MetaboliteExtractionandPlatformsforMetabolomicStudies

AndrewD.Patterson,PhDAssociateProfessorofMolecularToxicologyPennStateUniversityadp117@psu.edu

Resources• MetabolomicsWorkbench

– www.metabolomicsworkbench.org– Largeresourceofexperimentalprotocols,datasets,andotherresources

• XCMSInstitute– https://xcmsonline.scripps.edu/institute– Greattutorialsonchromatography,platforms,databases

• MetabolomicsSocietyForums– http://www.metabolomics-forum.com/

• Twitter– #metabolomics

ObjectivesAttheconclusionofthislesson,studentswillbeableto:

• Definefactorsthatinfluencemetaboliteextractionanddescribetheirimpactonmetabolomicstudies

• Explainthevalueoforthogonalapproachesforimprovedmetaboliteidentificationandquantitation

Whichpartsofthemetabolomicprocessmightinfluenceyourdata?

OtherClassesofMetabolites

• Wheremightyouhavetroubleextractingeverythingfromaparticularclassofmetabolites?

• Example:Areallbileacidsthesameintermsofgeneralsolubilityinaqueousororganicsolvents?

MatrixEffects• Challengeswithurine––

• Challengeswithblood,serum,orplasma––

• Challengeswithtissue––

ExtractionofMetabolites

NucleicAcidExtraction

Whatdoweknowaboutourtargetanalyte?

• Negativelychargedphosphatebackbone(polar)• Needtoremoveproteins,lipids,etc

AboutSolventsOH

PHENOL

1.07g/cm3

OH H

WATER

1.00g/cm3

Cl

Cl

HCl

CHLOROFORM

1.49g/cm3

POLARITY

ProteinChemistry

https://en.wikipedia.org/wiki/Proteinogenic_amino_acid

Denatureproteins

• Hydrophobicaminoacidsfacephenol:chloroform

• Phe,Tyr,Leu

LiquidLiquidExtraction

DNAorRNA(aqueous)

Protein

Lipids(phenol:chloroform)

DNAExtractionProtocol• Disrupttissuein

phenol:chloroform

**chloroformpreventssmallamountsofwaterinphenolfromdissolvingmRNA**adjustpHtofavorDNA(basic)orRNA(acidic)isolation

• Centrifugetoseparatelayers• Dehydratewithalcohol

Whatcouldgowrong?

• Solventsnotappropriateorpreparedincorrectly– pHincorrect– Ratiosincorrect

• Contaminationofsolventsorbuffers

• Whatelsecanyouthinkof?

StandardOperatingProcedures

• Savestimeandpreventsmistakes• Consistentresults

• Checkinginsamples(samplelists,location)• Labelingandstoringsamples(aliquot)• Metaboliteextraction(targetedorglobal)• Acquiringdataonvariousplatforms(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

• Relativelyinexpensivecomparedtoacetonitrile

• Notregulatedlikeethanol• Easytoevaporate

• Extractspolarand(some)non-polarmolecules– why?

Acetonitrile

• Advantagesmostlyforchromatography– ReducedabsorbanceforUVbasedmethods– Reducedpressurecomparedtomethanol– Greaterelutionstrength(generally)– HILICapplications

• Expensive– Isolatedasabyproductnotproduceddirectly– Shortagescaninfluencepriceandavailability

Chloroformvs MTBE

• Chloroformdensitiy – 1.49g/cm3

• MTBEdensity– 0.740g/cm3

• Toxicityofchloroform(checkoutATSDR.CDC.GOV)

• Stillmadeneedtotailorspecificextractionsforlipidclasses(hexaneforTAGsorMTBEforCer)– Moredetailcheckoutcyberlipid.org orlipidmaps.org

SOWHAT?

Metabolomics

Metabolomicsisthesystematicanalysisoftheuniquechemicalfingerprintsleftbehindbyspecificcellularprocesses

FOOD

PESTICIDES

COSMETICS

POLLUTANTS

DRUGS

XENOBIOT

IC

METAB

OLISM

ENDO

GENOUS

METAB

OLISMMETABOLOME

GUTMICROBIOTA

MetabolomicsAll “-omics” based scientific disciplines aim at the collectivecharacterization and measurement of their particularconstituent molecules

• A comprehensive approach to study complete pools ofbiological molecules

• Defines the structure, function and dynamics of anorganism

MetabolomicsVast chemical diversity among small molecule metaboliteshas made extended coverage of the metabolomechallenging

• Size (50 – 1500 Da)• Concentration ( pM – mM)• Physicochemical properties (diverse log P values)• Stereochemistry (distinct biological activity)

EHP2014Vol122Number8http://ehp.niehs.nih.gov/1308015/

http://journal.frontiersin.org/article/10.3389/fimmu.2016.00044/full

MetaboliteExtraction• Currently no analytical technique exists that is capable of

measurement of all classes of cellular metabolites

• Metabolite extraction is a crucial step in any metabolomics study– Critical to both targeted and global based profiling strategies

• Optimized extraction methodology should fulfill several criteria:– Extract the largest number of metabolites– Unbiased and non-selective - physical or chemical properties

of a molecule– Non-destructive - no modification of metabolites

organicphase

aqueousphase

KD

HAorg

HAaq+H20 H30+ +A-Ka

organicphase

aqueousphase

KD

HAorg

HAaq

SeparationofMetabolites• Massspectrometryusuallyrequiressomeformofchromatographicseparation

• Mostsystemsuseeitherliquidorgaschromatography

• Fractionationofsamplecomponentssimplifiestheresultingmassspectrawhileensuring moreaccuratecompoundidentification

• Capacityfactor(k)iscriticaltooptimizingresolution• IncreasedresolutionallowslongerMSdwelltimesresultinginbettersignal/noiseratios

• Inadequatechromatographicseparationofmetabolitesresultsin:• signalsuppression– ionsuppression• compromisedmetabolitequantification• reducedmetabolitecoverage

CeramidePhysicochemicalProperties

• Ceramidesareafamilyofwaxylipidmolecules.– Namederivedfromthelatin word:cera =waxy+amide

• Ceramidesarecomprisedof:– sphingosine:18carbonunsaturatedaminoalcohol– fattyacidmoiety – amidebond

• Ceramidesarenotwatersoluble:– Veryhydrophobic– Confinedtocellularmembranes– Participateinlipidraftformation– >200structurallydistinctspecieshavebeenidentifiedin

mammaliancells

CeramideGeneralStructure

• Ceramide(d18:1/16:0)

• 2-amino-1,3-octadec-4-ene-diol• Aminoalcohol(sphingoid)backbone

• Palmitic acid• Fattyacylgroup

• Ceramide(d18:1/24:1(15Z))• 2-amino-1,3-octadec-4-ene-diol

• Aminoalcohol(sphingoid)backbone

• 15-tetracosenoicacid• Fattyacylgroup

Mol. BioSyst., 2015, 11, 698--713 | 699

StructuresandNomenclature

CeramideBiosynthesis

Front.Endocrinol.,30July2014| https://doi.org/10.3389/fendo.2014.00127

CeramideBiochemistry

Ceramidesarefoundinhighconcentrationinthemembraneofcells

• Structuralcomponentofthelipidbilayer

• Bioactivelipid- implicatedinavarietyofphysiologicalfunctionsincluding:

• Apoptosisandcellgrowtharrest• differentiationandcellsenescence• cellmigrationandadhesion

Ceramides areconvertedrapidlytomorecomplexsphingolipids:

• Sphingomyelin• Glycosylceramides• Littleaccumulationobserved

– Exceptfortheskin(50%oftotallipidscanbeceramides)

BiosynthesisofCeramides

Denovobiosynthesis

• Ceramidesynthasescouplesphinganine +longchainfattyacidtoform

dihydroceramide

• Doublebondintroducedintoposition4ofthesphingoid base

§ ceramidesynthases5and6generatearespecificforpalmitic acid

§ ceramidesynthases1(brainandskeletalmuscle)specificforstearicacid§ ceramidesynthases2specificforverylongchainCoA-thioesters(C20-C26)

§ ceramidesynthases3unusualceramidesofskin&testes

BiosynthesisofCeramides

Catabolismofcomplexsphingolipids:

• Sphingomyelinases/phospholipaseCbreakdownsphingomyelin in

animaltissues

• Manyfactorscanstimulatethehydrolysisofsphingomyelin to

produceceramide:

§ Cytokines:TNF-a,IFN-g&variousinterleukins§ 1,25-dihydroxy-vitaminD3

§ endotoxin§ nervegrowthfactor§ ionizingradiation&heat

LCMethodDevelopmentWheretoStart?

• DesigningandoptimizinganLCmethodinvolveschoosingappropriate:

1. Separationmechanism:NPC,RPLC,HILIC,sizeexclusionion,exchangeetc

2. Columnchemistry:C2,C4,C8,C18,cyanopropyl,phenyl,biphenyl,amide,SiOH etc

3. Columnproperties:poresize,particlesize&columndimensions

4. Stationaryandmobilephasecombinations

• Criticaltooptimizingthechromatographicefficiency,retention,resolution&selectivityofanalytes

CeramideScoutingGradientsonWatersBEHC18

Steepgradient;largechangeinorganicmodifier.

Providesinitialinformationaboutanalyteretention&resolution.

AidsinexpeditingthedevelopmentofanoptimizedLCmethod

PoorResolution(R)LongRetention(Tr)Time

1. C16:02. C18:13. C18:04. C20:05. C24:16. C22:07. C24:0

1 2 3 4 75 6

FractionationofCeramideMetabolitesonWatersCSHC18Column

Column:100x2.1mm1.7uSolventSystem:A=MeCN:water (60:40v/v)B=2-propanol:MeCN(90:10v/v)10mMNH4OAc+0.1%formicacidBallisticgradient0.4ml/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

BallisticGradient:

• Ultra-fastgradient

• Rapidchangeinorganicmodifier

• Separationsperformedathighflowrates

• Highlinearvelocity

FractionationofCeramideMetabolitesonWatersCSHC18Column

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%formicacidBallisticgradient0.4ml/min@55oC

QQQMSDetector

CeramideExtraction

Extraction protocols and LC-MS methodsadapted from Shaner RL et al JLR 2009

• Add50mgoflivertissueto50%aqueousmethanol–Whynotchloroformdirectly?

• HomogenizeinBertin Precellys at6500rpmwith~10zirconiumbeadsfor30seconds

CeramideExtraction– cont’d• Add1mLofCHCl3:MeOH(2:1,v/v)containing20μl of

C17:0internalstandardsolution(use1mM stocksolution)– Whyinternalstandardatthispoint?– Shouldweuseglassorplastic?Doesitmatter?– Whatifyouswapchloroformforhexaneorisopropanol?

• Homogenizeagainandcentrifugeat18,000xgfor10mintoseparatephases

• Transferorganicphasetoanewtube(#2)andrepeatextractionofleftovermaterial– Whyrepeat?

CeramideExtraction– cont’d

• Combineorganicphasesanddrydowninavacuumcentrifuge

• Solubilizeresidualsin50μl ofCHCl3:MeOH(2:1,v/v)–Whychloroformhere?

• Saponificationoracidhydrolysisofresidualstoreleaseceramides

CeramideExtraction– cont’d

• Incubateresidualswith0.5mLof1MHCl inMeOH @50°Cfor1hr (orbaseforsapn)

• Coolsamplesandre-extract

• Solubilizewithin30μl ofCHCl3:MeOH(2:1,v/v),sonicate for5minutesinsonicatingwaterbath–Whysonicate?

CeramideExtraction– cont’d

• Dilute10foldwithacetonitrile:isopropanol:water (1:1:1,v/v)

• Centrifugetoremoveanyparticulatesandtransfertoautosampler tube

0

1´1 0 3

2´1 0 3

3´1 0 3

4´1 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´1 0 3

2´1 0 3

3´1 0 3

4´1 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

Ileum Serum

Lipidomics RevealsCeramides areDecreasedinFXRIntestine-nullMice

Conclusions• Extractionprotocolscanimpactmetabolomicdata

setsconsiderably

• SolventsystemcompositionandpHexhibitthemostdramaticeffectsonmetaboliterecovery– Themagnitudeoftheseeffectsdependonmetabolite

class– Someclassesofmetabolites

• Thenumberofextractionrepetitionsalsoplaysaroleinenhancingmetaboliterecovery– Tradeoff- longersamplepreptime– Largersamplevolumestoprocess(evaporate)

Conclusions

• TraditionalRPLCmethodscanprovideefficientseparationofacyl-carnitine,bileacidandCoAthioester mixtures.– Advancementsinhybridparticletechnologies– AllowingforextremesinmobilephasepHand

temperature– manipulateselectivity– Complexligandstationaryphaseinteractions

• HILICmethodsaresuperioratseparatinghighlypolarmetabolites.– Nucleotidesandderivatives– Smallpolarmetabolites– sugars,organicacids,amino

acids,hydrophilicvitamins

Conclusions– cont’d

• There’snoone“perfect”extractionorLCmethodavailablecapableofefficientlyextractingorresolving,respectively,allcomponentsorfeaturesinthemetabolome

• Advancedcolumnchemistries(amide,aminopropyl,biphenyl,graphite,phenyl-hexyl)andalternativechromatographicmethodologies(HILIC)canprovideenhancedcoverageofthemetabolome

• Differentplatformscanprovidegreaterconfidenceinmetabolitemeasurement

Acknowledgments

PennStateUniversity• ChrisChiaro• PhilipSmith• JaredCorrell• Jingwei Cai• JingtaoZhang

NationalCancerInstitute• FrankJ.Gonzalez• KrisKrausz• Changtao Jiang• Fei Li

NIEHSR01ES022186

MRC• JulianGriffin• ElizabethStanley