CN110308228A - A kind of analysis method of sea cucumber protein group - Google Patents

A kind of analysis method of sea cucumber protein group Download PDF

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CN110308228A
CN110308228A CN201910731910.XA CN201910731910A CN110308228A CN 110308228 A CN110308228 A CN 110308228A CN 201910731910 A CN201910731910 A CN 201910731910A CN 110308228 A CN110308228 A CN 110308228A
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sea cucumber
protein
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analysis method
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陈溪
林天野
吴慈
褚莹倩
贺舒文
黄大亮
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
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    • 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
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N30/14—Preparation by elimination of some components
    • 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
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    • 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
    • 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
    • G01N30/04—Preparation or injection of sample to be analysed
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    • G01N2030/067—Preparation by reaction, e.g. derivatising the sample
    • 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
    • G01N30/04—Preparation or injection of sample to be analysed
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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
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
    • G01N2030/8831—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving peptides or proteins

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Abstract

The present invention relates to the analysis method research field of Protein in Food group, specifically a kind of analysis method of sea cucumber protein group.The analysis method of sea cucumber protein group disclosed in this invention, by studying efficient Tissue lysates as Protein Extraction and dissolution system, the development holoprotein sample pretreatment new technology that extractability is strong, sample loss is low extracts protein in sea cucumber sample, and further digests；In turn, it is combined by liquid chromatogram classification and high resolution mass spectrum identification technology, protein in sea cucumber sample is quantified；The sensitivity for improving low abundance proteins quantitative analysis, to improve the quantitative coverage of protein group.The method of the present invention be efficiently and for sea cucumber sample protein group extract, enzymatic hydrolysis, classification, identification and analysis method, improve low abundance proteins quantitative analysis sensitivity and coverage, from proteomics in terms of in sea cucumber sample protein progress variance analysis.

Description

A kind of analysis method of sea cucumber protein group
Technical field
The present invention relates to the analysis method research field of Protein in Food group, specifically a kind of sea cucumber protein group Analysis method.
Background technique
Sea cucumber category Holothuroidea (Holothuroidea), is echinoderm.In wall of sea cucumber Stichopus japonicus rich in protein, polysaccharide and Biological saponin(e isoreactivity ingredient also contains niacin, vitamin A, B12, and iron, manganese, zinc, selenium and other trace elements can improve body Immunity, anti-aging, antitumor and anticancer, edible medical value with higher and economic value.Studies have reported that sea cucumber There is similar medicinal characteristic with ginseng, it has also become the rare food materials of the Asian countries such as China, Japan.(Aminin DL, Menchinskaya ES, Pisliagin EA, et al. Anticancer Activity of Sea Cucumber Triterpene Glycosides [J] Mar Drugs, 2015,13(3): 1202-1223. Zhang Weiwei, Lu Yin sea cucumber it is anti- Function of tumor progress [J] China traditional Chinese medicine magazine, 2010(1): 105-108.) living standard is needed with people The continuous improvement wanted especially there is the precious marine product of high nutritive value to expand food source, by pass using marine resources Note.
Currently, the research to sea cucumber proteomics, usually the manufacturing process of research sea cucumber to sea cucumber institutional framework and (Zheng Mingjing, Zhou Meiling, Chen Ni wait research of the ultra high pressure treatment to sea cucumber institutional framework and qualitative effects for the influence of quality [J] food industry science and technology .2016,37 (4): 187-210), or established for the nursery breeding, breeding, disease research work of sea cucumber Fixed basis (Zhang Peng, Li Chenghua, Li Ye, wait stichopus japonicus (Apostichopusjaponicus) the related egg of skin ulceration syndrome generation White separation and identification [J] Oceanologia et Limnologia Sinica .2013,44 (3): 1-6；Tian Yan, Mo Haibo, Chang Yaqing wait stichopus japonicus intestines group The foundation of textured protein dielectrophoresis system and optimization [J] fishery science progress .2013,34 (5): 74-81).Roc etc. is opened to use Lysate (7mol/L urea, 2mol/L thiocarbamide, 40mmol/L DDT, 4% W/V CHAPS, 2% V/V pharmalyte) is extracted The albumen of stichopus japonicus intestinal tissue identifies stichopus japonicus skin ulceration syndrome analysis of two-dimensional gel electrophoresis maps in albumen level, solidifying using dielectrophoresis Glue has taken differential protein spot and has analyzed and identified part variation albumen with MALDI-TOF/TOF, to grasp disease genesis mechanism Provide theoretical foundation.The preparation of sample is the key link of albumen of finding differences, and sample extraction should obtain all as far as possible Protein, in case subsequent separation and identification.The different Protein Extraction item such as buffer composition, pH value, salinity, temperature Part can all directly influence proteomics and obtain result of study, especially become apparent in Study on Different Proteomics.It is logical It crosses and studies efficient Tissue lysates as Protein Extraction and dissolution system, it is complete to develop that extractability is strong, sample loss is low Protein example, which pre-processes new technology, has far-reaching significance the research of sea cucumber sample protein group；And for analytical technology, Although Two dimensional Electrophoresis of Proteins is most widely used technology in current proteomics research, it is usually used in proteomics Research, but be not that protein content that each protein spots include is sufficient to protein identification, and resolution ratio compare it is lower, The overlapping for being easy to protein occur is unfavorable for the detection of low-abundance protein, hydrophobic proteins, operates also time-consuming and laborious.In addition, sharp Also it is more suitable for the Primary Study of sea cucumber albumen primary structure with MALDI-TOF-TOF mass-spectrometric technique, and Q-Exactive Orbitrap MS is a kind of technology with high-resolution, sensitivity, accuracy out newly developed in recent years, with common mass spectrum It compares, fragment ion scanning analysis identifies the superior ability of various lipid isomers.Currently, the technology has been applied successfully It is applied to analysis sea cucumber sample in different growths in having no in the identification of the polar lipid component difference identification of milk and people's milk Period, different geographical, different processing technology sea cucumber difference analysis.
Summary of the invention
The present invention is to propose that one kind is efficient, for sea cucumber sample to solve deficiencies of the prior art Protein group is extracted, enzymatic hydrolysis, is classified, identification and analysis method, and the sensitivity and coverage of low abundance proteins quantitative analysis are improved, Protein in sea cucumber sample is quantified.
The technical solution that the present invention takes to achieve the above object is: a kind of analysis method of sea cucumber protein group, It is characterized by: the method follows the steps below:
S1, take sea cucumber tissue (removing intestines) through over cleaning after, crushed, be put into mortar, quicklyed while grinding to grinding Liquid nitrogen is added in alms bowl, until sea cucumber tissue pieces are in solid powdered；
S2, sea cucumber powder is placed into microcentrifugal tube, 6 ~ 10 mol/L urea elements or detergent alkylate sulphur of 400 μ L is added Sour sodium or Triton X-100 Reverse Microemulsion System, and 4 ~ 6 μ L proteinase inhibitor C ocktail are added, by ultraphonic pipe Be placed in ice face, be ultrasonically treated under conditions of 4 DEG C, in ultrasonication the working time be 10s, intermittent time 10s, 3 ~ 6 min are run, then 20 ~ 30min of centrifugal treating at 2000g, 4 DEG C, takes supernatant, and wherein supernatant is sea cucumber tissue Soluble holoprotein solution；
S3, it is needed on conversion film with the concentration of BCA quantification kit measurement sea cucumber total protein according to the protein concentration that measurement obtains The volume (enzymatic hydrolysis can carry the albumen of about 200mg on general film) of loading, is added 50 mM ammonium hydrogen carbonate of certain volume (NH4HCO3) each centrifuge tube of trim, make overall 150 μ L of applied sample amount, and the 100mM DTT of 15 μ L, 95 DEG C of incubations are added It is centrifuged after 5min, the NH of 100 ~ 200 μ L 50mM is added on the ultrafiltration membrane of centrifugal ultrafiltration unit4HCO3After solution 14000g, 10 ~ 30min of centrifugation at 20 DEG C, 1 ~ 2 time；Waste liquid in collecting pipe is discarded, is added on ultrafiltration membrane and uses 50mM NH4HCO3Solution allocation 100 μ L of 20mM iodoacetamide solution, oscillation 1min be sufficiently mixed after, be protected from light be incubated for 20min, under the conditions of 14000g, 20 DEG C It is centrifuged 10 ~ 30min, 10 mM NH are added on ultrafiltration membrane4HCO3It is centrifuged 15min at 14000g, 20 DEG C, is repeated twice, is replaced Centrifugal ultrafiltration unit collecting pipe；
5 ~ 8 μ L trypsase (Trypsin) solution are added on S4, the ultrafiltration membrane into S3 step, with 10 mM NH4HCO3 50 μ L are supplemented to, sufficiently oscillation 1min, tightly wrap ultrafiltration membrane using Parafilm film, is placed in 4 ~ 18h of incubation at 37 DEG C；To ultrafiltration 50 ~ 150 μ L ultrapure waters are added on film, is centrifuged 10min at 14000g, 20 DEG C, is repeated once, obtaining can containing sea cucumber tissue Dissolubility holoprotein；
S5, it is marked again using TMT10,10 samples of label, and records 10 samples of each label substance markers, be incubated at room temperature 15 μ L HCl- azanol reaction 15min are added in 1h, terminate reaction, mark quantitative differences albumen with this, are lyophilized；
S6, for the sample of S5 step, be classified using liquid chromatogram, using C18 high PH be classified, 5 ~ 55min, every 2 ~ 3min Collect efflux, freeze-drying；With acetonitrile: the 200 μ L of mixed liquor that water volume ratio is 1:1 ~ 6:4 dissolves, and divides 4 ~ 8 groups, merges its classification Liquid is redissolved after freeze-drying with mobile phase；
S7, upper machine liquid chromatogram-high resolution mass spectrum combined instrument LC-MS/MS analysis.
Sea cucumber 0.08 ~ 0.12g of powder, is placed into 2mL microcentrifugal tube in the step S1.
Protein concentration operation is measured in the step S3 are as follows: the supernatant for obtaining step S2 adds ultrapure in centrifuge tube Water dilutes 40 times of sample to be tested, using BCA protein quantification kit measurement total protein concentration.
The analysis method of sea cucumber protein group disclosed in this invention, for sea cucumber sample protein group extract, enzymatic hydrolysis, Classification, identification and analysis method improve the sensitivity and coverage of low abundance proteins quantitative analysis, pass through high resolution mass spectrum, hair The quantification of protein technology of efficient protein example pretreatment and high precision, and the method for combining TMT labeled in vitro, benefit are opened up Polypeptide after generating after being digested with isotope-labelled protein carries out quantitative analysis in holoprotein level, realizes in sea cucumber The depth covering of protein is quantitative, and proteomic techniques are applied to analysis sea cucumber sample in different growing stage, difference Region, different processing technology sea cucumber difference analysis, have no relevant report.The present invention compared with the existing technology, has following Advantage:
Research using novel proteomic techniques to beche-de-mer products, can establish the pretreatment of efficient protein example and The protein of high accuracy is qualitative and quantitative technique, and in different growing stage, different geographical, different processing technology sea cucumbers The variation of protein group is studied, and carries out quantitative analysis to differential protein using TMT labelling method, from proteomics angle The differential protein of degree analysis sea cucumber sample.
Detailed description of the invention
Fig. 1 is the differential protein analysis of the South Pole black stichopus japonicus and Dalian Sea Area culturing sea cucumbers；
Fig. 2 is with sea area with the differential protein analysis of time salt marsh and instant holothurian；
Fig. 3 is that 1 year differential protein analysis for participating in 3 years ginsengs is broadcast with sea area bottom；
In figure: ▲ there is the albumen of significant difference, ● the not albumen of significant difference.
Specific embodiment:
Method provided by the invention is described in detail below with reference to embodiment, is followed the steps below, but not in any form The limitation present invention.
Embodiment 1
A kind of analysis method of sea cucumber protein group, the method follow the steps below:
S1, take sea cucumber tissue through over cleaning after, crushed, be put into mortar, while grinding into mortar annex solution Nitrogen, until sea cucumber tissue pieces are in solid powdered.
S2, sea cucumber powder 0.10115g is placed into 2mL ultraphonic pipe, the 8M urea element of 400 μ L is added, wherein urea element is used The dissolution of 50 mM ammonium bicarbonate solns, and 4 μ L protease inhibitors (Cocktail) are added, ultraphonic pipe is placed in ice face, 4 It is ultrasonically treated under conditions of DEG C, the working time is 10s in ultrasonication, intermittent time 10s, and ultrasonication is total 3min, then the centrifugal treating 20min at 2000g, 4 DEG C, takes supernatant, and wherein supernatant is that sea cucumber organizes soluble shell egg White solution.
S3, it takes the supernatant obtained in step in centrifuge tube, adds ultrapure water to dilute 40 times of sample to be tested, use BCA Protein quantification kit measurement total protein concentration.Bovine serum albumin(BSA) (BSA, Bovine serum is configured with pure water Albumin), the BSA for such as weighing 0.00201g adds 1mL secondary water, is configured to the BSA working solution of 2mg/mL, configuration standard albumen Concentration Linear Points are as follows: 0.01mg/mL, 0.02mg/mL, 0.05 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, BCA working solution is added according to the requirement of BCA kit in 0.4 mg/mL, and 37 DEG C of hatchings survey its absorbance, and then calculate urea The total protein concentration for the sea cucumber sample that element extracts is 11.031 mg/mL.
18.1 μ L sea cucumber protein extracts and the NH of 131.9 μ L, 50 mM are taken with pipettor4HCO3Solution is in ultrafiltration membrane On, and 15 μ L 100mM DTT are added, 10min is centrifuged at 95 DEG C of 5min, 14000g, 20 DEG C of incubations.To centrifugal ultrafiltration unit Ultrafiltration membrane on the NH of 100 μ L50 mM is added4HCO320min is centrifuged at 14000g, 20 DEG C after solution.100 are added again The NH of μ L50 mM4HCO3It is centrifuged 20min at 14000g, 20 DEG C after solution, and discards waste liquid in collecting pipe.Add on filter membrane Enter 100 μ L 20mM IAA(50mM NH4HCO3Solution allocation), oscillation 1min is sufficiently mixed, and it is protected from light and is incubated for 20min, 14000g is centrifuged 15min under the conditions of 20 DEG C.10 mM NH are added on ultrafiltration membrane4HCO3It is centrifuged at 14000g, 20 DEG C 15min is repeated twice.Replace centrifugal ultrafiltration unit collecting pipe.
S4,8 μ L Trypsin, 42 μ L10 mM NH are added on ultrafiltration membrane4HCO3, sufficiently oscillation 1min, use Parafilm film tightly wraps ultrafiltration membrane, is placed at 37 DEG C and is incubated overnight.50 μ L ultrapure waters are added on ultrafiltration membrane, in 14000g, 20 It is centrifuged 10min at DEG C, is repeated once.It obtains organizing soluble holoprotein containing sea cucumber.
S5, it is marked again using TMT10,10 samples of label, and records 10 samples of each label substance markers, such as use TMT10 130NTM1 year wild sea cucumber of label, TMT10 129CTM3 years wild sea cucumbers of label, TMT10-128NTM5 years wild seas of label Ginseng, etc. is incubated at room temperature 1h, and 15 μ L HCl- azanol reaction 15min are added, and terminates reaction.5 μ L are respectively taken to mix in equal volume It closes.Freeze-drying is done to close.And 50 μ L ultrapure waters are added, oscillation dissolution.
S6, S5 sample feed liquor phase chromatographic fractionation is taken, the every 2min of 5min ~ 55min collects efflux, with 200 μ L 6 after freeze-drying: 4(acetonitrile: water V/V) all classification liquid of solution dissolution, merge first group: 5 ~ 8min, 18 ~ 20min, 30 ~ 32min, 42 ~ 44min It is classified fraction；Merge second group of 8 ~ 10min, 20 ~ 22min, 32 ~ 34 min, 44 ~ 46 min and is classified fraction；Merging third group: 10 ~ 12min, 22 ~ 24 min, 34 ~ 36 min, 46 ~ 48 min are classified fraction；Merge the 4th group: 12 ~ 14min, 24 ~ 26 min, 36 ~ 38 min, 48 ~ 50 min are classified fraction；Merge the 5th group: 14 ~ 16min, 26 ~ 28 min, 38 ~ 40 min, 50 ~ 52 min divide Grade fraction；Merge the 6th group: 16 ~ 18min, 28 ~ 30 min, 38 ~ 40 min, 52 ~ 55 min are classified fraction.By above 6 groups of samples Product freeze-drying.With 200 μ L, 6:4(acetonitriles: water V/V) 6 groups of classification liquid of dissolution, are redissolved after freeze-drying with mobile phase.
S7, ultra performance liquid chromatography-level four bars/electrostatic field orbit trap high resolution mass spectrum (UPLC-Q-Exactive Orbitrap Mass, Thermo Scientific, USA), match nanoliter sampling system and heatable electric spray ion source (HESI) it measures, quantitative analysis is carried out to differential protein.
Chromatographic condition: chromatographic column: Accucore RP-MS (Thermo Scientific, 100 × 2.1 mm, 2.6 μ m)；Column temperature: 40 DEG C；Mobile phase A: 0.1% formic acid-aqueous solution；Mobile phase B: 2% formic acid-methanol solution；Flow velocity: 0.6 mL/ min；Sample volume: 6 μ L；Eluent gradient: 2% B(0 min) -7% B(0.1 min) -23% B(45.1min) -40% B (65.1min) -80% B(70.1 min) -80% B(85.1 min), runing time 85.1min.
Mass Spectrometry Conditions: scan pattern: the first mass spectrometric full scan based on high resolution mass spectrum adds the second order ms of data dependence Scan (Full MS/dd-MS2)；First mass spectrometric (MS) parameter setting: resolution ratio R=70000, the AGC target:3 of full scan ×106, maximum residence time (Maximum IT): 60 ms, scanning range m/z300 ~ 1800；Second order ms (MS/MS) parameter Setting: target:5 × 10 resolution ratio R=35000, AGC4, maximum residence time (Maximum IT): 60 ms；Capillary tube temperature Degree: 320 DEG C；Heating temperature: 320 DEG C；Sheath gas: 40 arb of N2 flow velocity；Assist gas: 10 arb of N2 flow velocity；Spray voltage is (Spray Voltage): 3200 V；Lens voltage: 50 V.
Search library condition: the mass spectrometric data file (* .raw) collected using Q-Exactive mass spectrograph is used MaxQuant(version 1.5.1.1) software search library, and database is the sea cucumber albumen database of the website UniProt downloading (Stichopus japonicus).Retrieval parameter setting are as follows: trypsase complete degestion at most allows 2 leakage enzyme sites: Gu Surely it is modified to cysteine residues (C)+57.0215Da of peptide fragment, the variable oxidation for being modified with methionine residue (M)+ Acetylation+the 42.0106Da of 15.9949Da and the end albumen N-；Parent ion quality tolerance is 10ppm, fragment ion Mass deviation is 20mmu.Controlling false positive rate in protein and peptide section level simultaneously is 1%.
The invention has the characteristics that:
(1) Protein Extraction: develop efficient Tissue Lysis system for sea cucumber sample, in conjunction with ultrasonication technology, to sample Middle holoprotein extracts, and urea element, neopelex (SDS), Triton X-100 system may be selected, and divide The influence to Protein Extraction efficiency such as various extract concentrations, ultrasonication condition is not investigated；
(2) it digests: extracted protein example being digested using enzymatic isolation method on free solution enzymatic isolation method or film, and to enzyme Solution condition (reductive alkylation condition, enzyme concentration, enzymolysis time etc.) optimizes；
(3) peptide fragment is classified: to improve protein identification overburden depth, using reverse-phase chromatography or ion-exchange chromatography to protein Enzymolysis liquid is classified, and optimizes the merging mode of the eluent gradient setting and collected fraction of liquid chromatogram classification, is reduced The complexity of protein example enzymolysis product；
(4) it Mass Spectrometric Identification: is reflected using UPLC-Q-Exactive Orbitrap Mass high resolution mass spectrum to classification peptide fragment It is fixed, peptide fragment is marked using light, heavy label method, and then quantitative, to disclose comprehensively different growing stage, different geographical, The change of divergence of related protein expression quantity in different processing technology sea cucumbers.By taking the black stichopus japonicus in the South Pole is compared with culturing sea cucumbers as an example (see figure 1) detects 611 albumen altogether, analyzes through UPLC-Q-Exactive Orbitrap that successful identification goes out 201 differences altogether M-band；(see Fig. 2,122 ° 583 ' ' of Huanghai Sea east longitude, 39 ° of 283 ' ' triennial of north latitude is extra large so that salt marsh is compared with instant holothurian as an example Ginseng), successful identification goes out 54 differential proteins altogether；By taking 1 year compared with 3 years wild sea cucumbers as an example (see Fig. 3,122 ° of Huanghai Sea east longitude 583 ' ', 39 ° of 283 ' ' triennial sea cucumber of north latitude), successful identification goes out 18 differential proteins altogether；And it is in three repeated experiments Existing similar expression pattern.
Embodiment 2
A kind of analysis method of sea cucumber protein group, the method follow the steps below:
S1, take sea cucumber tissue through over cleaning after, crushed, be put into mortar, while grinding into mortar annex solution Nitrogen, until sea cucumber tissue pieces are in solid powdered.
S2, sea cucumber powder 0.1195g is placed into 2mL ultraphonic pipe, the 6 mol/L detergent alkylates of 400 μ L is added Sodium sulfonate (SDS), wherein SDS is dissolved with 50 mM ammonium bicarbonate solns, and 6 μ L protease inhibitors (Cocktail) are added, Ultraphonic pipe is placed in ice face, is ultrasonically treated under conditions of 4 DEG C, the working time is 10s, interval in ultrasonication Time 10s, ultrasonication amount to 3min, and then the centrifugal treating 30min at 2000g, 4 DEG C, takes supernatant, wherein supernatant is Soluble holoprotein solution is organized for sea cucumber.
S3, it takes the supernatant obtained in step in centrifuge tube, adds ultrapure water to dilute 40 times of sample to be tested, use BCA Protein quantification kit measurement total protein concentration.Bovine serum albumin(BSA) (BSA, Bovine serum is configured with pure water Albumin), the BSA for such as weighing 0.00200g adds 1mL secondary water, is configured to the BSA working solution of 2mg/mL, configuration standard albumen Concentration Linear Points are as follows: 0.01mg/mL, 0.02mg/mL, 0.05 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, BCA working solution is added according to the requirement of BCA kit in 0.4 mg/mL, and 37 DEG C of hatchings survey its absorbance, and then calculate The total protein concentration for the sea cucumber sample that SDS is extracted is 3.4826 mg/mL.
57.4 μ L sea cucumber protein extracts and the NH of 92.6 μ L, 50 mM are taken with pipettor4HCO3Solution is in ultrafiltration membrane On, and 15 μ L 100mM DTT are added, 10min is centrifuged at 95 DEG C of 5min, 14000g, 20 DEG C of incubations.To centrifugal ultrafiltration unit Ultrafiltration membrane on the NH of 200 μ L50 mM is added4HCO330min is centrifuged at 14000g, 20 DEG C after solution.100 are added again The NH of μ L50 mM4HCO3It is centrifuged 20min at 14000g, 20 DEG C after solution, and discards waste liquid in collecting pipe, this step repeats Once.100 μ L 20mM IAA(50mM NH are added on filter membrane4HCO3Solution allocation), oscillation 1min is sufficiently mixed, and is protected from light 30min is centrifuged under the conditions of being incubated for 20min, 14000g, 20 DEG C.10 mM NH are added on ultrafiltration membrane4HCO3In 14000g, 20 DEG C Lower centrifugation 15min, is repeated twice.Replace centrifugal ultrafiltration unit collecting pipe.
S4,5 μ L Trypsin, 45 μ L10 mM NH are added on ultrafiltration membrane4HCO3, sufficiently oscillation 1min, use Parafilm film tightly wraps ultrafiltration membrane, is placed at 37 DEG C and is incubated for 16h.100 μ L ultrapure waters are added on ultrafiltration membrane, in 14000g, 20 It is centrifuged 10min at DEG C, is repeated once.It obtains organizing soluble holoprotein containing sea cucumber.
S5, it is marked again using TMT10,10 samples of label, and records 10 samples of each label substance markers, such as used TMT10- 131 wild sea cucumbers of label 1 year, TMT103 years wild sea cucumbers of -129C label, TMT105 years wild seas of -128N label Ginseng, etc. is incubated at room temperature 1h, and 15 μ L HCl- azanol reaction 15min are added, and terminates reaction.5 μ L are respectively taken to mix in equal volume It closes.Freeze-drying is done to close.And 50 μ L ultrapure waters are added, oscillation dissolution.
S6, S5 sample feed liquor phase chromatographic fractionation is taken, the every 2min of 5min ~ 55min collects efflux, with 200 μ L 6 after freeze-drying: 4(acetonitrile: water V/V) all classification liquid of solution dissolution, merge first group: 5 ~ 8min, 18 ~ 20min, 30 ~ 32min, 42 ~ 44min It is classified fraction；Merge second group of 8 ~ 10min, 20 ~ 22min, 32 ~ 34 min, 44 ~ 46 min and is classified fraction；Merging third group: 10 ~ 12min, 22 ~ 24 min, 34 ~ 36 min, 46 ~ 48 min are classified fraction；Merge the 4th group: 12 ~ 14min, 24 ~ 26 min, 36 ~ 38 min, 48 ~ 50 min are classified fraction；Merge the 5th group: 14 ~ 16min, 26 ~ 28 min, 38 ~ 40 min, 50 ~ 52 min divide Grade fraction；Merge the 6th group: 16 ~ 18min, 28 ~ 30 min, 38 ~ 40 min, 52 ~ 55 min are classified fraction.By above 6 groups of samples Product freeze-drying.With 200 μ L, 6:4(acetonitriles: water V/V) 6 groups of classification liquid of dissolution, are redissolved after freeze-drying with mobile phase.
S7, ultra performance liquid chromatography-level four bars/electrostatic field orbit trap high resolution mass spectrum (UPLC-Q-Exactive Orbitrap Mass, Thermo Scientific, USA), match nanoliter sampling system and heatable electric spray ion source (HESI) it measures, quantitative analysis is carried out to differential protein.Specific instrument condition is the same as embodiment 1.
Embodiment 3
S1, take sea cucumber tissue through over cleaning after, crushed, be put into mortar, while grinding into mortar annex solution Nitrogen, until sea cucumber tissue pieces are in solid powdered.
S2, sea cucumber powder 0.08002g is placed into 2mL ultraphonic pipe, the 6 mol/LTriton X- of 400 μ L is added 100 reverse micro emulsions, wherein Triton X-100 reverse micro emulsion is dissolved with 50 mM ammonium bicarbonate solns, and 6 μ L eggs are added White enzyme inhibitor (Cocktail), ultraphonic pipe is placed in ice face, is ultrasonically treated, was ultrasonically treated under conditions of 4 DEG C The working time is 10s, intermittent time 10s in journey, and ultrasonication amounts to 6min, then the centrifugal treating at 2000g, 4 DEG C 30min takes supernatant, and wherein supernatant is that sea cucumber organizes soluble holoprotein solution.
S3, it takes the supernatant obtained in step in centrifuge tube, adds ultrapure water to dilute 40 times of sample to be tested, use BCA Protein quantification kit measurement total protein concentration.Bovine serum albumin(BSA) (BSA, Bovine serum is configured with pure water Albumin), the BSA for such as weighing 0.00201g adds 1mL secondary water, is configured to the BSA working solution of 2mg/mL, configuration standard albumen Concentration Linear Points are as follows: 0.01mg/mL, 0.02mg/mL, 0.05 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, BCA working solution is added according to the requirement of BCA kit in 0.4 mg/mL, and 37 DEG C of hatchings survey its absorbance, and then calculate The total protein concentration for the sea cucumber sample that Triton X-100 is extracted is 5.1069 mg/mL.
39.2 μ L sea cucumber protein extracts and the NH of 110.8 μ L, 50 mM are taken with pipettor4HCO3Solution is in ultrafiltration membrane On, and 15 μ L 100mM DTT are added, 10min is centrifuged at 95 DEG C of 5min, 14000g, 20 DEG C of incubations.To centrifugal ultrafiltration unit Ultrafiltration membrane on the NH of 100 μ L50 mM is added4HCO310min is centrifuged at 14000g, 20 DEG C after solution.100 are added again The NH of μ L50 mM4HCO3It is centrifuged 10min at 14000g, 20 DEG C after solution, and discards waste liquid in collecting pipe.Add on filter membrane Enter 100 μ L 20mM IAA(50mM NH4HCO3Solution allocation), oscillation 1min is sufficiently mixed, and it is protected from light and is incubated for 10min, 14000g is centrifuged 30min under the conditions of 20 DEG C.10 mM NH are added on ultrafiltration membrane4HCO3It is centrifuged at 14000g, 20 DEG C 15min is repeated twice.Replace centrifugal ultrafiltration unit collecting pipe.
S4,8 μ L Trypsin, 45 μ L10 mM NH are added on ultrafiltration membrane4HCO3, sufficiently oscillation 1min, use Parafilm film tightly wraps ultrafiltration membrane, is placed at 37 DEG C and is incubated for 4h.150 μ L ultrapure waters are added on ultrafiltration membrane, in 14000g, 20 DEG C Lower centrifugation 10min, is repeated once.It obtains organizing soluble holoprotein containing sea cucumber.
S5, it is marked again using TMT10,10 samples of label, and records 10 samples of each label substance markers, such as used TMT10- 131 wild sea cucumbers of label 1 year, TMT103 years wild sea cucumbers of -129C label, TMT105 years wild seas of -128N label Ginseng, etc. is incubated at room temperature 1h, and 15 μ L HCl- azanol reaction 15min are added, and terminates reaction.5 μ L are respectively taken to mix in equal volume It closes.Freeze-drying is done to close.And 50 μ L ultrapure waters are added, oscillation dissolution.
S6, S5 sample feed liquor phase chromatographic fractionation is taken, the every 2min of 5min ~ 55min collects efflux, with 200 μ L 6 after freeze-drying: 4(acetonitrile: water V/V) all classification liquid of solution dissolution, merge first group: 5 ~ 8min, 18 ~ 20min, 30 ~ 32min, 42 ~ 44min It is classified fraction；Merge second group of 8 ~ 10min, 20 ~ 22min, 32 ~ 34 min, 44 ~ 46 min and is classified fraction；Merging third group: 10 ~ 12min, 22 ~ 24 min, 34 ~ 36 min, 46 ~ 48 min are classified fraction；Merge the 4th group: 12 ~ 14min, 24 ~ 26 min, 36 ~ 38 min, 48 ~ 50 min are classified fraction；Merge the 5th group: 14 ~ 16min, 26 ~ 28 min, 38 ~ 40 min, 50 ~ 52 min divide Grade fraction；Merge the 6th group: 16 ~ 18min, 28 ~ 30 min, 38 ~ 40 min, 52 ~ 55 min are classified fraction.By above 6 groups of samples Product freeze-drying.With 200 μ L, 1:1(acetonitriles: water V/V) 6 groups of classification liquid of dissolution, are redissolved after freeze-drying with mobile phase.
S7, ultra performance liquid chromatography-level four bars/electrostatic field orbit trap high resolution mass spectrum (UPLC-Q-Exactive Orbitrap Mass, Thermo Scientific, USA), match nanoliter sampling system and heatable electric spray ion source (HESI) it measures, quantitative analysis is carried out to differential protein.Specific instrument condition is the same as embodiment 1.

Claims (3)

1. a kind of analysis method of sea cucumber protein group, it is characterised in that: the method follows the steps below:
S1, take sea cucumber to organize, remove intestines, through over cleaning after, crushed, be put into mortar, quicklyed while grinding to grinding Liquid nitrogen is added in alms bowl, until sea cucumber tissue pieces are in solid powdered；
S2, sea cucumber powder is placed into microcentrifugal tube, 6 ~ 10 mol/L urea elements or detergent alkylate sulphur of 400 μ L is added Sour sodium or Triton X-100 Reverse Microemulsion System, and 4 ~ 6 μ L proteinase inhibitor C ocktail are added, by ultraphonic pipe Be placed in ice face, be ultrasonically treated under conditions of 4 DEG C, in ultrasonication the working time be 10s, intermittent time 10s, 3 ~ 6 min are run, then 20 ~ 30min of centrifugal treating at 2000g, 4 DEG C, takes supernatant, and wherein supernatant is sea cucumber tissue Soluble holoprotein solution；
S3, it is needed on conversion film with the concentration of BCA quantification kit measurement sea cucumber total protein according to the protein concentration that measurement obtains The volume of loading is subsequent centrifugation trim, 50 mM NH of certain volume is added4HCO3Solution makes overall 150 μ of applied sample amount L, and be added 15 μ L 100mM DTT, 95 DEG C of incubations 5min after be centrifuged, on the ultrafiltration membrane of centrifugal ultrafiltration unit addition 100 ~ The NH of 200 μ L 50mM4HCO3After solution at 14000g, 20 DEG C be centrifuged 10 ~ 30min, 1 ~ 2 time；Waste liquid in collecting pipe is discarded, It is added on ultrafiltration membrane and uses 50mM NH4HCO3The 100 μ L of 20mM iodoacetamide solution of solution allocation, oscillation 1min are sufficiently mixed Afterwards, it is protected from light and is incubated for 20min, 10 ~ 30min is centrifuged under the conditions of 14000g, 20 DEG C, 10 mM NH are added on ultrafiltration membrane4HCO3It is molten Liquid is centrifuged 15min at 14000g, 20 DEG C, is repeated twice, and replaces centrifugal ultrafiltration unit collecting pipe；
5 ~ 8 μ L trypsin solutions are added on S4, the ultrafiltration membrane into S3 step, with 10mM NH4HCO350 μ L are supplemented to, Sufficiently oscillation 1min, tightly wraps ultrafiltration membrane using Parafilm film, is placed in 4 ~ 18h of incubation at 37 DEG C；To on ultrafiltration membrane be added 50 ~ 150 μ L ultrapure waters, are centrifuged 10min at 14000g, 20 DEG C, are repeated once, and obtain organizing soluble holoprotein containing sea cucumber；
S5, it is marked again using TMT10,10 samples of label, and records 10 samples of each label substance markers, be incubated at room temperature 15 μ L HCl- azanol reaction 15min are added in 1h, terminate reaction, mark quantitative differences albumen with this, are lyophilized；
S6, it is classified, is classified using C18 high PH, 5 ~ 55min with liquid chromatogram for the sample of S5 step, every 2 ~ 3min is collected Efflux, freeze-drying；With acetonitrile: the 200 μ L of mixed liquor that water volume ratio is 1:1 ~ 6:4 dissolves, and divides 4 ~ 8 groups, merges its and is classified liquid, It is redissolved after freeze-drying with mobile phase；
S7, it is analyzed using liquid chromatogram-high resolution mass spectrum combined instrument LC-MS/MS.
2. a kind of analysis method of sea cucumber protein group according to claim 1, it is characterised in that: extra large in the step S1 Join 0.08 ~ 0.12g of powder, is placed into 2mL microcentrifugal tube.
3. a kind of analysis method of sea cucumber protein group according to claim 1, it is characterised in that: surveyed in the step S3 Determine protein concentration operation are as follows: the supernatant for obtaining step S2 adds ultrapure water to dilute 40 times of sample to be tested in centrifuge tube, uses BCA protein quantification kit measurement total protein concentration.
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