutton
1 (TPI1), pyruvate kinase (PKM), phosphoglyceric kinase 1 (PGK1), fructosediphosphate aldolase A (ALDOA) and glycerol
Aldehyde -3- phosphate dehydrogenase (GAPDH) constitutes the core of the protein-protein interaction network, these albumen also can be used as high-quality
The labelled protein of mutton.
As shown in figure 5, sharing 24 in low-quality mutton has the complicated phosphorylated protein to interact, in the protein phase
In interaction network, muscle skelemin such as tropomyosin 1 (TPM1), myoglobulin heavy chain 1 (MYH1), myosin weight
The interaction of chain 2 (MYH2), myoglobulin heavy chain 6 (MYH6) and titin (TTN) constitutes the core of the network, these
Albumen also can be used as the labelled protein of low-quality mutton.
6, using the series LC-MSn of Q-Exactive mass spectrograph joint Easy nLC1000 efficient liquid phase composition according to upper
The content for stating the different meat samples marker proteins to be measured of method detection of step 2~3, can sentence according to the content of labelled protein
The quality height to break between different mutton: if labelled protein is high-quality mutton labelled protein, labelled protein content is higher
Illustrate that meat samples quality to be measured is better；If labelled protein is low-quality mutton labelled protein, labelled protein content is higher
Illustrate that meat samples quality to be measured is poorer.
Claims (6)
1. a kind of method of the labelled protein evaluation quality of mutton based on phosphated peptide section screening, it is characterised in that by following steps
Composition:
(1) 12 hours high-quality mutton and low-quality meat samples after acquisition is killed；
(2) phosphorylated protein in extraction step (1) acquisition sample, and the phosphorylated protein of acquisition is digested, obtain phosphorylation
Peptide fragment；
(3) the qualitative and quantitative detection of phosphated peptide section progress step (2) obtained using biological mass spectrometry, with the phosphoric acid after qualitative
Change peptide fragment in high-quality mutton relative quantification value and the logarithm conduct of the ratio between relative quantification value in low-quality mutton sentence
The foundation of disconnected peptide fragment phosphorylation level variation, the i.e. logarithm are greater than 1 phosphorylation level up-regulation, which is less than -1 phosphorus
Be acidified horizontal down-regulation, the logarithm be between 1 and -1 then the phosphated peptide section in high-quality mutton and low-quality meat samples
In phosphorylation level without significant changes；
(4) the corresponding phosphorylated protein of phosphated peptide section after qualitative to step (3) carries out GO enrichment and phosphorylated protein KEGG
Path analysis；
(5) according to the analysis result of step (4) and conjugated protein interactive network, by high-quality meat samples and low-quality
The significant phosphorylated protein of content difference is as labelled protein in meat samples；
(6) content of the different meat samples marker proteins to be measured of detection, according to the content of labelled protein judge different mutton it
Between quality height: if labelled protein is high-quality mutton labelled protein, the higher explanation of labelled protein content mutton to be measured
Sample quality is better；If labelled protein is low-quality mutton labelled protein, the higher explanation mutton to be measured of labelled protein content
Sample quality is poorer.
2. the method for the labelled protein evaluation quality of mutton according to claim 1 based on phosphated peptide section screening, special
Sign is: in step (1), the high-quality mutton is the longissimus dorsi muscle meat of sheep, and low-quality mutton is the abdominal muscles of sheep.
3. the method for the labelled protein evaluation quality of mutton according to claim 1 based on phosphated peptide section screening, special
Sign is: in step (3), the detection includes the detection of peptide fragment phosphorylation site and phosphorylation level.
4. the method for the labelled protein evaluation quality of mutton according to claim 1 based on phosphated peptide section screening, special
Sign is: in step (5), the relative quantification value of phosphated peptide section and low high-quality mutton sample in the high-quality meat samples
The logarithm of the ratio between relative quantification value of phosphated peptide section is greater than 1 in product or less than -1, then the phosphated peptide section is in high-quality sheep
Content difference in meat sample product and low high-quality mutton is significant.
5. the method for the labelled protein evaluation quality of mutton according to claim 1 based on phosphated peptide section screening, special
Sign is: in step (5), the labelled protein of the high-quality mutton is phosphofructokinase, triphosphoric acid isomerase 1, third
Pyruvate kinase, phosphoglyceric kinase 1, fructosediphosphate aldolase A and glyceraldehyde-3-phosphate dehydrogenase.
6. the method for the labelled protein evaluation quality of mutton according to claim 1 based on phosphated peptide section screening, special
Sign is: in step (5), the labelled protein of the low-quality mutton is tropomyosin 1, myoglobulin heavy chain 1, flesh ball
Ferritin heavy chain 2, myoglobulin heavy chain 6 and titin.
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