LC-MS Based Metabolomics AnalysingAnalysing thethe METABOLOMEMETABOLOME 1.1. MetaboliteMetabolite ExtractionExtraction 2.2. MetaboliteMetabolite detectiondetection (with(with oror withoutwithout separation)separation) 3.3. DataData analysisanalysis MetaboliteMetabolite DetectionDetection GC-MS: Naturally volatile or made volatile (any organic- flavors, sugars, lipids, acids) NMR – any compound containing hydrogen HP Liquid - Chromatography + detector Comon detectors- - UV-detector (phenolics) - MASS SPECTROMETER (MS) as detector (LC-MS) MetaboliteMetabolite DetectionDetection MASS SPECTROMETER (MS) as detector (LC-MS): Compounds that are not well characterized by other methods: Non volatile High molecular weight Too sensitive to heat to be analyzed by GC Your Result Sample LC/MS Sample Sample Efficient Separation Preparation Introduction Gradient (column) Data (computer) LC- MS Ions Ions Ionization UV Detection Separation MS Interface Spectra Today ComponentsComponents inin LCLC--MSMS Ion Ion Formation Sorting PeptiPeptiddee && ProteinProtein SeqSequuenciencinngg Compound ID APcI Analyzer Software LC Structure Elucidation ESI & Detector Quantitation Interface: Chemical Triple Quadrupole Atmospheric Results Separation Quadrupole -Time Of Flight Pressure Quadrupole - Ion-Trap Ionization FT-MS MassMass SpectrometerSpectrometer 1. Breaks up constituents into molecular ions and other fragments 2. The ions then pass through an electric and/or magnetic field that separates them according to their mass-to-charge ratio (m/z) 3. Measures masses MassMass SpectrometerSpectrometer 4. Universal detection method * compared to UV/VIS (PDA), fluorescence etc. * more specific than NMR 5. More sensitive for most compounds 6. Structural information on metabolite * fragmentation pattern * accurate mass 7. For both LC and GC TechnologyTechnology ofof LCLC--MSMS andand LCLC--MSMS--MSMS –– InterfacesInterfaces-- IonizationIonization (elimination(elimination ofof solventsolvent andand generationgeneration ofof gasgas--phasephase ions)ions) –– e.g.e.g. ZZ SpraySpray –– AnalyzersAnalyzers –– QuadrupolesQuadrupoles (Q)(Q) andand TimeTime ofof FlightFlight (TOF)(TOF) LCLC--MSMS InterfacesInterfaces InIn MSMS-- ** MeasuringMeasuring thethe massmass ofof aa hugehuge varietyvariety ofof compounds,compounds, inin aa hugehuge varietyvariety ofof matricesmatrices ** NeedNeed rangerange ofof methodsmethods toto IONISEIONISE allall thethe differentdifferent compoundscompounds AlternativeAlternative IonizationIonization ModesModes AlternativeAlternative IonizationIonization ModesModes • EI or CI, Electron (impact) OR Chemical Ionization (in GC-MS) • Gas-phase ionization methods • Small volatile molecules are heated and enter the gas phase Not always suitable: • Difficult to get large or involatile molecules into the gas phase • Laser desorption • Matrix-assisted laser desorption ionization (MALDI) • Particle bombardment • Fast atomic bombardment (FAB) • Secondary ion mass spectrometry (SIMS) • Field desorption Ionization AlternativeAlternative IonizationIonization ModesModes • EI or CI, Electron (impact) OR Chemical Ionization (in GC-MS) • Gas-phase ionization methods • Small volatile molecules are heated and enter the gas phase Not always suitable: • Difficult to get large or involatile molecules into the gas phase • Heating the non-volatile molecules degrades them AlternativeAlternative IonizationIonization ModesModes Ionization for Non-Volatiles: Early ones- • Particle bombardment • Fast atomic bombardment (FAB) • Secondary ion mass spectrometry (SIMS) • Field desorption Ionization • Thermospray ionization AlternativeAlternative IonizationIonization ModesModes Ionization for Non-Volatiles: Early ones- • Particle bombardment - • Fast atomic bombardment (FAB) • Secondary ion mass spectrometry (SIMS) • single experiments, background signal from matrix • • Field desorption - complex, single experiments at once • Thermospray - temprature degrades sample AlternativeAlternative IonizationIonization ModesModes •Atmospheric Pressure Ionization (API), in LC-MS • Electrospray Ionisation (ESI): polar and semi-polar • Atmospheric Pressure Chemical Ionization (APCI): less polar APCI ES lipids water polarity of analyte molecule AtmosphericAtmospheric PressurePressure IonisationIonisation (API)(API) TechniquesTechniques ESIESI andand APCIAPCI differdiffer in…in… •• HowHow ionsions areare generatedgenerated ••ESIESI -- ssolutionolution phasephase ionizationionization ••APCIAPCI -- gasgas phasephase ionizationionization •• AnalyteAnalyte compatibilitycompatibility ••ESIESI -- ppolarolar compoundscompounds andand largelarge biomoleculesbiomolecules ••APCIAPCI -- llessess polar,polar, smallersmaller compoundscompounds (relative(relative toto thosethose ionizedionized byby ESI)ESI) thatthat havehave somesome volatilityvolatility •• FlowFlow raterate compatibilitycompatibility ••ESIESI -- 00.001.001 toto 11 mL/minmL/min ••APCIAPCI -- 00.2.2 toto 22 mL/minmL/min IonizationIonization MethodsMethods •Electrospray (ESI) •Atmospheric Pressure Chemical Ionization (APCI) “Soft” •Laser Desorption (MALDI) Ionization •Chemical Ionization ESI, APCI and MALDI can be used with LC •Fast Atom Bombardment (FAB or SIMS) “Hard” •Electron Impact Ionization EI ionization can be used with GC HowHow dodo thethe analytesanalytes becomebecome charged?charged? -- WhileWhile inin EI,EI, lossloss ofof anan electronelectron producingproducing aa radicalradical molecularmolecular ionion -- InIn softsoft ionisationionisation techniquestechniques,, analyteanalyte moleculesmolecules are:are: protonatedprotonated [M[M ++ H]H]+ or:or: dede--protenoatedprotenoated [M[M -- H]H]- -- CouldCould alsoalso bebe sodiated,sodiated, potassiatedpotassiated etc..etc.. (adducts)(adducts) IonIon FormationFormation inin ESIESI High positive or High negative charge HowHow dodo thethe analytesanalytes becomebecome charged?charged? High postive or negative charge Reppeled positive (or negative) ions PositivePositive oror NegativeNegative Modes?Modes? TheThe formationformation ofof positivepositive oror negativenegative ionsions dependsdepends onon thethe signsign ofof thethe appliedapplied electricalelectrical fieldfield ES+:ES+: (M+H)(M+H)++ GoodGood ionizationionization ofof basicbasic ccompoundsompounds (get(get proton)proton) E.g.E.g. amino,amino, amide,amide, ester,ester, aldehyde/ketoaldehyde/keto functionalfunctional groupsgroups (formic(formic acidacid inin samplesample solutionsolution toto helphelp ionize)ionize) ES-:ES-: (M-H)(M-H)-- AcidicAcidic CompoundsCompounds (give(give proton)proton) E.g.E.g. organicorganic acids,acids, containingcontaining OHOH (ammonium(ammonium bufferbuffer inin samplesample solutionsolution toto helphelp ionize)ionize) SolventSolvent LossLoss inin ESIESI && IonIon FormationFormation High postive or negative charge ElectrosprayElectrospray IonizationIonization ESIESI ElectrosprayElectrospray TheoryTheory SummarySummary ESIESI ESIESI isis anan atmosphericatmospheric pressurepressure ionion sourcesource SmallSmall moleculesmolecules singlysingly chragedchraged HighHigh MWMW samplessamples becomebecome multiplymultiply chareschares (e.g.(e.g. proteins)proteins) MWsMWs ofof 150,000150,000 DaDa (amu)(amu) cabcab bebe measuredmeasured accuratelyaccurately AtmosphericAtmospheric PressurePressure ChemicalChemical IonizationIonization -- APCIAPCI AtmosphericAtmospheric PressurePressure IonizationIonization InterfaceInterface APcIAPcI Ionization of solvent APcI & solvent transfers the charge to analyte SourceSource Sample is vaporised APcIAPcI TheoryTheory AtmosphericAtmospheric PressurePressure ChemicalChemical IonisationIonisation ((APcI)APcI) LowLow molecularmolecular weightweight (<1000(<1000 Da)Da) SinglySingly chargedcharged speciesspecies ESIESI vsvs APcIAPcI Technique Flow Rate MW Range Species (ml/min) Produced ESI 0.001 – 0.3 <200,000 Da (M+H)+ (M-H)- (M+nH)n+ APcI 0.2 – 2.0 <1000 Da (M+H)+ (M-H)- TMTM ZZ SPRAYSPRAY SourceSource What happens from here? ZZ--SpraySpray InterfaceInterface MassMass AnalyzersAnalyzers IonIon SortingSorting ComponentsComponents inin LCLC--MSMS Ion Ion Formation Sorting PeptiPeptiddee && ProteinProtein SeqSequuenciencinngg Compound ID APcI Analyzer Software LC Structure Elucidation ESI & Detector Quantitation Interface: Triple Quadrupole Chemical Atmospheric Quadrupole -Time Of Flight Results Separation Pressure Quadrupole - Ion-Trap Ionization FT-MS QuadrupoleQuadrupole andand TandemTandem QuadrupoleQuadrupole IonIon SeparationSeparation AnalyzersAnalyzers QuadrupoleQuadrupole TheoryTheory BenefitsBenefits ofof TimeTime--OfOf--FlightFlight MSMS highhigh massmass resolutionresolution (up(up toto 1010 oror 55 ppmppm)) exactexact massmass Resolution & Accuracy of a Mass-spectrometer ResolutionResolution Resolution, (or Resolving Power)of a mass sectrometer: A measure of its ability to separate adjacent ions At higher resolution, small differences may be detected. Determining Resolution Single Ion method Double Ion method Full Width at Half Maximum ∆mr 2 adjacent ion peaks (50%, FWHM) with a 10% valley max or at 5% of the peak height mave m R = R = ∆m ∆mr MassMass AnalyzersAnalyzers •Ion Cyclotron (FT-ICR-MS) “High Resolution” •Time of Flight Instruments (TOF) •Magnetic Sector •Quadrupole Ion Trap “Low Resolution” •Quadrupole Instruments HighHigh ResolutionResolution vs.vs. LowLow ResolutionResolution 129 131 130 Low Resolution High Resolution ResolutionResolution C20H9+ C19H7N+ C H N O + 13 19 3 2 C20H9+C19H7N+ C13H19N3O2+ 3 different compounds 3 different compounds