CN105646655B - A method of identifying Holothuria mexicana using specificity peptide fragment group - Google Patents
A method of identifying Holothuria mexicana using specificity peptide fragment group Download PDFInfo
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- 241001024242 Holothuria mexicana Species 0.000 title claims abstract description 46
- 102000007079 Peptide Fragments Human genes 0.000 title claims abstract description 24
- 108010033276 Peptide Fragments Proteins 0.000 title claims abstract description 24
- 238000000034 method Methods 0.000 title claims abstract description 18
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 37
- 229920001184 polypeptide Polymers 0.000 claims abstract description 36
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 36
- 241000251511 Holothuroidea Species 0.000 claims description 21
- 239000012528 membrane Substances 0.000 claims description 9
- 239000000243 solution Substances 0.000 claims description 9
- 239000000284 extract Substances 0.000 claims description 7
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 claims description 6
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 claims description 6
- 235000012538 ammonium bicarbonate Nutrition 0.000 claims description 6
- 239000001099 ammonium carbonate Substances 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 238000001819 mass spectrum Methods 0.000 claims description 6
- 239000012460 protein solution Substances 0.000 claims description 6
- 238000000108 ultra-filtration Methods 0.000 claims description 6
- 102000004142 Trypsin Human genes 0.000 claims description 5
- 108090000631 Trypsin Proteins 0.000 claims description 5
- 238000001514 detection method Methods 0.000 claims description 5
- 239000000706 filtrate Substances 0.000 claims description 5
- 239000004615 ingredient Substances 0.000 claims description 5
- 102000004169 proteins and genes Human genes 0.000 claims description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 5
- 238000002203 pretreatment Methods 0.000 claims description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 3
- XBJFCYDKBDVADW-UHFFFAOYSA-N acetonitrile;formic acid Chemical compound CC#N.OC=O XBJFCYDKBDVADW-UHFFFAOYSA-N 0.000 claims description 3
- 239000004202 carbamide Substances 0.000 claims description 3
- 150000001768 cations Chemical class 0.000 claims description 3
- 230000007071 enzymatic hydrolysis Effects 0.000 claims description 3
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims description 3
- 238000011010 flushing procedure Methods 0.000 claims description 3
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 claims description 3
- 238000000751 protein extraction Methods 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 230000003595 spectral effect Effects 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims 1
- 230000009514 concussion Effects 0.000 claims 1
- 241000894007 species Species 0.000 abstract description 4
- 150000002500 ions Chemical class 0.000 description 12
- 241000965254 Apostichopus japonicus Species 0.000 description 7
- 241000208340 Araliaceae Species 0.000 description 7
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 7
- 235000003140 Panax quinquefolius Nutrition 0.000 description 7
- 235000008434 ginseng Nutrition 0.000 description 7
- 239000008367 deionised water Substances 0.000 description 4
- 229910021641 deionized water Inorganic materials 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 2
- 241000219112 Cucumis Species 0.000 description 2
- 235000015510 Cucumis melo subsp melo Nutrition 0.000 description 2
- UNXHWFMMPAWVPI-QWWZWVQMSA-N D-threitol Chemical compound OC[C@@H](O)[C@H](O)CO UNXHWFMMPAWVPI-QWWZWVQMSA-N 0.000 description 2
- 241000258955 Echinodermata Species 0.000 description 2
- 241000919871 Eleutherozoa Species 0.000 description 2
- UNXHWFMMPAWVPI-UHFFFAOYSA-N Erythritol Natural products OCC(O)C(O)CO UNXHWFMMPAWVPI-UHFFFAOYSA-N 0.000 description 2
- 229920002683 Glycosaminoglycan Polymers 0.000 description 2
- FJJCIZWZNKZHII-UHFFFAOYSA-N [4,6-bis(cyanoamino)-1,3,5-triazin-2-yl]cyanamide Chemical compound N#CNC1=NC(NC#N)=NC(NC#N)=N1 FJJCIZWZNKZHII-UHFFFAOYSA-N 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 125000002228 disulfide group Chemical group 0.000 description 2
- PGLTVOMIXTUURA-UHFFFAOYSA-N iodoacetamide Chemical compound NC(=O)CI PGLTVOMIXTUURA-UHFFFAOYSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 235000013372 meat Nutrition 0.000 description 2
- 239000012452 mother liquor Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 2
- 229930182490 saponin Natural products 0.000 description 2
- 150000007949 saponins Chemical class 0.000 description 2
- 108010020366 Arginine kinase Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 241001188169 Isostichopus badionotus Species 0.000 description 1
- 244000061458 Solanum melongena Species 0.000 description 1
- 235000002597 Solanum melongena Nutrition 0.000 description 1
- 102000005937 Tropomyosin Human genes 0.000 description 1
- 108010030743 Tropomyosin Proteins 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 102000006602 glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 239000002955 immunomodulating agent Substances 0.000 description 1
- 229940121354 immunomodulator Drugs 0.000 description 1
- 230000002584 immunomodulator Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000001151 other effect Effects 0.000 description 1
- 235000014102 seafood Nutrition 0.000 description 1
- 238000011179 visual inspection Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- 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
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/43504—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates
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Abstract
The present invention relates to a kind of methods for identifying Holothuria mexicana (Holothuria mexicana) using specificity peptide fragment group, and one group of control polypeptide being used for through mass spectrography identification Holothuria mexicana, the sequence of the polypeptide are as follows: NO.1~6 SEQ ID；The peptide fragment is the exclusive peptide fragment of Holothuria mexicana, and expression is had no in other species.
Description
Technical field
The invention belongs to field of biotechnology, identify Mexico sea using specificity peptide fragment group in particular to a kind of
Join the method for (Holothuria mexicana).
Background technique
Sea cucumber is rich in acidic mucopolysaccharide, has immunomodulator, anticoagulation, control blood glucose and reducing blood lipid and other effects to human body.
So sea cucumber enjoys the good reputation of " nutrition treasure-house ", modern is described as sea cucumber " first of seafood delights eight delicacies ".
Sea cucumber is subordinate to Echinodermata (Echinodermata), Eleutherozoa (Eleutherozoa), Holothuroidea
It (Holothuroidea), is the general name of a major class invertebrate in ocean, type belongs to 160 at 1400 kinds or more
Belong to, be distributed in the vast sea area in intertidal zone and ocean seabed, wherein Tropical Ocean Area type is abundant.
The sea cucumber of different cultivars compares, and some glycosaminoglycan contents are high, beneficial to cardiovascular and cerebrovascular；Some saponin contents
Height is beneficial to anticancer.Sea cucumber is the same with ginseng, if saponin content is high, medical value is high, but may smell bad；And have
Sea cucumber protein content it is high, such as eggplant ginseng and stichopus japonicus, can largely on hotel and Domestic dining table, protein is lacked, battalion
Support nutriment needed for undesirable crowd provides body.
Holothuria mexicana (Holothuria mexicana) is the common sea cucumber kind in american torrid zone sea area, nutriture value
Value, medical value and the sea cucumber kind for originating in China's temperate zone marine site have very big difference, therefore, on the market to Related product
Differentiation and identify just seem particularly significant.
Method due to lacking particularly effective identification sea cucumber kind at present identifies the method for sea cucumber type also only by passing
The visual inspection of system lacks the support of technical level, and many bad businessman is caused to adulterate, pass a fake product off as a genuine one and cheat consumer,
Therefore a kind of method of the identification sea cucumber of efficiently and accurately is needed.
Summary of the invention
Polypeptide of the present invention to Holothuria mexicana (Holothuria mexicana) Isostichopus Badionotus
It is studied, establishes the technology identified from peptide level to Holothuria mexicana, filled up what domestic and international sea cucumber identified
Blank.
Present invention firstly relates to one group for detecting the polypeptide of Holothuria mexicana, the sequence of the polypeptide alone or in combination
Are as follows:
SEQ ID NO.1:AGIALSDNFVK；
SEQ ID NO.2:AQQTAENEMANLQR；
SEQ ID NO.3:DAAEAAMK；
SEQ ID NO.4:LTVTEEELQR；
SEQ ID NO.5:DVYTPSGYTLDR；
SEQ ID NO.6:YLTADMYAR.
The invention further relates to a kind of methods for detecting Holothuria mexicana, and described method includes following steps:
(1) mass spectrum pre-treatment is carried out to sample to be tested, obtains candidate polypeptide filtrate:
(2) polypeptide moiety that sample to be tested is examined by mass spectrography, analyzes the Holothuria mexicana ingredient in sample.
The mass spectrum pre-treatment step is as follows:
(1) 1g sea cucumber sample homogeneous is weighed into pulverulence, and 10mL protein extract (8M urea, 50mM is added
NH4HCO3) shake and extract albumen, high-speed low temperature is centrifuged (20000r/min), supernatant 200ul is taken to be transferred in 1ml EP pipe；
(2) 2 μ L 1mol/L DTT (0.154g disulfide group threitol DTT is dissolved in 1mL deionized water) is taken to be added to 100 μ
It is reacted 1 hour for 37 DEG C in the above-mentioned protein solution of L；
(3) IAA for taking 10 μ L now to match (accurately weighs 0.185g iodo-acetamide IAA to be dissolved in 1mL deionized water, configure
Spare at the IAA mother liquor of 1mol/L, configuration process need to be protected from light, matching while using) it is added to the above-mentioned reaction for having cooled down to room temperature
In liquid, room temperature is protected from light 1 hour；
(4) it uses 10K filter membrane 20000g ultrafiltration 30 minutes, uses 200 μ L 50mmol/L ammonium bicarbonate soln repeated flushing
Behind filter membrane upper layer 3 times, it is transferred in new EP pipe；
(5) 200 μ L ammonium bicarbonate solns are added and repeat this step, merges solution and completes protein extraction under film；
(6) 5ul Trypsin enzyme solutions (the Trypsin enzyme solutions of 1 μ g/ μ L) is taken to be added in above-mentioned protein solution 37 DEG C
Enzymatic hydrolysis 16-18 hours；
(7) it uses 10K filter membrane 14000g ultrafiltration 20 minutes, the peptide fragment filtrate of lower layer is collected, to upper machine testing.
The mass spectrography is detected as, using AB SCIEX5600 detections,
Mobile phase A: 0.1% formic acid-acetonitrile, Mobile phase B: 0.1% formic acid-water,
Flow velocity: 0.25mL/min,
TOF scanning range: 350-1500Da,
Cation reaction pattern, GS1:35, GS2:45, Curtain Gas:35, ISVF:5500, TEM:500, DP:100,
CE:10.
Holothuria mexicana ingredient in the analysis sample is that the mass spectral results of sample to be tested are compared SEQ ID
There is the mass spectrum inspection of each specific polypeptide of NO.1~6 SEQ ID in the mass spectrogram of each specific polypeptide of NO.1~6
When surveying spectrogram, that is, it can determine whether that the tissue samples are Holothuria mexicana sample.
Detailed description of the invention
Fig. 1, AGIALSDNFVK the peptide fragment fracture on AB 5600
The mass spectrogram of Fig. 2, AGIALSDNFVK in Holothuria mexicana
Fig. 3, AQQTAENEMANLQR the peptide fragment fracture on AB 5600
The mass spectrogram of Fig. 4, AQQTAENEMANLQR in Holothuria mexicana
Fig. 5, DAAEAAMK the peptide fragment fracture on AB 5600
The mass spectrogram of Fig. 6, DAAEAAMK in Holothuria mexicana
Fig. 7, LTVTEEELQR the peptide fragment fracture on AB 5600
The mass spectrogram of Fig. 8, LTVTEEELQR in Holothuria mexicana
Fig. 9, DVYTPSGYTLDR the peptide fragment fracture on AB 5600
The mass spectrogram of Figure 10, DVYTPSGYTLDR in Holothuria mexicana
Figure 11, YLTADMYAR the peptide fragment fracture on AB 5600
The mass spectrogram of Figure 12, YLTADMYAR in Holothuria mexicana
Specific embodiment
Embodiment 1, the sequence source of specific polypeptide and comparison information
1. the source of the exclusive polypeptide A GIALSDNFVK of Holothuria mexicana
The exclusive polypeptide A GIALSDNFVK of Holothuria mexicana is from albumen glyceraldehyde-3-phosphate
Dehydrogenase, partial [Apostichopus japonicus], the Accesion number on NCBI is gi
| 334821165, shown in the following SEQ ID NO.7 of sequence, analysis finds that the 6th Asn of AGIALNENFVK has been mutated into Ser, the 7th
Position Glu has been mutated into Asp, has obtained polypeptide A GIALSDNFVK.It is compared by NCBI blast, although discovery has plant, bacterium
Equal species are consistent with its, but compare stichopus japonicus, Haiti melon, U.S.'s meat ginseng, Mexico's ginseng, and polypeptide A GIALSDNFVK is Mexico sea
Join exclusive:
SEQ ID NO.7:
ATQKTVDGPSAKKWRDGRGAAQNIIPASTGAAKAVGKVIPDLNGKLTGMAFRVPVPDVSVVDLTVRLKK
KASYDDIKAVIKKAASSTSLQSILGYTEDQVVSQDFRGDTRSSIFDAKAGIALNENFVKLVSWYDNEYGYSHRVIDL
ILYMAKK
Fig. 1 is AGIALSDNFVK peptide fragment fracture on AB 5600
Fig. 2 is mass spectrogram of the AGIALSDNFVK in Holothuria mexicana,
Its parent ion is 567.811,
Daughter ion is respectively as follows: 893.473,822.436,709.352 and 622.320.
2. the exclusive source polypeptide A QQTAENEMANLQR, DAAEAAMK, LTVTEEELQR of Holothuria mexicana
Holothuria mexicana exclusive polypeptide A QQTAENEMANLQR, DAAEAAMK, LTVTEEELQR are both from albumen
Tropomyosin [Apostichopus japonicus], the Accesion number on NCBI is gi |
302340969, shown in the following SEQ ID NO.8 of sequence, analysis finds that the 7th Glu of AQQTAEEEMANLQR has been mutated into Asn,
Polypeptide A QQTAENEMANLQR is obtained；The 2nd Val of DVAEASMK has been mutated into Ala, and the 6th Ser has been mutated into Ala, has obtained
Polypeptide DAAEAAMK；The 9th Ala of LTVTEEELAR has been mutated into Gln, has obtained polypeptide LTVTEEELQR；By NCBI
Blast is compared, although polypeptide DAAEAAMK discovery has, bacterium is consistent with its, comparison stichopus japonicus, Haiti melon, U.S.'s meat ginseng, Mo Xi
Brother's ginseng, polypeptide DAAEAAMK is that Holothuria mexicana is exclusive；Polypeptide A QQTAENEMANLQR, LTVTEEELQR are not found and other
Any species are consistent, are the exclusive polypeptide of Holothuria mexicana:
SEQ ID NO.8:
MDTIKKKLSQLKADKEKALDEKDVAEASMKEAMERVEQVNDENKELQTRIKQLETELDDTSEKLNTTVI
KCEAAEKAQQTAEEEMANLQRKLQLTEEELSRSEERVADLQSKYTDIEQSSEENERQRKVLESRSAADDERMSELET
QVMSSKTSLEDSDRKYDEASRKLTVTEEELARSEERSAAFESSLSQMKEELHQLHNNVKSLEAQEEKFTENEEMYEK
KVRDLEDKLKVAEDRADIAEESLKSLKTSLDQLEDELMIEKEKVREMTEEMERTIQELNFEV
Using AB SCIEXThe fracture and mass spectrogram of 5600 mass spectrographs (AB 5600) detection target peptide fragment
Fig. 3 is AQQTAENEMANLQR peptide fragment fracture on AB 5600
Fig. 4 is mass spectrogram of the AQQTAENEMANLQR in Holothuria mexicana,
Its parent ion is 802.378,
Daughter ion is respectively as follows: 1175.547,975.468,861.425 and 872.375.
Fig. 5 is DAAEAAMK peptide fragment fracture on AB 5600
Fig. 6 is mass spectrogram of the DAAEAAMK in Holothuria mexicana,
Its parent ion is 403.689,
Daughter ion is respectively as follows: 691.344,620.307,549.270 and 420.228.
Fig. 7 is LTVTEEELQR peptide fragment fracture on AB 5600
Fig. 8 is mass spectrogram of the LTVTEEELQR in Holothuria mexicana,
Its parent ion is 609.322,
Daughter ion is respectively as follows: 1003.505,904.437,803.389 and 674.347.
3. the source of Holothuria mexicana exclusive polypeptide DVYTPSGYTLDR, YLTADMYAR
Holothuria mexicana exclusive polypeptide DVYTPSGYTLDR, YLTADMYAR are from albumen arginine kinase
[Apostichopus japonicus], the Accesion number on NCBI is gi | 4586462, the following SEQ of sequence
Shown in ID NO.9, analysis finds that the 3rd Ala of DVATPSGYTLDR has been mutated into Tyr, has obtained polypeptide DVYTPSGYTLDR；
The 9th Lys of YLTADMYAK has been mutated into Arg, has obtained polypeptide YLTADMYAR.It is compared by NCBI blast, polypeptide
DVYTPSGYTLDR, YLTADMYAR do not have found consistent with other any species, are the exclusive polypeptide of Holothuria mexicana:
SEQ ID NO.9:
MANLNQKKYPAKDDFPNFEGHKSLLSKYLTADMYAKLRDVATPSGYTLDRAIQNGVDNPDFHLGLLAGD
EETYTVFADLFDPVIEEYHNGFKKTDNHKTDLDASKILDDVLDPAYVISSRVRTGRNIRGMALSPHVCRSERRAIEK
MVSEALNSLAADLKGKYYSLMKMDEKTQQQLIDDHFLFDRPVSRHFTSGGMARDFPDGRGIWHNDKKNFLVWINEED
HTRIISMQMGGNMKEVFERFTRGLTEVEKHIKDKTGKEFMKNDHLGFVLTCPSNLGTGVRCSVHAKLPHMAKDKRFE
EICTKMRLQKRGTSGEFTESVGGVYDISNLDRLGSSEVEQVNCVIKGVKVLIEMEKKLEKGESIDDLVPK
Fig. 9 is DVYTPSGYTLDR peptide fragment fracture on AB 5600
Figure 10 is mass spectrogram of the DVYTPSGYTLDR in Holothuria mexicana,
Its parent ion is 693.830,
Daughter ion is respectively as follows: 1172.558,1009.495,908.447 and 811.395.
Figure 11 is YLTADMYAR peptide fragment fracture on AB 5600
Figure 12 is mass spectrogram of the YLTADMYAR in Holothuria mexicana,
Its parent ion is 552.263,
Daughter ion is respectively as follows: 940.456,827.372,726.324 and 655.287.
Embodiment 2, sea cucumber sample process and detecting step
The step of analyzing sea cucumber sample to be measured include
(1) Sample pretreatment step:
(1) 1g sea cucumber sample homogeneous is weighed into pulverulence, and 10mL protein extract (8M urea, 50mM is added
NH4HCO3) shake and extract albumen, high-speed low temperature is centrifuged (20000r/min), supernatant 200ul is taken to be transferred in 1ml EP pipe；
(2) 2 μ L 1mol/L DTT (0.154g disulfide group threitol DTT is dissolved in 1mL deionized water) is taken to be added to 100 μ
It is reacted 1 hour for 37 DEG C in the above-mentioned protein solution of L；
(3) IAA for taking 10 μ L now to match (accurately weighs 0.185g iodo-acetamide IAA to be dissolved in 1mL deionized water, configure
Spare at the IAA mother liquor of 1mol/L, configuration process need to be protected from light, matching while using) it is added to the above-mentioned reaction for having cooled down to room temperature
In liquid, room temperature is protected from light 1 hour；
(4) it uses 10K filter membrane 20000g ultrafiltration 30 minutes, uses 200 μ L 50mmol/L ammonium bicarbonate soln repeated flushing
Behind filter membrane upper layer 3 times, it is transferred in new EP pipe；
(5) 200 μ L ammonium bicarbonate solns are added and repeat this step, merges solution and completes protein extraction under film；
(6) 5ul Trypsin enzyme solutions (the Trypsin enzyme solutions of 1 μ g/ μ L) is taken to be added in above-mentioned protein solution 37 DEG C
Enzymatic hydrolysis 16-18 hours；
(7) it uses 10K filter membrane 14000g ultrafiltration 20 minutes, the peptide fragment filtrate of lower layer is collected, to upper machine testing.
(2) machine testing on,
Using AB SCIEX5600 detections,
Mobile phase A: 0.1% formic acid-acetonitrile, Mobile phase B: 0.1% formic acid-water,
Flow velocity: 0.25mL/min,
TOF scanning range: 350-1500Da,
Cation reaction pattern, GS1:35, GS2:45, Curtain Gas:35, ISVF:5500, TEM:500, DP:100,
CE:10.
The mass spectrogram of each specific polypeptide in testing result comparative example 1, mass spectrum described in current embodiment 1 is examined out
When surveying spectrogram, that is, it can determine whether that the tissue samples are Holothuria mexicana sample.
It is further identified, the peptide fragment is the proprietary peptide fragment of Holothuria mexicana, in different sea cucumbers such as other stichopus japonicus, plum blossom ginsengs
Kind kind has no expression.
Claims (4)
1. one group of polypeptide for independent or any combination detection Holothuria mexicana (Holothuria mexicana), described
The sequence of polypeptide are as follows:
SEQ ID NO.1:AGIALSDNFVK；
SEQ ID NO.2:AQQTAENEMANLQR；
SEQ ID NO.3:DAAEAAMK；
SEQ ID NO.4:LTVTEEELQR；
SEQ ID NO.5:DVYTPSGYTLDR；
SEQ ID NO.6:YLTADMYAR.
2. a kind of method for detecting Holothuria mexicana, described method includes following steps:
(1) mass spectrum pre-treatment is carried out to sample to be tested, obtains candidate polypeptide filtrate:
(2) polypeptide moiety that sample to be tested is examined by mass spectrography, analyzes the Holothuria mexicana ingredient in sample；
Holothuria mexicana ingredient in the analysis sample are as follows: compare the mass spectral results of sample to be tested such as claim 1 institute
There is SEQ ID NO.1 as described in claim 1 in the mass spectrogram of each specific polypeptide of NO.1~6 SEQ ID stated
When the Mass Spectrometer Method spectrogram of~6 any bar or a plurality of specific polypeptide, that is, it can determine whether that the tissue samples are Holothuria mexicana sample
This.
3. according to the method described in claim 2, it is characterized in that, the mass spectrum pre-treatment step is as follows:
(1) sea cucumber sample homogeneous is weighed into pulverulence, and protein extract concussion is added and extracts albumen, high-speed low temperature centrifugation takes
Supernatant is transferred in EP pipe, the ingredient of the protein extract are as follows: 8M urea, 50mM NH4HCO3；
(2) DTT is taken to be added in above-mentioned protein solution, 37 DEG C are reacted 1 hour；
(3) IAA that enchashment is matched is added in the above-mentioned reaction solution for having cooled down to room temperature, and room temperature is protected from light 1 hour；
(4) it uses 10K ultrafiltration through membranes 30 minutes, using behind ammonium bicarbonate soln repeated flushing filter membrane upper layer 3 times, is transferred to new
In EP pipe；
(5) it adds ammonium bicarbonate soln and repeats this step, merge solution and complete protein extraction under film；
(6) Trypsin enzyme solutions is taken to be added to above-mentioned protein solution, in 37 DEG C enzymatic hydrolysis 16-18 hours；
(7) it uses 10K ultrafiltration through membranes 20 minutes, the peptide fragment filtrate of lower layer is collected, to upper machine testing.
4. according to the method described in claim 3, it is characterized in that, the mass spectrography is detected as, using AB SCIEX5600 detections,
Mobile phase A: 0.1% formic acid-acetonitrile, Mobile phase B: 0.1% formic acid-water,
Flow velocity: 0.25mL/min,
TOF scanning range: 350-1500Da,
Cation reaction pattern, GS1:35, GS2:45, Curtain Gas:35, ISVF:5500, TEM:500, DP:100, CE:
10。
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