Bi-2013-Optimization of harv

optimization of havard

AnalBioanalChem(2013)405:5279–5289DOI10.1007/s00216-013-6927-9

RESEARCHPAPER

Optimizationofharvesting,extraction,andanalyticalprotocolsforUPLC-ESI-MS-basedmetabolomicanalysisofadherentmammaliancancercells

HuichangBi&KristopherW.Krausz&

SoumenK.Manna&FeiLi&CarolineH.Johnson&FrankJ.Gonzalez

Received:8February2013/Revised:15March2013/Accepted:18March2013/Publishedonline:19April2013#Springer-VerlagBerlinHeidelberg2013

AbstractInthisstudy,aliquidchromatographymassspec-trometry(LC/MS)-basedmetabolomicsprotocolwasoptimizedforquenching,harvesting,andextractionofmetabolitesfromthehumanpancreaticcancercelllinePanc-1.Trypsin/ethylenediaminetetraaceticacid(EDTA)treatmentandcellscrapinginwaterwerecomparedforsampleharvesting.Fourdifferentextractionmethodswerecomparedtoinvestigatetheefficiencyofintracellularmetaboliteextraction,includingpureacetonitrile,methanol,methanol/chloroform/H2O,andmethanol/chloroform/acetonitrile.Theseparationefficienciesofhydrophilicinteractionchromatography(HILIC)andreversed-phaseliquidchromatography(RPLC)withUPLC-QTOF-MSwerealsoevaluated.Globalmetabolomicsprofileswerecompared;thenumberoftotaldetectedfeaturesandtherecoveryandrelativeextractionefficienciesoftargetmetaboliteswereassessed.Trypsin/EDTAtreatmentcausedsubstantialme-taboliteleakageprovingitinadequateformetabolomicsstudies.DirectscrapingafterflashquenchingwithliquidnitrogenwaschosentoharvestPanc-1cellswhichallowedforsamplestobe

ElectronicsupplementarymaterialTheonlineversionofthisarticle(doi:10.1007/s00216-013-6927-9)containssupplementarymaterial,whichisavailabletoauthorizedusers.

H.Bi(*)

SchoolofPharmaceuticalSciences,SunYat-senUniversity,132#WaihuandongRoad,GuangzhouUniversityCity,Guangzhou510006,China

e-mail:bihchang@mail.sysu.edu.cn

H.Bi:K.W.Krausz:S.K.Manna:F.Li:C.H.Johnson:F.J.Gonzalez

LaboratoryofMetabolism,CenterforCancerResearch,NationalCancerInstitute,NIH,Bethesda,MD20892,USA

C.H.Johnson

CenterforMetabolomicsandMassSpectrometry,TheScrippsResearchInstitute,10550NorthTorreyPinesRoad,LaJolla,CA92037,USA

storedbeforeextraction.Methanol/chloroform/H2Owaschosenastheoptimalextractionsolventtorecoverthehighestnumberofintracellularfeatureswiththebestreproducibility.HILIChadbetterresolutionforintracellularmetabolitesofPanc-1cells.ThisoptimizedmethodthereforeprovideshighsensitivityandreproducibilityforavarietyofcellularmetabolitesandcanbeapplicabletofurtherLC/MS-basedglobalmetabolomicsstudyonPanc-1celllinesandpossiblyothercancercelllineswithsimilarchemicalandphysicalproperties.

KeywordsMetabolomics.Samplepreparation.Metaboliteextraction.Panc-1cellline.HILIC

Introduction

Metabolismiseitherdirectlyorindirectlyinvolvedineveryaspectofcellfunction.Cellularmetabolomicscanuncoverinformationreflectingchangestobiochemicalreactions,metabolicpathways,andprocessesoccurringwithinthecells[1].Thus,metabolomicanalysisofcellshasemergedasanimportantapproachforstudyingcellularbiochemistry.Metabolicactivityofcells,inparticularcentralcarbonme-tabolism,fattyacid,andlipidmetabolism,havebeenshowntoplayanimportantroleincancerprogressionanddrugresponse[2].Therefore,characterizationofcancercell-specificmetabolomicsignaturescouldbeofgreatvalueintheearlydiagnosisofcanceraswellasinfollowingthera-peuticresponse[3].Pancreaticcancerisoneofthemostaggressiveandlethalcancersandisthefourthleadingcauseofcancer-relateddeathsworldwide[4].Thehumanpancre-aticcancercelllinePanc-1hasbeenusedextensivelyasacellculturemodeltostudyadvancedstageofpancreaticcancer.TheanalysisofmetabolicprofilesofPanc-1cellsusingmassspectrometry(MS)-basedmetabolomicscould