CN105646655A - Specific peptide fragment group method for identification of Holothuria mexicana - Google Patents
Specific peptide fragment group method for identification of Holothuria mexicana Download PDFInfo
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- CN105646655A CN105646655A CN201510968453.8A CN201510968453A CN105646655A CN 105646655 A CN105646655 A CN 105646655A CN 201510968453 A CN201510968453 A CN 201510968453A CN 105646655 A CN105646655 A CN 105646655A
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- 241001024242 Holothuria mexicana Species 0.000 title claims abstract description 47
- 238000000034 method Methods 0.000 title claims abstract description 17
- 102000007079 Peptide Fragments Human genes 0.000 title abstract 3
- 108010033276 Peptide Fragments Proteins 0.000 title abstract 3
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 57
- 229920001184 polypeptide Polymers 0.000 claims abstract description 37
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 37
- 238000004949 mass spectrometry Methods 0.000 claims abstract description 5
- 241000251511 Holothuroidea Species 0.000 claims description 21
- 238000001819 mass spectrum Methods 0.000 claims description 17
- 150000002500 ions Chemical class 0.000 claims description 15
- 239000000243 solution Substances 0.000 claims description 11
- 239000012528 membrane Substances 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 8
- 238000001514 detection method Methods 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
- 239000000203 mixture Substances 0.000 claims description 6
- 239000012460 protein solution Substances 0.000 claims description 6
- 238000000108 ultra-filtration Methods 0.000 claims description 6
- 239000000706 filtrate Substances 0.000 claims description 5
- 238000002203 pretreatment Methods 0.000 claims description 4
- 102000004142 Trypsin Human genes 0.000 claims description 3
- 108090000631 Trypsin Proteins 0.000 claims description 3
- 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
- 230000009514 concussion Effects 0.000 claims description 3
- 238000000605 extraction 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
- 102000004169 proteins and genes Human genes 0.000 claims description 3
- 108090000623 proteins and genes Proteins 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 230000003595 spectral effect Effects 0.000 claims description 2
- 241000894007 species Species 0.000 abstract description 4
- 241000965254 Apostichopus japonicus Species 0.000 description 7
- 241000208340 Araliaceae Species 0.000 description 6
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 6
- 235000003140 Panax quinquefolius Nutrition 0.000 description 6
- 235000008434 ginseng Nutrition 0.000 description 6
- 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
- 239000000047 product Substances 0.000 description 3
- 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
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 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
- 125000002228 disulfide group Chemical group 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- PGLTVOMIXTUURA-UHFFFAOYSA-N iodoacetamide Chemical compound NC(=O)CI PGLTVOMIXTUURA-UHFFFAOYSA-N 0.000 description 2
- 235000013372 meat Nutrition 0.000 description 2
- 239000012452 mother liquor Substances 0.000 description 2
- 230000000050 nutritive effect Effects 0.000 description 2
- 238000012797 qualification Methods 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
- 241000919871 Eleutherozoa Species 0.000 description 1
- 241000196324 Embryophyta Species 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
- 241000529895 Stercorarius Species 0.000 description 1
- 241000941620 Thelenota ananas Species 0.000 description 1
- 102000005937 Tropomyosin Human genes 0.000 description 1
- 108010030743 Tropomyosin Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 230000000694 effects Effects 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
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 235000014102 seafood Nutrition 0.000 description 1
- 239000000126 substance Substances 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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- 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
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- 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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- 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
- G01N33/48—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 invention relates to a specific peptide fragment group method for identification of Holothuria mexicana, and a contrast polypeptide group for the identification of Holothuria mexicana by mass spectrometry. The polypeptide has a sequence of SEQ ID NO. 1-6; the peptide fragment is unique in Holothuria Mexicana and does not express in other species.
Description
Technical field
The invention belongs to biological technical field, specifically, it relates to a kind of method using specificity peptide section group to differentiate Holothuria mexicana (Holothuriamexicana).
Background technology
Sea cucumber is rich in acid mucopolysaccharide, and human body has the effects such as immunomodulator, anticoagulation, control blood sugar and reducing blood-fat. So, sea cucumber enjoys the good reputation of " nutrition treasure-house ", and modern is described as sea cucumber " first of seafood delights eight treasure ".
Sea cucumber is subordinate to Echinodermata (Echinodermata), trip is at subphylum (Eleutherozoa), sea cucumber guiding principle (Holothuroidea), it it is the general name of a big class invertebrates in ocean, kind is more than 1400 kinds, belonging to 160 genus, be distributed in the vast marine site in tideland and seabed, ocean, wherein marine site, torrid zone kind is enriched.
The sea cucumber of different varieties is compared, some glycosaminoglycan content height, is beneficial to by cardiovascular and cerebrovascular; Some saponin content height, to anticancer useful. Sea cucumber is the same with ginseng, if saponin content height, then pharmaceutical use height, but possibility smell bad; And some sea cucumber protein content height, such as eggplant ginseng and stichopus japonicus, it is possible to be used on hotel and family's dining table in a large number, provide the nutritive substance needed for health to potein deficiency, malnutritive crowd.
Holothuria mexicana (Holothuriamexicana) is the common sea cucumber kind in american torrid zone marine site, its nutritive value, pharmaceutical use and the sea cucumber kind originating in marine site, China temperate zone have very big difference, therefore, commercially to the differentiation of related products with differentiate just to seem very important.
The method especially effectively identifying sea cucumber kind owing to lacking at present, differentiate that the method for sea cucumber kind is also just by traditional visual inspection, lack the support of state of the art, cause many bad businessman to adulterate, pass a fake product off as a genuine one deception human consumer, it is thus desirable to the method for the qualification sea cucumber of a kind of efficiently and accurately.
Summary of the invention
The polypeptide of Holothuria mexicana (Holothuriamexicana) IsostichopusBadionotus has been studied by the present invention, establishes the technology differentiated by Holothuria mexicana from polypeptide level, has filled up the blank that domestic and international sea cucumber differentiates.
First the present invention relates to one group for detecting the polypeptide of Holothuria mexicana alone or in combination, and the sequence of described polypeptide is:
SEQIDNO.1:AGIALSDNFVK;
SEQIDNO.2:AQQTAENEMANLQR;
SEQIDNO.3:DAAEAAMK;
SEQIDNO.4:LTVTEEELQR;
SEQIDNO.5:DVYTPSGYTLDR;
SEQIDNO.6:YLTADMYAR.
The present invention also relates to a kind of method detecting Holothuria mexicana, and described method comprises the steps:
(1) sample to be tested is carried out mass spectrum pre-treatment, obtains polypeptide filtrate to be measured:
(2) the polypeptide composition of sample to be tested is checked by mass spectroscopy, the Holothuria mexicana composition in analyzing samples.
Described mass spectrum pre-treatment step is as follows:
(1) take the 1g sea cucumber powdered state of sample homogeneous, add 10mL protein extract (8M urea, 50mMNH4HCO3) concussion extraction albumen, high-speed low temperature centrifugal (20000r/min), gets supernatant liquor 200ul and transfers in 1mlEP pipe;
(2) get 2 �� L1mol/LDTT (0.154g disulfide group threitol DTT is dissolved in 1mL deionized water) and join in the 100 above-mentioned protein solutions of �� L 37 DEG C of reactions 1 hour;
(3) (accurately taking 0.185g iodo-acetamide IAA is dissolved in 1mL deionized water to get the IAA that 10 �� L now join, the IAA mother liquor being configured to 1mol/L is for subsequent use, configuration process needs lucifuge, matching while using) join in the above-mentioned reaction solution being cooled to room temperature, room temperature lucifuge reacts 1 hour;
(4) 10K filter membrane 20000g ultrafiltration 30 minutes is adopted, it may also be useful to 200 �� L50mmol/L ammonium bicarbonate solns are transferred in new EP pipe after repeatedly rinsing filter membrane upper strata 3 times;
(5) add 200 �� L ammonium bicarbonate solns again and repeat this step, merge solution and complete protein extraction under film;
(6) get 5ulTrypsin enzyme solution (the Trypsin enzyme solution of 1 �� g/ �� L) and join in above-mentioned protein solution 37 DEG C of enzymolysis 16-18 hour;
(7) adopt 10K filter membrane 14000g ultrafiltration 20 minutes, collect the peptide section filtrate of lower floor, treat machine testing.
Described mass spectroscopy is detected as, and adopts ABSCIEX5600 detections,
Mobile phase A: 0.1% formic acid-acetonitrile, Mobile phase B: 0.1% formic acid-water,
Flow velocity: 0.25mL/min,
TOF sweep limit: 350-1500Da,
Positive ion reaction pattern, GS1:35, GS2:45, CurtainGas:35, ISVF:5500, TEM:500, DP:100, CE:10.
Holothuria mexicana composition in described analyzing samples is, by the mass spectrogram of each bar specific polypeptide of the mass spectral results of testing sample contrast SEQIDNO.1��6, when the mass spectrometric detection spectrogram of each bar specific polypeptide of SEQIDNO.1��6 occurs, can judge that this tissue samples is Holothuria mexicana sample.
Accompanying drawing explanation
Fig. 1, AGIALSDNFVK be peptide section fracture on AB5600
Fig. 2, the AGIALSDNFVK mass spectrum in Holothuria mexicana
Fig. 3, AQQTAENEMANLQR be peptide section fracture on AB5600
Fig. 4, the AQQTAENEMANLQR mass spectrum in Holothuria mexicana
Fig. 5, DAAEAAMK be peptide section fracture on AB5600
Fig. 6, the DAAEAAMK mass spectrum in Holothuria mexicana
Fig. 7, LTVTEEELQR be peptide section fracture on AB5600
Fig. 8, the LTVTEEELQR mass spectrum in Holothuria mexicana
Fig. 9, DVYTPSGYTLDR be peptide section fracture on AB5600
Figure 10, the DVYTPSGYTLDR mass spectrum in Holothuria mexicana
Figure 11, YLTADMYAR be peptide section fracture on AB5600
Figure 12, the YLTADMYAR mass spectrum in Holothuria mexicana
Embodiment
Embodiment 1, the sequence source of specific polypeptide and comparison information
1. the source of the polypeptide A GIALSDNFVK that Holothuria mexicana is exclusive
The exclusive polypeptide A GIALSDNFVK of Holothuria mexicana comes from albumen glyceraldehyde-3-phosphatedehydrogenase, partial [Apostichopusjaponicus], its Accesionnumber on NCBI is gi | 334821165, shown in the following SEQIDNO.7 of sequence, analyze and find that AGIALNENFVK the 6th Asn has been mutated into Ser, 7th Glu has been mutated into Asp, obtains polypeptide A GIALSDNFVK. Through NCBIblast comparison, although it is consistent with it to find that there is the species such as plant, bacterium, but contrast stichopus japonicus, Haiti melon, U.S.'s meat ginseng, Mexico's ginseng, polypeptide A GIALSDNFVK is that Holothuria mexicana is exclusive:
SEQIDNO.7:
ATQKTVDGPSAKKWRDGRGAAQNIIPASTGAAKAVGKVIPDLNGKLTGMAFRVPVPD VSVVDLTVRLKKKASYDDIKAVIKKAASSTSLQSILGYTEDQVVSQDFRGDTRSSIFDAKAGIALNENFVKLVSWYDNEYGYSHRVIDLILYMAKK
Fig. 1 is AGIALSDNFVK peptide section fracture on AB5600
Fig. 2 is the mass spectrum of AGIALSDNFVK in Holothuria mexicana,
Its parent ion is 567.811,
Daughter ion is respectively: 893.473,822.436,709.352 and 622.320.
2. polypeptide A QQTAENEMANLQR, DAAEAAMK, LTVTEEELQR that Holothuria mexicana is exclusive originates
Exclusive polypeptide A QQTAENEMANLQR, DAAEAAMK, the LTVTEEELQR of Holothuria mexicana all comes from albumen tropomyosin [Apostichopusjaponicus], its Accesionnumber on NCBI is gi | 302340969, shown in the following SEQIDNO.8 of sequence, analyze and find that AQQTAEEEMANLQR the 7th Glu has been mutated into Asn, obtain polypeptide A QQTAENEMANLQR; DVAEASMK the 2nd Val has been mutated into Ala, and the 6th Ser has been mutated into Ala, obtains polypeptide DAAEAAMK; LTVTEEELAR the 9th Ala has been mutated into Gln, obtains polypeptide LTVTEEELQR; Through NCBIblast comparison, although to find that there is bacterium consistent with it for polypeptide DAAEAAMK, but contrast stichopus japonicus, Haiti melon, U.S.'s meat ginseng, Mexico's ginseng, polypeptide DAAEAAMK is that Holothuria mexicana is exclusive; Polypeptide A QQTAENEMANLQR, LTVTEEELQR do not find that species any with other are consistent, are the polypeptide that Holothuria mexicana is exclusive:
SEQIDNO.8:
MDTIKKKLSQLKADKEKALDEKDVAEASMKEAMERVEQVNDENKELQTRIKQLETELDDTSEKLNTTVIKCEAAEKAQQTAEEEMANLQRKLQLTEEELSRSEERVADLQSKYTDIEQSSEENERQRKVLESRSAADDERMSELETQVMSSKTSLEDSDRKYDEASRKLTVTEEELARSEERSAAFESSLSQMKEELHQLHNNVKSLEAQEEKFTENEEMYEKKVRDLEDKLKVAEDRADIAEESLKSLKTSLDQLEDELMIEKEKVREMTEEMERTIQELNFEV
Adopt ABSCIEX5600 mass spectrographs (AB5600) detect fracture and the mass spectrum of target peptide section
Fig. 3 is AQQTAENEMANLQR peptide section fracture on AB5600
Fig. 4 is the mass spectrum of AQQTAENEMANLQR in Holothuria mexicana,
Its parent ion is 802.378,
Daughter ion is respectively: 1175.547,975.468,861.425 and 872.375.
Fig. 5 is DAAEAAMK peptide section fracture on AB5600
Fig. 6 is the mass spectrum of DAAEAAMK in Holothuria mexicana,
Its parent ion is 403.689,
Daughter ion is respectively: 691.344,620.307,549.270 and 420.228.
Fig. 7 is LTVTEEELQR peptide section fracture on AB5600
Fig. 8 is the mass spectrum of LTVTEEELQR in Holothuria mexicana,
Its parent ion is 609.322,
Daughter ion is respectively: 1003.505,904.437,803.389 and 674.347.
3. the source of polypeptide DVYTPSGYTLDR, YLTADMYAR that Holothuria mexicana is exclusive
Exclusive polypeptide DVYTPSGYTLDR, the YLTADMYAR of Holothuria mexicana comes from albumen argininekinase [Apostichopusjaponicus], its Accesionnumber on NCBI is gi | 4586462, shown in the following SEQIDNO.9 of sequence, analyze and find that DVATPSGYTLDR the 3rd Ala has been mutated into Tyr, obtain polypeptide DVYTPSGYTLDR; YLTADMYAK the 9th Lys has been mutated into Arg, obtains polypeptide YLTADMYAR. Through NCBIblast comparison, polypeptide DVYTPSGYTLDR, YLTADMYAR do not find that species any with other are consistent, are the polypeptide that Holothuria mexicana is exclusive:
SEQIDNO.9:
MANLNQKKYPAKDDFPNFEGHKSLLSKYLTADMYAKLRDVATPSGYTLDRAIQNGVDNPDFHLGLLAGDEETYTVFADLFDPVIEEYHNGFKKTDNHKTDLDASKILDDVLDPAYVISSRVRTGRNIRGMALSPHVCRSERRAIEKMVSEALNSLAADLKGKYYSLMKMDEKTQQQLIDDHFLFDRPVSRHFTSGGMARDFPDGRGIWHNDKKNFLVWINEEDHTRIISMQMGGNMKEVFERFTRGLTEVEKHIKDKTGKEFMKNDHLGFVLTCPSNLGTGVRCSVHAKLPHMAKDKRFEEICTKMRLQKRGTSGEFTESVGGVYDISNLDRLGSSEVEQVNCVIKGVKVLIEMEKKLEKGESIDDLVPK
Fig. 9 is DVYTPSGYTLDR peptide section fracture on AB5600
Figure 10 is the mass spectrum of DVYTPSGYTLDR in Holothuria mexicana,
Its parent ion is 693.830,
Daughter ion is respectively: 1172.558,1009.495,908.447 and 811.395.
Figure 11 is YLTADMYAR peptide section fracture on AB5600
Figure 12 is the mass spectrum of YLTADMYAR in Holothuria mexicana,
Its parent ion is 552.263,
Daughter ion is respectively: 940.456,827.372,726.324 and 655.287.
Embodiment 2, sea cucumber sample process and detecting step
The step that sea cucumber sample to be measured is analyzed is comprised
(1) Sample pretreatment step:
(1) take the 1g sea cucumber powdered state of sample homogeneous, add 10mL protein extract (8M urea, 50mMNH4HCO3) concussion extraction albumen, high-speed low temperature centrifugal (20000r/min), gets supernatant liquor 200ul and transfers in 1mlEP pipe;
(2) get 2 �� L1mol/LDTT (0.154g disulfide group threitol DTT is dissolved in 1mL deionized water) and join in the 100 above-mentioned protein solutions of �� L 37 DEG C of reactions 1 hour;
(3) (accurately taking 0.185g iodo-acetamide IAA is dissolved in 1mL deionized water to get the IAA that 10 �� L now join, the IAA mother liquor being configured to 1mol/L is for subsequent use, configuration process needs lucifuge, matching while using) join in the above-mentioned reaction solution being cooled to room temperature, room temperature lucifuge reacts 1 hour;
(4) 10K filter membrane 20000g ultrafiltration 30 minutes is adopted, it may also be useful to 200 �� L50mmol/L ammonium bicarbonate solns are transferred in new EP pipe after repeatedly rinsing filter membrane upper strata 3 times;
(5) add 200 �� L ammonium bicarbonate solns again and repeat this step, merge solution and complete protein extraction under film;
(6) get 5ulTrypsin enzyme solution (the Trypsin enzyme solution of 1 �� g/ �� L) and join in above-mentioned protein solution 37 DEG C of enzymolysis 16-18 hour;
(7) adopt 10K filter membrane 14000g ultrafiltration 20 minutes, collect the peptide section filtrate of lower floor, treat machine testing.
(2) upper machine testing,
Adopt ABSCIEX5600 detections,
Mobile phase A: 0.1% formic acid-acetonitrile, Mobile phase B: 0.1% formic acid-water,
Flow velocity: 0.25mL/min,
TOF sweep limit: 350-1500Da,
Positive ion reaction pattern, GS1:35, GS2:45, CurtainGas:35, ISVF:5500, TEM:500, DP:100, CE:10.
The mass spectrogram of each bar specific polypeptide in detected result comparative example 1, when going out mass spectrometric detection spectrogram described in current embodiment 1, can judge that this tissue samples is Holothuria mexicana sample.
Through further qualification, described peptide section is the proprietary peptide section of Holothuria mexicana, and at other stichopus japonicus, Thelenota ananas (Jaeger)�� etc., different sea cucumber product are all has no expression.
Claims (5)
1. one group is used for separately or the polypeptide of arbitrary combination detection Holothuria mexicana (Holothuriamexicana), and the sequence of described polypeptide is:
SEQIDNO.1:AGIALSDNFVK;
SEQIDNO.2:AQQTAENEMANLQR;
SEQIDNO.3:DAAEAAMK;
SEQIDNO.4:LTVTEEELQR;
SEQIDNO.5:DVYTPSGYTLDR;
SEQIDNO.6:YLTADMYAR.
2. detecting a method for Holothuria mexicana, described method comprises the steps:
(1) sample to be tested is carried out mass spectrum pre-treatment, obtains polypeptide filtrate to be measured:
(2) the polypeptide composition of sample to be tested is checked by mass spectroscopy, the Holothuria mexicana composition in analyzing samples.
3. method according to claim 2, it is characterised in that, described mass spectrum pre-treatment step is as follows:
(1) take the sea cucumber powdered state of sample homogeneous, add protein extract (8M urea, 50mMNH4HCO3) concussion extraction albumen, high-speed low temperature is centrifugal, gets supernatant liquor and transfers in EP pipe;
(2) getting DTT joins in above-mentioned protein solution, and 37 DEG C are reacted 1 hour;
(3) IAA that enchashment is joined joins in the above-mentioned reaction solution being cooled to room temperature, and room temperature lucifuge reacts 1 hour;
(4) 10K filter membrane ultrafiltration 30 minutes is adopted, it may also be useful to ammonium bicarbonate soln is transferred in new EP pipe after repeatedly rinsing filter membrane upper strata 3 times;
(5) add ammonium bicarbonate soln again and repeat this step, merge solution and complete protein extraction under film;
(6) get Trypsin enzyme solution and join above-mentioned protein solution, in 37 DEG C of enzymolysis 16-18 hour;
(7) adopt 10K filter membrane ultrafiltration 20 minutes, collect the peptide section filtrate of lower floor, treat machine testing.
4. according to the method in claim 2 or 3, it is characterised in that, described mass spectroscopy is detected as, and adopts ABSCIEX5600 detections,
Mobile phase A: 0.1% formic acid-acetonitrile, Mobile phase B: 0.1% formic acid-water,
Flow velocity: 0.25mL/min,
TOF sweep limit: 350-1500Da,
Positive ion reaction pattern, GS1:35, GS2:45, CurtainGas:35, ISVF:5500, TEM:500, DP:100, CE:10.
5. according to the method in claim 2 or 3, it is characterized in that, Holothuria mexicana composition in described analyzing samples is, by the mass spectrogram of each bar specific polypeptide of the mass spectral results of testing sample contrast SEQIDNO.1��6, when the mass spectrometric detection spectrogram of each bar specific polypeptide of SEQIDNO.1��6 occurs, can judge that this tissue samples is Holothuria mexicana sample.
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