，，，，*

(1.南華大學(xué)腫瘤研究所，湖南 衡陽421001；2.南華大學(xué)醫(yī)學(xué)院解剖教研室)

·基礎(chǔ)醫(yī)學(xué)·

胃癌差異表達(dá)蛋白的蛋白質(zhì)組學(xué)分析

李素云1,2，陳蘇瓊1，謝遠(yuǎn)杰1，張志偉1，賀修勝1*

(1.南華大學(xué)腫瘤研究所，湖南 衡陽421001；2.南華大學(xué)醫(yī)學(xué)院解剖教研室)

目的篩選與鑒定胃癌差異表達(dá)蛋白質(zhì)分子。方法采用定量蛋白組學(xué)方法，通過同位素iTRAQ標(biāo)記胃癌與正常胃黏膜組織總蛋白，然后用強陽離子交換/反相液相色譜(SCX/nanoHPLC)進(jìn)行電噴霧串聯(lián)質(zhì)譜分析(ESI-MS/MS)，獲得兩組樣本的多肽及相對豐度信息，通過數(shù)據(jù)庫搜索與比對，鑒定差異表達(dá)蛋白質(zhì)。

結(jié)果共鑒定出319個蛋白質(zhì)，其中表達(dá)差異在2倍以上的88個，在胃癌中呈高表達(dá)的12個、呈低表達(dá)的76個。按蛋白質(zhì)功能分為七大類：細(xì)胞骨架蛋白、分子伴侶、信號傳導(dǎo)、生物代謝、凋亡，蛋白質(zhì)合成與代謝類及其他蛋白質(zhì)。結(jié)論成功篩選了胃癌差異表達(dá)蛋白質(zhì)分子，這些蛋白質(zhì)的表達(dá)改變，可能參與了胃癌的發(fā)生和發(fā)展。

胃癌； iTRAQTM同位素標(biāo)記相對和絕對定量； 多維液相色譜； 串聯(lián)質(zhì)譜

我國是胃癌的高發(fā)區(qū)，每年死于胃癌的人數(shù)約為16萬人，因此，提高胃癌的診療水平，是我國腫瘤基礎(chǔ)及臨床研究工作者所面臨的挑戰(zhàn)[1]?，F(xiàn)今臨床所使用的腫瘤診斷標(biāo)記物有CA19-9、CEA、CA72-4[2-3]，但這些標(biāo)記物對于早期胃癌檢測缺乏特異性，僅能用作為術(shù)后療效監(jiān)測[4]。

本研究將LCM技術(shù)純化后的組織，胰酶裂解，iTRAQ試劑分別標(biāo)記多肽，強陽離子反相液相色譜進(jìn)行肽段分離，結(jié)合電噴霧串聯(lián)質(zhì)譜對多肽進(jìn)行定性定量，通過數(shù)據(jù)庫搜索鑒定胃腺癌的差異表達(dá)蛋白質(zhì)，尋找胃癌早期診斷的特異分子標(biāo)志物，對胃癌的早期診斷、防治及預(yù)后評估、發(fā)病機制提供理論和實驗基礎(chǔ)。

1 材料與方法

1.1 材料

1.1.1 標(biāo)本 20例手術(shù)切除的新鮮胃腺癌組織及配對的正常胃黏膜上皮組織，取自湖南省衡陽市南華大學(xué)附屬第一醫(yī)院腫瘤外科，所有取材嚴(yán)格按照實驗要求操作，所取組織經(jīng)激光捕獲顯微切割純化。

1.1.2 試劑 trypsin為 promega Madison WI，乙腈(ACN )為Fisher scientific pittsburg,PA，0.1%甲酸購自sigma公司，三氟乙酸(TFA，色譜純) 購自Merck公司，iTRAQ Reagentkit購自Applied Biosystems公司。

1.2 胃癌細(xì)胞和正常胃黏膜上皮細(xì)胞蛋白質(zhì)的消化和標(biāo)記

1.2.1 蛋白沉淀還原與半胱氨酸封閉 每組樣品100 μg蛋白質(zhì)，分別置于潔凈的微量離心管中，分別加入4倍體積的丙醇(-20 ℃)和12%三氯乙酸(TCA)沉淀蛋白，將兩組樣品離心管置于-20 ℃孵育過夜； 兩組樣品離心管離心 12 000 g,4 ℃,30 min,棄上清，同樣的試劑再次沉淀，-20 ℃孵育2 h,棄上清讓沉淀自然干燥，再溶解在緩沖溶液中；分別加入2 μL還原劑[三(2-羰基乙基)磷鹽酸鹽]； 60 ℃中孵育2.5 h，離心使樣品沉于管底，棄上清液；分別加入2 μL封閉劑半胱氨酸 (硫甲磺酸S-甲醋)，渦旋，室溫下孵育20 min。

1.2.2 胰蛋白酶消化蛋白 每個樣品管中按1∶20(胰酶∶蛋白質(zhì))的比例加入胰蛋白酶，旋渦混合，在37 ℃下過夜(12～16 h)。

1.2.3 iTRAQ試劑標(biāo)記消化蛋白 標(biāo)記使用4標(biāo)試劑盒進(jìn)行2標(biāo)標(biāo)記，使用116,117標(biāo)記。向116、117報告基團的iTRAQ試劑瓶內(nèi)加入70 μL無水乙醇，混勻，使iTRAQ試劑充分溶解備用；將含116報告基團的iTRAQ試劑與胃正常黏膜上皮細(xì)胞組蛋白樣品管混合，117報告基團的iTRAQ試劑與胃癌細(xì)胞蛋白樣品管混合，室溫下孵育l h；將標(biāo)記好的消化蛋白樣品混合入統(tǒng)一管中，旋轉(zhuǎn)真空離心濃縮干燥(Speed Vac)混合后的蛋白。

1.3 全自動2D-Nano-LC-ESI-MS/MS分析肽段

肽段使用強陽離子交換/反相納升級液相色譜(SCX/nanoHPLC)進(jìn)行電噴霧串聯(lián)質(zhì)譜分析(ESI-MS/MS)。

1.4 數(shù)據(jù)分析

所有的串級譜圖通過SEQUEST搜索引擎 [v.28 (revision 12),Thermo Electron Corp.]進(jìn)行數(shù)據(jù)庫檢索。數(shù)據(jù)庫為Swiss-Prot人種屬數(shù)據(jù)庫(Release 2010_04，20331條序列)。為了減少假陽性結(jié)果，在該數(shù)據(jù)庫中增加一個含有所有蛋白反轉(zhuǎn)序列的誘餌數(shù)據(jù)庫。搜索參數(shù)設(shè)置如下：胰蛋白酶(Trypsin)，半酶切方式，最大漏切為2。固定修飾為半胱氨酸(Cysteine)的甲基硫代磺酸甲酯化(methyl methanethiosulfonate,MMTS)，可變修飾為甲硫氨酸(Methionine)的氧化，賴氨酸(lysine)和肽段N端的iTRAQ試劑標(biāo)記。單一同位素模式，肽段質(zhì)量誤差50 ppm，碎片離子質(zhì)量誤差0.05 Da。

使用Trans Proteomic Pipeline 軟件(revision 4.0)(Institute of Systems Biology,Seattle,WA)，通過Peptide Prophet(P≥0.90)和Protein Prophet(P≥0.95)來過濾肽段和蛋白結(jié)果，獲得置信度在0.95以上的蛋白結(jié)果。根據(jù)肽段結(jié)果通過定量計算方法，計算得到各個蛋白的定量結(jié)果。

2 結(jié) 果

2.1 蛋白質(zhì)的質(zhì)譜分析

所有的串級譜圖通過SEQUEST搜索引擎進(jìn)行Swiss-Prot人種屬數(shù)據(jù)庫檢索。使用Trans Proteomic Pipeline 軟件來過濾肽段和蛋白，獲得置信度在0.95以上的蛋白結(jié)果。共發(fā)現(xiàn)鑒定了319個蛋白，其中差異蛋白為88個，12個蛋白在胃腺癌中高表達(dá)，76個蛋白在胃腺癌中低表達(dá)(表1)。

2.2 已鑒定的差異蛋白的亞細(xì)胞定位和功能分類

根據(jù)UniprotKB/Swiss-prot數(shù)據(jù)庫中蛋白質(zhì)的相對定位和功能信息，對人胃低分化腺癌所鑒定得到的蛋白質(zhì)進(jìn)行亞細(xì)胞定位和功能分類。從圖1中可以看出已鑒定出的蛋白有17.4%位于細(xì)胞漿中；27.7%定位于線粒體，大部分都是氧化還原和呼吸鏈相關(guān)的酶類；5.1%為核糖體蛋白；12.2%定位于細(xì)胞外基質(zhì)，基本上都是分泌蛋白；14.2%為細(xì)胞骨架蛋白；4.0%的蛋白僅定位于細(xì)胞核；位于細(xì)胞漿和細(xì)胞核的蛋白質(zhì)為9.1%；10.2%定位于細(xì)胞膜上，與細(xì)胞連接和信號傳導(dǎo)相關(guān)。

對于蛋白質(zhì)的功能分類見圖2，已鑒定的蛋白質(zhì)中27.4%與生物代謝相關(guān)如Creatine kinase B-type等；另一大類(17.9%)為信號傳導(dǎo)通路蛋白質(zhì)如Ras-related protein Rab-44等；蛋白合成與代謝類也占16.8%如60S ribosomal protein L8等；還有14.7%是細(xì)胞骨架類蛋白質(zhì)如Myosin-13；4.2%的分子伴侶類；8.4%的細(xì)胞凋亡類。

表1Nano-RPLC-MS/MS鑒定的GAC和NGEC差異在2倍以上的蛋白質(zhì)

AcessionnumberProteinnamePercentcoverage116rati-o117rati-o116∶117rati-olocalizationfunctionUp-regulationproteinsO94876SorbinandSH3domain-containingprotein21.90.310.680.46cytoplasmsignalP21980Protein-glutaminegam-maglutamyltransferase24.10.310.680.46apoptoticP51888Prolargin8.90.310.670.46extracellularapoptoticP08670Vimentin52.60.310.660.47cytosolCellularcomponentmove-mentP07951Tropomyosinbetachain36.30.320.680.47cytoplasmcytoskelectonP20774Mimecan21.80.320.680.47extracellularGrowthfactoractivityP51884Lumican33.70.320.670.48extracellularcollagenfibrilorganizationQ86VW7Keratin,typeIcytoskeletal181.20.320.670.48P21793Decorin12.50.320.670.48extracellularproteinbindingQ9UKX3Myosin-131.20.320.670.48cytoplasmmusclecontractionO05787Keratin,typeIIcytoskel-etal855.30.330.660.50CytoplasmcytoskelectonP12814Alpha-actinin-117.80.330.660.50cytoplasmcytoskelectonregulationofapoptosisDown-regulationproteinP491894-trimethylaminobutyr-aldehydedehydrogenase4.70.660.332.00cytoplasmcarnitinebiosyntheticprocess;Fattyacidmetab-olismp2539840SribosomalproteinS127.60.660.332.00cytoplasmtranslationalelongationQ16891Mitochondrialinnermembraneprotein12.50.670.332.03Mitochondrioninnermembr-ane.aerobicrespirationP30041Peroxiredoxin-613.40.670.332.03cytoplasmcellredoxhomeostasisQ04941Proteolipidprotein28.60.670.332.03MembraneChemotaxis;cytokine-me-diatedsignalingpathwayP34931Heatshock70kDaprotein12.40.670.322.09Cytoplasm;NucleusmolecularchaperonesP34931Carbonicanhydrase98.30.670.322.09Membranone-carbonmetabolicprocessP15311Ezrin17.40.670.322.09CytoplasmCytoskeletonP21796Voltage-dependentanion-selectivechannelprotein35.70.680.322.13MitochondrionapoptoticP07195L-lactatedehydrogenaseBchain16.80.680.322.13CytoplasmGlycolysis;L-lactatede-hydrogenaseactivityP6282960SribosomalproteinL2321.40.680.322.13RibosomeribosomalproteinimportintonucleusP31930Cytochromeb-c1com-plexsubunit1,mito-chondrial15.80.660.312.13Mitochondrioninnermem-braneElectrontransport;Re-spiratorychainP07737Profilin-127.10.680.312.19CytoplasmcytoskelectonP53597Succinyl-CoAligasesub-unitalpha,mitochondrial19.10.690.312.23MitochondriontricarboxylicacidcycleQ9VHD8Septin-92.60.690.312.23CytoplasmCellcycleQ03403Trefoilfactor236.40.690.312.23ExtracellulardigestionO75390Citratesynthase,mito-chondrial9.90.690.312.23MitochondriontricarboxylicacidcycleP3901940SribosomalproteinS1927.60.690.312.23Cytoplasm;NucleusRibonucleoprotein

AcessionnumberProteinnamePercentcoverage116rati-o117rati-o116∶117rati-olocalizationfunctionQ66K74Microtubule-associatedprotein1S2.70.690.690.312.23Cytoplasm;NucleusapoptoticO75964ATPsynthasesubunitg,mitochondrial23.30.690.312.23Mitochondrioninnermem-branecellredoxhomeostasisP1080960kDaheatshockpro-tein,mitochondrial300.690.32.30MitochondrionmolecularchaperonesP08727Keratin,typeIcytoskele-tal19CK1946.50.690.32.30CytoplasmcytoskelectonQ9H4GOBand4.1-likeprotein11.40.70.32.30CytoplasmcytoskelectonA8MW06Thymosinbeta-4-likeprotein343.20.70.32.33CytoplasmcytoskelectonP45880Voltage-dependentanion-selectivechannelprotein28.20.70.32.33MitochondrionTransmembrane;signalP3091010kDaheatshockpro-tein,mitochondrial19.60.70.292.41MitochondrionmolecularchaperonesP00352Retinaldehydrogenase123.20.70.292.41CytoplasmRasGTPaseactivatorac-tivity;signalQ06830Peroxiredoxin-132.20.70.292.41CytoplasmcellredoxhomeostasisP12277CreatinekinaseB-type570.680.282.43CytoplasmcellredoxhomeostasisQ8NC51Plasminogenactivatorin-hibitor1RNAbindingprotein8.10.710.292.45Cytoplasm;NucleusregulationofmRNAsta-bilityP08238HeatshockproteinHSP90-beta17.30.710.292.45MitochondrionmolecularchaperonesP0C0S8HistoneH2Atype1500.710.292.45Nucleus;nucleosomeassemblyP4678240SribosomalproteinS511.30.710.292.45RibosomaltranslationalelongationQ13884Beta-1-syntrophin4.10.710.292.45membranecytoskelectonP30049ATPsynthasesubunitdelta13.70.710.282.54MitochondrioncellredoxhomeostasisP55084Trifunctionalenzymesubunitbeta,mitochon-drial5.50.710.282.54Mitochondrionfattyacidbeta-oxidationP08758AnnexinA512.20.710.282.54Cytoplasmanti-apoptosis;signaltransductionP05141ADP/ATPtranslocase217.80.720.282.57Mitochondrioninnermem-braneenergyreservemetabolicprocessP20648Potassium-transportingATPasealphachain112.90.720.282.57MembranePro-teinsignalP40925Malatedehydrogenase,cytoplasmic12.60.720.282.57CytoplasmcellredoxhomeostasisP35900Keratin,typeIcytoskele-tal208.30.720.282.57Cytoplasmcytoskelecton;apoptosisQ9UBU3Appetite-regulatinghor-mone12.80.720.272.67ExtracellularsignalP61769Beta-2-microglobulin8.40.730.272.70Cytoplasminterferon-gamma-media-tedsignalingpathwayQ9H2P0Activity-dependentneu-roprotectorhomeoboxprotein4.40.730.272.70Nucleusregulationoftranscrip-tion,DNA-dependentO14745Ezrin-radixin-moesin-bind-ingphosphoprotein50(EBP50)3.90.730.272.70CytoplasmMembranesignalingpathwayQ13797Integrinalpha-91.00.730.272.70MembranePro-teinsignalingpathwayP38117Electrontransferflavo-proteinsubunitbeta9.80.710.262.73MitochondrionrespiratoryelectrontransportchainP07954Fumaratehydratase,mi-tochondrial10.20.730.730.262.81MitochondrionTricarboxylicacidcycle

AcessionnumberProteinnamePercentcoverage116rati-o117rati-o116∶117rati-olocalizationfunctionP23528Cofilin-129.50.740.262.85CytoplasmRhoproteinsignaltrans-duction;cytoskelectonP14091CathepsinE3.70.740.252.96Endosomeaspartic-typeendopepti-daseactivityP30046D-dopachromedecarbox-ylase220.740.252.96CytoplasmmelaninbiosyntheticprocessQ13423NAD(P)transhydroge-nase7.10.740.252.96MitochondrionTricarboxylicacidcycleP09525AnnexinA48.20.740.252.96CytoplasmsignaltransductionP98088Mucin-5AC(Fragments)9.80.740.252.96SecretedcelladhesionP30044Peroxiredoxin-5,mito-chondrial42.10740.252.96MitochondrioncellredoxhomeostasisP48735Isocitratedehydrogenase[NADP],mitochondrial37.40.730.243.04Mitochondrion2-oxoglutaratemetabolicprocessP08263GlutathioneS-transferaseA17.20.740.243.08CytoplasmglutathionemetabolicprocessP06703ProteinS100-A68.90.760.243.17Cytoplasm;NucleusS100betabinding;signaltransductionP36957Dihydrolipoyllysine-resi-duesuccinyltransferasecomponentof2-oxoglut-aratedehydrogenasecomplex,mitochondrial4.60.750.233.26MitochondriontricarboxylicacidcycleP6291760SribosomalproteinL86.20.770.233.35CytoplasmTranslationalelongationP00403Cytochromecoxidasesubunit24.40.770.233.35Mitochondrioninnermem-branemitochondrialelectrontransport,cytochromectooxygenQ86XP6Gastrokine-219.60.770.223.50SecretedsignalP07098Gastrictriacylglycerolli-pase17.80.760.213.62SecretedtriglyceridemetabolicprocessP13073Cytochromecoxidasesubunit4isoform1,mi-tochondrial12.40.780.213.71Mitochondrioninnermem-branecytochrome-coxidaseac-tivity,proteinbindingP20142Gastricsin14.70.760.23.8Secreteddigestion,proteolysisP10606Cytochromecoxidasesubunit5B47.30.770.23.85Mitochondrioninnermem-branerespiratoryelectrontransportchainP00790PepsinA17.50.710.164.44Secreteddigestion,proteolysisO60218Aldo-ketoreductasefam-ily116.80.80.184.44Cytoplasmdigestion,oxidation-reduc-tionprocessP25815ProteinS100-P28.40.810.184.50Nucleus.Cyto-plasmendothelialcellmigrationQ92522HistoneH1x12.70.770.164.81Nucleus.Cyto-plasmnucleosomeassembly;DNAbindingP56856Claudin-183.80.840.165.25Cellmembranecalcium-independentcell-celladhesion,tightjunc-tionassemblyQ96NY7Chlorideintracellularchannelprotein618.00.850.146.07Cytoplasmmembranechloridechannelcomplex;IontransportQ9NS71Gastrokine-138.20.820.136.31SecretedDigestion;signalQ7Z673Ras-relatedproteinRab-441.90.90.0910.00CellmembranesmallGTPasemediatedsignaltransductionQ5D1E8ZincfingerCCCHdo-main-containingprotein12A2.50.890.0811.13Cytoplasm;Nu-cleusApoptosis;Differentiation

圖1 被鑒定蛋白質(zhì)的亞細(xì)胞定位

圖2 Nano-RPLC-MS/MS鑒定的GAC和NGEC差異蛋白質(zhì)功能分類

3 討 論

蛋白質(zhì)系基因功能的最終執(zhí)行者，基因作用的發(fā)揮通過其表達(dá)產(chǎn)物蛋白質(zhì)實現(xiàn)，所以蛋白質(zhì)組學(xué)(Proteomics)的研究受到研究者們高度關(guān)注。比較蛋白質(zhì)組學(xué)是蛋白質(zhì)組學(xué)最重要的研究策略，有研究報道，采用比較蛋白質(zhì)組學(xué)技術(shù)，通過比較腫瘤與其起源的正常組織，或腫瘤發(fā)展不同階段組織中蛋白質(zhì)在表達(dá)水平和修飾狀態(tài)上的差異，發(fā)現(xiàn)了諸多癌變相關(guān)的特異性蛋白質(zhì)[5-8]。

程愛蘭等[9]人運用2-DE技術(shù)分別從鼻咽癌和正常的鼻咽上皮組織中純化出鼻咽癌細(xì)胞和正常鼻咽上皮細(xì)胞，發(fā)現(xiàn)了29 個差異蛋白質(zhì)，其中 15 個蛋白質(zhì)僅在 NPC 中表達(dá)或者高表達(dá)，而14 個蛋白質(zhì)在 NPC 中表達(dá)下調(diào)或者缺失。Neubauer等[10]應(yīng)用2DE技術(shù)，發(fā)現(xiàn)了區(qū)別 (ER)+/(PR)+和ER+/PR-乳腺預(yù)后的相關(guān)蛋白質(zhì)transgelin、cydophilinA及Neudesin，這些蛋白質(zhì)的差異表達(dá)，并發(fā)現(xiàn)ER+/PR+乳腺癌預(yù)后好于ER+/PR-的乳腺癌。

蛋白質(zhì)組技術(shù)，是后基因時代基因功能研究的重要技術(shù)，近年來在蛋白質(zhì)組研究中新興的多維液相色譜( MD-LC)技術(shù)，是一種非膠的分離方法，利用兩種或多種分離機理不同的色譜模式組合對樣品進(jìn)行正交分離。因便于接口控制、易與質(zhì)譜連接、自動化程度高等優(yōu)勢，已經(jīng)作為與2DE互補的技術(shù)在大規(guī)模蛋白質(zhì)組研究中發(fā)揮了重要作用[11]。

本實驗中采用的是強陽離子交換色譜(SCX)和反相高效液相色譜(HP-RPLC)多分離模式來分離蛋白質(zhì)，很大程度上克服了2DE的局限性，其與質(zhì)譜的聯(lián)用是目前蛋白質(zhì)組學(xué)研究技術(shù)中發(fā)展較快的領(lǐng)域。采用基于非膠路線的液相色譜分離方法和iTRAQ標(biāo)記定量蛋白質(zhì)組學(xué)技術(shù)，所需樣本量少，可以很好與LCM技術(shù)結(jié)合運用。

本研究樣本經(jīng)過SCX的預(yù)處理后，復(fù)雜程度大大降低，同時iTRAQ標(biāo)記的引入，保證了分離過程中化學(xué)行為的一致性，可直接對差異蛋白進(jìn)行對比，提高了實驗結(jié)果的準(zhǔn)確性。這種實驗技術(shù)的聯(lián)用可替代傳統(tǒng)的2-DE、DIGE技術(shù)，是對胃癌等實體瘤進(jìn)行差異分析的有效方法[12]。

采用iTRAQ同位素4標(biāo)試劑盒進(jìn)行2標(biāo)116、117標(biāo)記，116標(biāo)記正常胃黏膜上皮的總蛋白，117標(biāo)記胃低分化腺癌的總蛋白，通過比較標(biāo)記同一蛋白的116、117的相對豐強度來進(jìn)行相對定量。肽段使用強陽離子交換/反相納升級液相色譜(SCX/nanoHPLC)進(jìn)行電噴霧串聯(lián)質(zhì)譜分析(ESI-MS/MS)，使用Trans Proteomic Pipeline 軟件通過Peptide Prophet(P≥0.90)和Protein Prophet(P≥0.95)來過濾肽段和蛋白結(jié)果，可獲得置信度在0.95以上的蛋白結(jié)果，鑒定出胃癌差異表達(dá)蛋白質(zhì)分子，包括高表達(dá)和低表達(dá)蛋白質(zhì)分子。高表達(dá)的蛋白主要是細(xì)胞骨架蛋白和分泌蛋白比如Vimentin、Myosin-13、Decorin、Lumican，低表達(dá)的蛋白集中在信號傳導(dǎo)、生物代謝、蛋白合成與代謝等生物學(xué)功能上如Ras-related protein Rab-44、NAD(P) transhydrogenase、60S ribosomal protein L8等。這些蛋白的差異表達(dá)都與腫瘤的發(fā)生發(fā)展相關(guān)，表明胃癌發(fā)病機制的復(fù)雜性。

胃癌的發(fā)生發(fā)展的過程是一個多因素、多步驟、多分子參與的復(fù)雜事件，可能包括相關(guān)癌基因激活和(或)抑癌基因失活，上調(diào)和(或)下調(diào)相關(guān)蛋白質(zhì)的表達(dá)，經(jīng)其相應(yīng)信號傳導(dǎo)途徑，導(dǎo)致胃癌的發(fā)生與發(fā)展[13-15]。

綜上所述，人低分化胃癌組織與正常胃黏膜上皮組織的蛋白質(zhì)表達(dá)存在差異。但胃癌發(fā)生發(fā)展的確切分子機制、蛋白質(zhì)間相互作用、及其蛋白質(zhì)表達(dá)調(diào)控網(wǎng)絡(luò)，有待今后進(jìn)一步深入研究。

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ProteomicsAnalysisofDifferentialExpressionProteinsinGastricCarcinoma

LI Suyun,CHEN Suqiong,XIE Yuanjie,et al

(CancerResearchInstitute，UniversityofSouthChina，Hengyang,Hunan421001，China)

ObjectiveTo identify the differential expression proteins between gastric adenocarcinoma cells (GAC) and normal gastric adenoepithelial cells (NGAC),and to look for early diagnosis of Gastric carcinoma (GC )specific proteins.MethodsThe total proteins were extracted from the tissues.The total proteins labeled with different iTRAQ stable isotope were segregated with Nano-RPLC-MS/MS,to obtain relative abundance of peptides information.The differential expression proteins were identified through SWISSPORT database.ResultsThrough Quantitative proteomic analysis of GAC and NGAC,319 proteins were identified with MS,88 of which were differential expression proteins.There were 12 Up-regulation and 76 Down-regulation proteins in GAC.The identified proteins can be divided into seven groups according to protein function:biological oxidation,signal transduction,protein composition and metabolism,molecular chaperones,cytoskeleton,apoptotic and others.ConclusionThe differentially expressed protein molecules were successfully screened and identified in gastric carcinoma,and the expression changing of these proteins may be involved in the genesis and development of gastric carcinoma.

gastric carcinoma; iTRAQTM; multidimensional liquid chromatography; tandem Mass spectrometry

10.15972/j.cnki.43-1509/r.2015.01.004

2014-05-30；

2014-10-20

湖南省自然科學(xué)基金資助項目(12JJ3102)，湖南省衛(wèi)生廳科研基金資助項目(B2012-046),衡陽市科技局資助項目(2013HKJ19)，湖南省高校創(chuàng)新平臺開放基金資助項目(13K082)，地方高校國家級大學(xué)生創(chuàng)新創(chuàng)業(yè)訓(xùn)練計劃資助項目(2012105555017).

*通訊作者，E-mail：hexiusheng@hotmail.com.

R735.2

A

(此文編輯：蔣湘蓮)