MetaboliteExtrac.onforMetabolomicStudies
AndrewD.Pa;erson,PhDAssociateProfessorofMolecularToxicologyPennStateUniversityadp117@psu.edu
Resources
• MetabolomicsWorkbench– www.metabolomicsworkbench.org– Largeresourceofexperimentalprotocols,datasets,andotherresources
• XCMSIns.tute– h;ps://xcmsonline.scripps.edu/ins.tute– Greattutorialsonchromatography,plaQorms,databases
• Twi;er– #metabolomics
Objec.vesAttheconclusionofthislesson,studentswillbeableto:• Definefactorsthatinfluencemetaboliteextrac.onanddescribetheirimpactonmetabolomicstudies
• Explainthevalueofchromatographyforimprovedmetaboliteiden.fica.onandquan.ta.on
Extrac.onofMetabolites
NucleicAcidExtrac.on
Whatdoweknowaboutourtargetanalyte?• Nega.velychargedphosphatebackbone(polar)• Needtoremoveproteins,lipids,etc
AboutSolventsOH
PHENOL
1.07g/cm3
OH H
WATER
1.00g/cm3
Cl
Cl
HCl
CHLOROFORM
1.49g/cm3
POLARITY
ProteinChemistry
h;ps://en.wikipedia.org/wiki/Proteinogenic_amino_acid
Denatureproteins• Hydrophobicamino
acidsfacephenol:chloroform
• Phe,Tyr,Leu
LiquidLiquidExtrac.on
DNAorRNA(aqueous)
Protein
Lipids(phenol:chloroform)
DNAExtrac.onProtocol• Disrupt.ssuein
phenol:chloroform
**chloroformpreventssmallamountsofwaterinphenolfromdissolvingmRNA**adjustpHtofavorDNA(basic)orRNA(acidic)isola.on• Centrifugetoseparatelayers• Dehydratewithalcohol
Whatcouldgowrong?
• Solventsnotappropriateorpreparedincorrectly– pHincorrect– Ra.osincorrect
• Contamina.onofsolventsorbuffers
• Whatelsecanyouthinkof?
StandardOpera.ngProcedures
• Saves.meandpreventsmistakes• Consistentresults
• Checkinginsamples(samplelists,loca.on)• Labelingandstoringsamples(aliquot)• Metaboliteextrac.on(targetedorglobal)• AcquiringdataonvariousplaQorms(MS,NMR)
EvenwithSOPs…
Noteinfluenceofindividualpreparingpooledsamples.Easytoseewhopreparedwhat.
DECAFCOFFEE
SUMATRACOFFEE COLUMBIAN
COFFEE
CommonSolventsforMetabolomics
• Methanol
• Acetonitrile• Chloroform
• Methyltert-butylether(MTBE)
• Water
NH3C
CH3HO
Cl
Cl
HCl
H3C
H3C
CH3
CH3O
OH
H
Listisnotinclusive
Methanol
• Rela.velyinexpensivecomparedtoacetonitrile
• Notregulatedlikeethanol• Easytoevaporate
• Extractspolarand(some)non-polarmolecules–why?
Acetonitrile
• Advantagesmostlyforchromatography– ReducedabsorbanceforUVbasedmethods– Reducedpressurecomparedtomethanol– Greaterelu.onstrength(generally)– HILICapplica.ons
• Expensive– Isolatedasabyproductnotproduceddirectly– Shortagescaninfluencepriceandavailability
ChloroformvsMTBE
• Chloroformdensi.y–1.49g/cm3• MTBEdensity–0.740g/cm3
• Toxicityofchloroform(checkoutATSDR.CDC.GOV)
• S.llmadeneedtotailorspecificextrac.onsforlipidclasses(hexaneforTAGsorMTBEforCer)– Moredetailcheckoutcyberlipid.orgorlipidmaps.org
SOWHAT?
Metabolomics
Metabolomicsisthesystema.canalysisoftheuniquechemicalfingerprintslekbehindbyspecificcellularprocesses
FOOD
PESTICIDES
COSMETICS
POLLUTANTS
DRUGS
XENOBIOTIC
METAB
OLISM
ENDO
GENOUS
METAB
OLISM
METABOLOME
GUTMICROBIOTA
MetabolomicsAll“-omics”basedscien.ficdisciplinesaimatthecollec.vecharacteriza.on and measurement of their par.cularcons.tuentmolecules
• Acomprehensiveapproachtostudycompletepoolsofbiologicalmolecules
• Defines the structure, func.on and dynamics of anorganism
MetabolomicsVast chemicaldiversityamong smallmoleculemetaboliteshas made extended coverage of the metabolomechallenging
• Size(50–1500Da)• Concentra.on(pM–mM)• Physicochemicalproper.es(diverselogPvalues)• Stereochemistry(dis.nctbiologicalac.vity)
EHP2014Vol122Number8h;p://ehp.niehs.nih.gov/1308015/
h;p://journal.fron.ersin.org/ar.cle/10.3389/fimmu.2016.00044/full
MetaboliteExtrac.on• Currently no analy.cal technique exists that is capable of
measurementofallclassesofcellularmetabolites
• Metaboliteextrac.onisacrucialstepinanymetabolomicsstudy– Cri.caltobothtargetedandglobalbasedprofilingstrategies
• Op.mizedextrac.onmethodologyshouldfulfillseveralcriteria:– Extractthelargestnumberofmetabolites– Unbiasedandnon-selec.ve-physicalorchemicalproper.es
ofamolecule– Non-destruc.ve-nomodifica.onofmetabolites
organicphase
aqueousphase
KD
HAorg
HAaq+H20 H30++A-Ka
organicphase
aqueousphase
KD
HAorg
HAaq
Separa.onofMetabolites• Massspectrometryusuallyrequiressomeformofchromatographicsepara.on
• Mostsystemsuseeitherliquidorgaschromatography
• Frac.ona.onofsamplecomponentssimplifiestheresul.ngmassspectrawhileensuringmoreaccuratecompoundiden.fica.on
• Capacityfactor(k)iscri.caltoop.mizingresolu.on• Increasedresolu.onallowslongerMSdwell.mesresul.nginbe;ersignal/noisera.os
• Inadequatechromatographicsepara.onofmetabolitesresultsin:
• signalsuppression–ionsuppression• compromisedmetabolitequan.fica.on• reducedmetabolitecoverage
CeramidePhysicochemicalProper.es
• Ceramidesareafamilyofwaxylipidmolecules.– Namederivedfromthela.nword:cera=waxy+amide
• Ceramidesarecomprisedof:– sphingosine:18carbonunsaturatedaminoalcohol– fa;yacidmoiety–amidebond
• Ceramidesarenotwatersoluble:– Veryhydrophobic– Confinedtocellularmembranes– Par.cipateinlipidrakforma.on– >200structurallydis.nctspecieshavebeeniden.fiedinmammaliancells
CeramideGeneralStructure
• Ceramide(d18:1/16:0)
• 2-amino-1,3-octadec-4-ene-diol• Aminoalcohol(sphingoid)backbone
• Palmi.cacid• Fa;yacylgroup
• Ceramide(d18:1/24:1(15Z))• 2-amino-1,3-octadec-4-ene-diol
• Aminoalcohol(sphingoid)backbone
• 15-tetracosenoicacid• Fa5yacylgroup
Mol. BioSyst., 2015, 11, 698--713 | 699
StructuresandNomenclature
CeramideBiochemistry
Ceramidesarefoundinhighconcentra.oninthemembraneofcells
• Structuralcomponentofthelipidbilayer
• Bioac.velipid-implicatedinavarietyofphysiologicalfunc.onsincluding:• Apoptosisandcellgrowtharrest• differen.a.onandcellsenescence• cellmigra.onandadhesion
Ceramidesareconvertedrapidlytomorecomplexsphingolipids:• Sphingomyelin• Glycosylceramides• Li;leaccumula.onobserved
– Exceptfortheskin(50%oftotallipidscanbeceramides)
BiosynthesisofCeramides
Denovobiosynthesis
• Ceramidesynthasescouplesphinganine+longchainfa;yacidtoform
dihydroceramide
• Doublebondintroducedintoposi.on4ofthesphingoidbase
§ ceramidesynthases5and6generatearespecificforpalmi.cacid § ceramidesynthases1(brainandskeletalmuscle)specificforstearicacid§ ceramidesynthases2specificforverylongchainCoA-thioesters(C20-C26)
§ ceramidesynthases3unusualceramidesofskin&testes
BiosynthesisofCeramides
Catabolismofcomplexsphingolipids:
• Sphingomyelinases/phospholipaseCbreakdownsphingomyelinin
animalJssues
• Manyfactorscans.mulatethehydrolysisofsphingomyelinto
produceceramide:
§ Cytokines:TNF-a,IFN-g&variousinterleukins§ 1,25-dihydroxy-vitaminD3§ endotoxin§ nervegrowthfactor§ ionizingradia.on&heat
LCMethodDevelopmentWheretoStart?
• Designingandop.mizinganLCmethodinvolveschoosingappropriate:
1. Separa.onmechanism:NPC,RPLC,HILIC,sizeexclusionion,exchangeetc
2. Columnchemistry:C2,C4,C8,C18,cyanopropyl,phenyl,biphenyl,amide,SiOHetc
3. Columnproper.es:poresize,par.clesize&columndimensions
4. Sta.onaryandmobilephasecombina.ons
• Cri.caltoop.mizingthechromatographicefficiency,reten.on,resolu.on&selec.vityofanalytes
CeramideScou.ngGradientsonWatersBEHC18
Steepgradient;largechangeinorganicmodifier.Providesini.alinforma.onaboutanalytereten.on&resolu.on.Aidsinexpedi.ngthedevelopmentofanop.mizedLCmethod
PoorResoluJon(R)LongRetenJon(Tr)Time
1. C16:02. C18:13. C18:04. C20:05. C24:16. C22:07. C24:0
1 2 3 4 75 6
Frac.ona.onofCeramideMetaboliteson
WatersCSHC18Column
Column:100x2.1mm1.7uSolventSystem:A=MeCN:water(60:40v/v)B=2-propanol:MeCN(90:10v/v)10mMNH4OAc+0.1%formicacidBallisJcgradient0.4ml/min@55oC
UPLC-ESI-QTOF-MS
0 5 1 0 1 5 2 0
2 0
4 0
6 0
8 0
1 0 0
G ra d ie n t C o m p a r is o n
tim e (m in )
% o
rgan
ic
b a llis t ics ta n d a r d
BallisJcGradient:
• Ultra-fastgradient
• Rapidchangeinorganicmodifier
• SeparaJonsperformedathighflowrates
• Highlinearvelocity
Frac.ona.onofCeramideMetaboliteson
WatersCSHC18Column
1. C16:02. C18:13. C18:04. C20:05. C24:16. C22:07. C24:0
UPLC-ESI-MS-MS
Column:100x2.1mm1.7uSolventSystem:A=MeCN:water(60:40v/v)B=2-propanol:MeCN(90:10v/v)10mMNH4OAc+0.1%formicacidBallisJcgradient0.4ml/min@55oC
QQQMSDetector
CeramideExtrac.on
Extrac.on protocols and LC-MS methodsadaptedfromShanerRLetalJLR2009
• Add50mgofliver.ssueto50%aqueousmethanol– Whynotchloroformdirectly?
• HomogenizeinBer.nPrecellysat6500rpmwith~10zirconiumbeadsfor30seconds
CeramideExtrac.on–cont’d• Add1mLofCHCl3:MeOH(2:1,v/v)containing20μlof
C17:0internalstandardsolu.on(use1mMstocksolu.on)– Whyinternalstandardatthispoint?– Shouldweuseglassorplas.c?Doesitma;er?– Whatifyouswapchloroformforhexaneorisopropanol?
• Homogenizeagainandcentrifugeat18,000xgfor10mintoseparatephases
• Transferorganicphasetoanewtube(#2)andrepeatextrac.onoflekovermaterial– Whyrepeat?
CeramideExtrac.on–cont’d
• Combineorganicphasesanddrydowninavacuumcentrifuge
• Solubilizeresidualsin50μlofCHCl3:MeOH(2:1,v/v)– Whychloroformhere?
• Saponifica.onoracidhydrolysisofresidualstoreleaseceramides
CeramideExtrac.on–cont’d
• Incubateresidualswith0.5mLof1MHClinMeOH@50°Cfor1hr(orbaseforsapn)
• Coolsamplesandre-extract
• Solubilizewithin30μlofCHCl3:MeOH(2:1,v/v),sonicatefor5minutesinsonica.ngwaterbath– Whysonicate?
CeramideExtrac.on–cont’d
• Dilute10foldwithacetonitrile:isopropanol:water(1:1:1,v/v)
• Centrifugetoremoveanypar.culatesandtransfertoautosamplertube
0
1 1́ 0 3
2 1́ 0 3
3 1́ 0 3
4 1́ 0 3
E ffe c t o f S o lv e n t S y s te m o n C 1 6 :0 C e ra m id e R e c o v e ry fro m M u r in e L iv e r
C 1 6 :0 C e ra m id e
pe
ak
are
a/m
g l
ive
r ti
ss
ue
C H C l3 :M e O HE tO A cH e x a n e :E tO A cH e x a n e :IP AM tB E
0
1 1́ 0 3
2 1́ 0 3
3 1́ 0 3
4 1́ 0 3
E ffe c t o f S o lv e n t S y s te m o n C 2 4 :0 C e ra m id e R e c o v e ry fro m M u r in e L iv e r
C 2 4 :0 C e ra m id e
pe
ak
are
a/m
g l
ive
r ti
ss
ue
C H C l3 :M e O HE tO A cH e x a n e :E tO A cH e x a n e :IP AM tB E
C 1 6 : 0 C 1 8 : 0 C 1 8 : 1 C 2 0 : 0 C 2 2 : 0 C 2 4 : 0 C 2 4 : 10
1 1́ 0 5
2 1́ 0 5
3 1́ 0 5
4 1́ 0 5
C e ra m id e S a p o n if ic a t io n R x n T e s t in gp
ea
k a
rea
E x tra c t io n
E x tra c t io n + 3 7 o C
E x tra c t io n + N a O H
E x tra c t io n + H C l
Ileum Serum
LipidomicsRevealsCeramidesareDecreasedinFXRIntes.ne-nullMice
OtherClassesofMetabolites
• Wheremightyouhavetroubleextrac.ngeverythingfromapar.cularclassofmetabolites?
• Example:Areallbileacidsthesameintermsofgeneralsolubilityinaqueousororganicsolvents?
MatrixEffects• Challengeswithurine
– –
• Challengeswithblood,serum,orplasma– –
• Challengeswith.ssue– –
Conclusions• Extrac.onprotocolscanimpactmetabolomicdatasets
considerably
• Solventsystemcomposi.onandpHexhibitthemostdrama.ceffectsonmetaboliterecovery
– Themagnitudeoftheseeffectsdependonmetaboliteclass– Someclassesofmetabolites
• Thenumberofextrac.onrepe..onsalsoplaysaroleinenhancingmetaboliterecovery
– Tradeoff-longersampleprep.me– Largersamplevolumestoprocess(evaporate)
Conclusions
• Tradi.onalRPLCmethodscanprovideefficientsepara.onofacyl-carni.ne,bileacidandCoAthioestermixtures.
– Advancementsinhybridpar.cletechnologies– AllowingforextremesinmobilephasepHand
temperature–manipulateselec.vity– Complexligandsta.onaryphaseinterac.ons
• HILICmethodsaresuperioratsepara.nghighlypolarmetabolites.
– Nucleo.desandderiva.ves– Smallpolarmetabolites–sugars,organicacids,amino
acids,hydrophilicvitamins
Conclusions–cont’d
• There’snoone“perfect”extrac.onorLCmethodavailablecapableofefficientlyextrac.ngorresolving,respec.vely,allcomponentsorfeaturesinthemetabolome
• Advancedcolumnchemistries(amide,aminopropyl,biphenyl,graphite,phenyl-hexyl)andalterna.vechromatographicmethodologies(HILIC)canprovideenhancedcoverageofthemetabolome
Acknowledgments
PennStateUniversity• ChrisChiaro• PhilipSmith• JaredCorrell
Na.onalCancerIns.tute• FrankJ.Gonzalez• KrisKrausz• ChangtaoJiang• FeiLi
NIEHSR01ES022186
MRC• JulianGriffin• ElizabethStanley
Metastarswww.metastars.org