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