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 Same nominal mass 3 different exact masses Low resolution High resolution

249 249.0700 249.0580 249.1479 MassMass AnalyzersAnalyzers Resolving Mass Power Accuracy •Ion Cyclotron (FT-ICR-MS) 200,000 <1ppm

•Time of Flight 20,000 3-10ppm (TOF)

•Magnetic Sector 60,000 2-5ppm

•Quadrupole Ion Trap 1,000 n/a

•Quadrupole 1,000 n/a MassMass Accuracy-Accuracy- FFTMSTMS - Instrument Calibration - ExactExact ExperimentalExperimental DifferenceDifference CalibrantCalibrant MassMass MassMass ((ppmppm)) AlanineAlanine 90.0590.055050 90.0590.0549984998 0.01750.0175 LysineLysine 147.11147.112828 147.11147.1127832783 0.11730.1173 GlutamateGlutamate 148.06148.060404 148.06148.0604200420 0.13530.1353 MethionineMethionine 150.05150.058383 150.05150.0583178317 0.11090.1109 TyrosineTyrosine 182.08182.081212 182.08182.0811841184 0.08660.0866 TryptophanTryptophan 205.09205.097272 205.09205.0971927192 0.03800.0380 MassMass AccuracyAccuracy DeterminingDetermining EmpiricalEmpirical Formula,Formula, StructuralStructural ElucidationElucidation 150.058317 Methionine

C5H12NO2S 0.06 ppm

# 12C 1H 16O 14N 31P 32S 23Na 39K mass error

1 5 12 2 1 0 1 0 0 150.0583257 5.826e-08 2 1 15 2 2 1 1 0 0 150.0586364 2.128e-06 3 3 15 0 2 0 1 0 1 150.0587525 2.902e-06 4 9 11 0 0 1 0 0 0 150.0592883 6.473e-06 5 3 15 0 1 2 0 1 0 150.0571936 7.486e-06 6 5 14 0 1 2 0 0 0 150.0595989 8.543e-06 7 4 16 1 0 1 0 0 1 150.0570349 8.544e-06 8 3 10 1 4 0 1 0 0 150.0569831 8.889e-06 9 2 16 3 0 2 0 0 0 150.0569188 9.318e-06 Mass Accuracy

Ability of a mass analyzer to assign the mass of an ion close to its true value (exact mass)

∆m accuracy = mreal - mmeasured ∆m accuracy

In ppm = 106 * ∆m accuracy / mmeasured Mass Accuracy

High mass accuracy (exact mass measurement) is usually associated to high resolution analyzers

Unknown compound determination Exact mass helps to define its atomic composition ScanScan SpeedSpeed (or(or rate)rate)

•• The rate at which we can acquire a mass spectrum, (mass units/sec).

•• Is an essential acquisition parameter for MS

•• Will affect the amount ofof informationinformation (qualitative(qualitative andand quantitative) that can reasonably be attained with a given mass analyzer. MassMass AnalyzersAnalyzers

Analyzer Range Accuracy Resolution Speed

amu amu amu/s

Quad < 4000 0.1 1000-2000 4000

Ion Trap < 20 000 0.1 1000-2000 4000

TOF 1 000 000 0.0001 500-10 000 1 000 000 NextNext ClassClass

DataData afterafter ionion detectiondetection inin LCLC--MSMS