Metabolite Extraction and Platforms for Metabolomic … … · · 2017-01-17Metabolite Extraction...
Transcript of Metabolite Extraction and Platforms for Metabolomic … … · · 2017-01-17Metabolite Extraction...
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
OtherClassesofMetabolites
• Wheremightyouhavetroubleextractingeverythingfromaparticularclassofmetabolites?
• Example:Areallbileacidsthesameintermsofgeneralsolubilityinaqueousororganicsolvents?
MatrixEffects• Challengeswithurine––
• Challengeswithblood,serum,orplasma––
• Challengeswithtissue––
NucleicAcidExtraction
Whatdoweknowaboutourtargetanalyte?
• Negativelychargedphosphatebackbone(polar)• Needtoremoveproteins,lipids,etc
ProteinChemistry
https://en.wikipedia.org/wiki/Proteinogenic_amino_acid
Denatureproteins
• Hydrophobicaminoacidsfacephenol:chloroform
• Phe,Tyr,Leu
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)
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
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
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
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