Introduction to Walk-Up Introduction to Walk-Up Mass SpectrometryMass Spectrometry

Jonathan A. Karty, Ph.D.Jonathan A. Karty, Ph.D.

July 21, 2008July 21, 2008

Topics CoveredTopics Covered

Introduction to MS and the MSFIntroduction to MS and the MSFMolecular Weight and Isotope Molecular Weight and Isotope

DistributionsDistributionsAccuracy and ResolutionAccuracy and ResolutionSources for Walk-Up MSSources for Walk-Up MSMass Analyzers for Walk-Up MSMass Analyzers for Walk-Up MSUpcoming Application SeminarsUpcoming Application Seminars

Mass Spectrometry FacilityMass Spectrometry Facility Located in A411Located in A411 Staffed from 9:30-5:30, M-F except holidaysStaffed from 9:30-5:30, M-F except holidays Staff includes:Staff includes:

Jonathan A. Karty, Ph.D. (Jon), facility managerJonathan A. Karty, Ph.D. (Jon), facility manager Angela M. Hansen (Angie), Sr. Mass SpectrometristAngela M. Hansen (Angie), Sr. Mass Spectrometrist Undergraduate technicians for 2008-2009Undergraduate technicians for 2008-2009

Derek ZipkinDerek Zipkin LaDasa JonesLaDasa Jones

Instruments for walk-up useInstruments for walk-up use Agilent 6890/5973 GC-MSAgilent 6890/5973 GC-MS Bruker Biflex III MALDI-TOFBruker Biflex III MALDI-TOF 2 Waters LCT Classic ESI-TOF2 Waters LCT Classic ESI-TOF 1 Agilent ESI-Quadrupole (coming soon?!?)1 Agilent ESI-Quadrupole (coming soon?!?)

Why Mass SpectrometryWhy Mass Spectrometry

Information is composition-specificInformation is composition-specific Very selective analytical techniqueVery selective analytical technique Most other spectroscopies can describe Most other spectroscopies can describe

functionality present, but not absolute formulafunctionality present, but not absolute formula

MS is VERY sensitiveMS is VERY sensitive MSF personnel dilute NMR samples 1:500MSF personnel dilute NMR samples 1:500 Picomole sensitivity is common in the MSFPicomole sensitivity is common in the MSF

Mass spectrometers have become MUCH Mass spectrometers have become MUCH easier to use in the last 15 yearseasier to use in the last 15 years

Three QuestionsThree Questions Did I make my compound?Did I make my compound?

Molecular weight is an intrinsic property of a Molecular weight is an intrinsic property of a substancesubstance

Molecular weight can therefore confirm identityMolecular weight can therefore confirm identity

Did I make anything else?Did I make anything else? Mass spectrometry is readily coupled to Mass spectrometry is readily coupled to

chromatographic techniqueschromatographic techniques Not all compounds ionize easily (cf. UV-VIS)Not all compounds ionize easily (cf. UV-VIS)

How much of it did I make?How much of it did I make? Response in the mass spectrometer is proportional to Response in the mass spectrometer is proportional to

analyte concentration (R = analyte concentration (R = αα[M])[M])Each compound has a unique response factor, Each compound has a unique response factor, αα

Common MS ApplicationsCommon MS Applications

Quick product identification (TLC plate)Quick product identification (TLC plate)Confirmation of elemental compositionConfirmation of elemental composition

Much more precise then EAMuch more precise then EASelective detector for GC/HPLCSelective detector for GC/HPLC

MS provides molecular weight information MS provides molecular weight information about each chromatographic peakabout each chromatographic peak

Reaction monitoringReaction monitoringCrude reaction mixture MSCrude reaction mixture MSStable isotope labelingStable isotope labelingStability studiesStability studies

Mass Spectrometer ComponentsMass Spectrometer Components

InletInlet Get samples into the instrumentGet samples into the instrument

SourceSource Ionize the molecules in a useful wayIonize the molecules in a useful way

Mass AnalyzerMass Analyzer Separates the ions by mass to charge (m/z) ratioSeparates the ions by mass to charge (m/z) ratio

DetectorDetector Converts ions into electronic signal or photonsConverts ions into electronic signal or photons

Data systemData system Photographic plates to computer clustersPhotographic plates to computer clusters

Important Concepts to RememberImportant Concepts to Remember Mass spectrometers analyze gas-phase ions, not Mass spectrometers analyze gas-phase ions, not

neutral moleculesneutral molecules Neutrals don’t respond to electric and magnetic fieldsNeutrals don’t respond to electric and magnetic fields If your molecule cannot ionize, MS cannot helpIf your molecule cannot ionize, MS cannot help

MS is not a “magic bullet” techniqueMS is not a “magic bullet” technique MS can describe atomic composition of an ionMS can describe atomic composition of an ion Connectivity of the atoms is much more challengingConnectivity of the atoms is much more challenging

Although MS requires a vacuum, it cannot be Although MS requires a vacuum, it cannot be performed in a vacuum of informationperformed in a vacuum of information Deriving useful information from MS data often requires Deriving useful information from MS data often requires

some foreknowledge of the system under investigationsome foreknowledge of the system under investigation

Molecular Weight CalculationsMolecular Weight Calculations

The molecular weight of a compound is The molecular weight of a compound is computed by summing the masses of all computed by summing the masses of all atoms that comprise the compound.atoms that comprise the compound.Morphine: CMorphine: C1717HH1919NONO33 = 12.011(17) = 12.011(17)

+1.008(19)+ 14.007 + 15.999(3) = 285.34 Da+1.008(19)+ 14.007 + 15.999(3) = 285.34 DaYet this is not the mass we observeYet this is not the mass we observe

285.136 is observed by EI-MS285.136 is observed by EI-MSMolecular weight is calculated assuming a Molecular weight is calculated assuming a

natural distribution of isotopesnatural distribution of isotopes

Monoisotopic vs. Average MassesMonoisotopic vs. Average Masses Most elements have a variety of isotopesMost elements have a variety of isotopes

C C 1212C is 98.9% abundant, C is 98.9% abundant, 1313C is 1.1% abundantC is 1.1% abundant For CFor C2020, 80% chance , 80% chance 1313CC00, 18% chance , 18% chance 1313CC11, 2% chance , 2% chance 1313CC22

Sn has 7 naturally occurring isotopes @ >5% ab.Sn has 7 naturally occurring isotopes @ >5% ab. F, P, Na, Al, Co, I, Au have only 1 natural isotopeF, P, Na, Al, Co, I, Au have only 1 natural isotope

Mass spectrometers can often resolve these Mass spectrometers can often resolve these isotopic distributionsisotopic distributions

Monoisotopic masses must be considered Monoisotopic masses must be considered Monoisotopic masses for multi-isotope species are Monoisotopic masses for multi-isotope species are

computed using most intense isotopes of all elements computed using most intense isotopes of all elements ((1212C, C, 11H, H, 3535Cl, Cl, 3232S, S, 7979Br, Br, 5858Ni)Ni)

For morphine, monoisotopic mass = 285.1365For morphine, monoisotopic mass = 285.1365 12.0000(17) + 1.0078(19) + 14.0031 + 15.9949(3)12.0000(17) + 1.0078(19) + 14.0031 + 15.9949(3)

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M ass [amu]285 286 287 288 289 290

CC1717HH1919NONO33 Mass Spectrum Mass Spectrum

13C0, 15N0

13C1

or 15N1 13C2 or13C1+15N1

or 15N2

Isotopic EnvelopesIsotopic EnvelopesMass spectrometers measure ion populationsMass spectrometers measure ion populations

Any single ion only has 1 isotopic compositionAny single ion only has 1 isotopic composition101022 – 10 – 1066 or more ions in a reliable peak or more ions in a reliable peak

The observed mass spectrum represents the The observed mass spectrum represents the sum of all those different compositionssum of all those different compositions

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Mass [amu]285 286 287 288 289 290

“M+ peak”

“M+1 peak”

“M+2 peak”

Isotopic Envelopes 2Isotopic Envelopes 2 Isotope envelopes can be used to preclude Isotope envelopes can be used to preclude

some elements from ionic compositionssome elements from ionic compositionsLack of intense M+2 peak precludes Cl or BrLack of intense M+2 peak precludes Cl or BrMany metals have unique isotopic signaturesMany metals have unique isotopic signatures

M+1/M+ ratio can be used to count carbonsM+1/M+ ratio can be used to count carbons [(M+1)/M+]/0.011 [(M+1)/M+]/0.011 ≈ # carbon atoms≈ # carbon atomsFor morphine: (0.1901/1)/0.011 = 17.28 For morphine: (0.1901/1)/0.011 = 17.28 17 17

Isotope table can be found on NIST websiteIsotope table can be found on NIST websiteLink from MSF “Useful Information” pageLink from MSF “Useful Information” page

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Mass [amu]1759 1760 1761 1762 1763 1764 1765

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Mass [amu]362 364 366 368 370 372 374 376 378

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Mass [amu]131 132 133 134 135 136 137 138 139

A few isotope patternsA few isotope patterns

C2H3Cl3trichloroethane

C12H27SnBrtributyltin bromide

C83H122N24O19

A 14-mer peptide

A little more on molecular ionsA little more on molecular ionsBe aware of ionization mechanismBe aware of ionization mechanism

EI, LDI, and CI generate radical cationsEI, LDI, and CI generate radical cationsMM++•• is an odd electron ionis an odd electron ionNitrogen rule is normalNitrogen rule is normal

Even parent ion mass implies even # of N atomsEven parent ion mass implies even # of N atoms M+ for morphine by EI is 285.136, odd # N (1)M+ for morphine by EI is 285.136, odd # N (1)

ESI, MALDI, and CI generate cation adductsESI, MALDI, and CI generate cation adductsM+H and M+Na are even electron ionsM+H and M+Na are even electron ionsNitrogen rule is inverted for odd mass cationsNitrogen rule is inverted for odd mass cations

Even parent ion mass implies odd # of N atomsEven parent ion mass implies odd # of N atoms M+Na for morphine by ESI is 308.126, odd # N (1)M+Na for morphine by ESI is 308.126, odd # N (1)

Metal atoms and pre-existing ions or radicals Metal atoms and pre-existing ions or radicals can alter observationscan alter observations

Charge State DeterminationCharge State Determination

Mass spectrometrists use 2 units of massMass spectrometrists use 2 units of mass Dalton Dalton 1 Da = 1 amu (1/12 of a 1 Da = 1 amu (1/12 of a 1212C atom)C atom) Thompson Thompson 1 Th = 1 Da/z (z is electron charge) 1 Th = 1 Da/z (z is electron charge)

Thompson is more correct when referring to data Thompson is more correct when referring to data from a mass spectrumfrom a mass spectrum For a +1 ion, m/z in Th For a +1 ion, m/z in Th ≈≈ mass in Da mass in Da

High molecular weight ions generated by ESI High molecular weight ions generated by ESI and MALDI often carry more than one chargeand MALDI often carry more than one charge Determined by measuring spacing between adjacent Determined by measuring spacing between adjacent

isotopes (e.g. isotopes (e.g. 1313CC11 and and 1313CC22) (charge = 1/spacing)) (charge = 1/spacing) 0.33 Th between isotopes, +3 charge0.33 Th between isotopes, +3 charge

Charge State ExamplesCharge State Examples

protein_modelingLCTmix of 6 proteins

m/z915 916 917 918

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prot_mix_0724a 350 (5.837) Sm (SG, 2x6.00); Cm (343:374) TOF MS ES+ 1.86e3915.7363

915.4818

915.2274

915.9765

916.2311

916.4857

916.7402

protein_modelingLCTmix of 6 proteins

m/z1084 1085 1086 1087 1088 1089 1090

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0

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prot_mix_0724a 655 (10.923) Sm (SG, 2x6.00); Cm (645:675) TOF MS ES+ 4541086.5515

1086.0433

1087.0444

1087.5529

1088.0460

protein_modelingLCTmix of 6 proteins

m/z500 501 502 503 504 505 506 507 508 509 510 511 512

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0

100

prot_mix_0724a 651 (10.856) Sm (SG, 2x6.00); Cm (648:651) TOF MS ES+ 783505.3506

506.3584

507.3566

915.2247

915.4818 915.7363

915.9765

916.2311

916.4857

505.3506

506.3584

507.3566

1086.0433

1086.5515

1087.0444

1087.5529

1088.0460

+1

1.01

0.51 +2

0.25

+4

Mass AccuracyMass Accuracy Mass accuracy reported as a relative valueMass accuracy reported as a relative value

ppm = parts per million (1 ppm = 0.0001%)ppm = parts per million (1 ppm = 0.0001%) 5 ppm @ m/z 300 = 300 * (5/105 ppm @ m/z 300 = 300 * (5/1066) = ) = ±0.0015 Th±0.0015 Th 5 ppm @ m/z 3,000 = 5 ppm @ m/z 3,000 = 3,000 * (5/103,000 * (5/1066) = ) = ±0.015 Th±0.015 Th

High resolving power facilitates precise mass High resolving power facilitates precise mass measurementsmeasurements

Mass accuracies for MSF instrumentsMass accuracies for MSF instruments LCT: <50 ppm (ext. calib.), <5 ppm (int. calib.)LCT: <50 ppm (ext. calib.), <5 ppm (int. calib.) Biflex MALDI-TOF: depends on mass rangeBiflex MALDI-TOF: depends on mass range

Under 3,000 Da w/ internal calibration: 60 ppmUnder 3,000 Da w/ internal calibration: 60 ppm Over 3,000 Da w/ internal calibration: 200 ppmOver 3,000 Da w/ internal calibration: 200 ppm

Quadrupole (GC-MS): ±0.2 Th (absolute)Quadrupole (GC-MS): ±0.2 Th (absolute)

Resolution is the ability to separate ions of Resolution is the ability to separate ions of nearly equal mass/chargenearly equal mass/charge e.g. Ce.g. C66HH55Cl and CCl and C66HH55OF @ 112 m/zOF @ 112 m/z

CC66HH55Cl = 112.00798 amu (all Cl = 112.00798 amu (all 1212C, C, 3535Cl, Cl, 11H)H)

CC66HH55OF = 112.03244 amu (all OF = 112.03244 amu (all 1212C, C, 1616O, O, 11H, H, 1919F)F) Resolving power >4700 required to resolve these twoResolving power >4700 required to resolve these two

Two definitions Two definitions Resolution = Resolution = ΔΔm/m (0.024/112.03 = 0.00022 or 2.2*10m/m (0.024/112.03 = 0.00022 or 2.2*10-4-4)) Resolving power = m/Resolving power = m/ΔΔm (112.03/0.024 = 4668)m (112.03/0.024 = 4668)

Walk-up instrument capabilitiesWalk-up instrument capabilities Biflex is capable of 10,000 resolving powerBiflex is capable of 10,000 resolving power LCT is capable of 5,000 resolving powerLCT is capable of 5,000 resolving power All peaks in GC-MS are about 0.6 Th wideAll peaks in GC-MS are about 0.6 Th wide

What is Resolution?What is Resolution?

Resolving Power ExampleResolving Power Example

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Mass [amu]111.95 112.00 112.05 112.10

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Mass [amu]111.95 112.00 112.05 112.10

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Mass [amu]111.95 112.00 112.05 112.10

RP= 3,000 RP= 5,000 RP= 7,000

All resolving powers are FWHM

C6H5OFC6H5Cl

Some useful software toolsSome useful software tools The “exact mass” feature in ChemDraw will give The “exact mass” feature in ChemDraw will give

you a monoisotopic massyou a monoisotopic mass IsisDraw exact mass is not correct for large IsisDraw exact mass is not correct for large

(>2,000 Da) compounds(>2,000 Da) compounds IsoPro (freeware) can be used to predict isotopic IsoPro (freeware) can be used to predict isotopic

envelopesenvelopes See MS Links page for URLSee MS Links page for URL

MassLynx “Isotope Model” can be used to MassLynx “Isotope Model” can be used to predict isotope patternspredict isotope patterns

BioLynx module of MassLynx can be used to BioLynx module of MassLynx can be used to predict oligopeptide, oligosaccharide, and predict oligopeptide, oligosaccharide, and oligonucleotide massesoligonucleotide masses

Electron Ionization (EI)Electron Ionization (EI)

Gas phase molecules are irradiated by Gas phase molecules are irradiated by beam of electronsbeam of electrons

Interaction between molecule and beam Interaction between molecule and beam results in electron ejectionresults in electron ejectionM + eM + e-- M M++•• + 2e + 2e--

Radical species dominateRadical species dominateEI is a very energetic processEI is a very energetic process

Molecules often fragment right after ionizationMolecules often fragment right after ionization

EI DiagramEI Diagram

Image from http://www.noble.org/Plantbio/MS/iontech.ei.html

EI AdvantagesEI Advantages

Simplest source design of allSimplest source design of allVery high yield (up to 0.1% ionization)Very high yield (up to 0.1% ionization)Simple, robust ionization mechanismSimple, robust ionization mechanism

Even noble gases are ionized by EIEven noble gases are ionized by EIFragmentation patterns can be used to Fragmentation patterns can be used to

identify speciesidentify speciesNIST ’08 library has over 220,000 spectraNIST ’08 library has over 220,000 spectra Interpretation allows functionalities to be Interpretation allows functionalities to be

deduced in novel compoundsdeduced in novel compounds

EI DisadvantagesEI Disadvantages

Fragmentation often makes intact Fragmentation often makes intact molecular ion difficult to observemolecular ion difficult to observe

Analytes must be in the gas phaseAnalytes must be in the gas phaseNot applicable to most saltsNot applicable to most saltsLabile compounds not amenable to EILabile compounds not amenable to EI

Databases are very limitedDatabases are very limitedNIST’08 has 192,000 unique compoundsNIST’08 has 192,000 unique compounds Interpreting EI spectra Interpreting EI spectra de novode novo is an art is an art

EI only generates positive ionsEI only generates positive ions

EI Mass SpectrumEI Mass Spectrum

Figure from Mass Spectrometry Principles and ApplicationsE. De Hoffmann, J. Charette, V. Strooband, eds., ©1996

Electrospray Ionization (ESI)Electrospray Ionization (ESI) Dilute solution of analyte (<1 mg/L) infused through Dilute solution of analyte (<1 mg/L) infused through

a fine needle in a high electric fielda fine needle in a high electric field Very small, highly charged droplets are createdVery small, highly charged droplets are created Solvent evaporates, droplets split and/or ions Solvent evaporates, droplets split and/or ions

evaporate to lower charge/area ratio evaporate to lower charge/area ratio Warm nebulizing gas accelerates dryingWarm nebulizing gas accelerates drying Free ions are directed into the vacuum chamberFree ions are directed into the vacuum chamber Ion source voltage depends on solventIon source voltage depends on solvent

Usually Usually ±±2500 – 2500 – ±±4500 V4500 V +HV makes positive ions, -HV makes negative ions+HV makes positive ions, -HV makes negative ions

ESI PictureESI Picture

Characteristics of ESI IonsCharacteristics of ESI Ions ESI is a thermal process (1 atm in source)ESI is a thermal process (1 atm in source)

Little fragmentation due to ionization (cf EI) Little fragmentation due to ionization (cf EI) Solution-phase ions are preserved in MSSolution-phase ions are preserved in MS

e.g. organometallic saltse.g. organometallic salts ESI ions are generated by ion transferESI ions are generated by ion transfer

(M+H)(M+H)++, (M+Na), (M+Na)++, or (M-H), or (M-H)--, rarely M, rarely M++•• or M or M--••

ESI often generates multiply charged ionsESI often generates multiply charged ions (M+2H)(M+2H)2+2+ or (M+10H) or (M+10H)10+10+

Most ions are 500-1500 m/zMost ions are 500-1500 m/z ESI spectrum x-axis must be mass/charge (m/z ESI spectrum x-axis must be mass/charge (m/z

or Th, not amu or Da)or Th, not amu or Da)

Advantages of ESIAdvantages of ESI

Gentlest ionization processGentlest ionization processGreatest chance of observing molecular ionGreatest chance of observing molecular ionVery labile analytes can be ionizedVery labile analytes can be ionized

Molecule need not be volatileMolecule need not be volatileProteins/peptides easily analyzed by ESIProteins/peptides easily analyzed by ESISalts can be analyzed by ESISalts can be analyzed by ESI

Easily coupled with HPLCEasily coupled with HPLCBoth positive and negative ions can be Both positive and negative ions can be

generated by the same sourcegenerated by the same source

ESI DisadvantagesESI Disadvantages Analyte must have an acidic or basic siteAnalyte must have an acidic or basic site

Hydrocarbons and steroids not readily ionized by ESIHydrocarbons and steroids not readily ionized by ESI

Analyte must be soluble in polar, volatile solventAnalyte must be soluble in polar, volatile solvent ESI is less efficient than other sourcesESI is less efficient than other sources

Most ions don’t make it into the vacuum systemMost ions don’t make it into the vacuum system

ESI is very sensitive to contaminantsESI is very sensitive to contaminants Solvent clusters can dominate spectraSolvent clusters can dominate spectra

Distribution of multiple charge states can make Distribution of multiple charge states can make spectra of mixtures hard to interpretspectra of mixtures hard to interpret e.g. polymer mass spectrae.g. polymer mass spectra

ESI ExamplesESI Examples10495LCT KC366js-29-1

m/z200 300 400 500 600 700 800 900 1000 1100 1200 1300

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js-29-1 54 (1.086) Cm (54:60) 1: TOF MS ES+ 6.40e3395.1219

304.0758

396.1333

397.1367

calib_0731LCT KC36620 pmol myo on col

m/z600 800 1000 1200 1400 1600 1800 2000 2200

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myo_0731a 721 (7.505) Sm (SG, 2x6.00); Cm (721:743) 1: TOF MS ES+ 577893.1618

848.5577

693.8809

693.6229

689.6234

808.1948

694.3848

942.7415

998.1490

1060.4785

1131.1024

1211.8010

1304.9185

1413.5582 1696.13731541.90811884.4519

2119.7839

C26H18O4

(M+H)+

myoglobin

(M+

10

H)1

0+

(M+

13

H)1

3+

Matrix-Assisted Laser Desorption/Ionization Matrix-Assisted Laser Desorption/Ionization (MALDI)(MALDI)

Analyte is mixed with UV-absorbing matrixAnalyte is mixed with UV-absorbing matrix ~10,000:1 matrix:analyte ratio~10,000:1 matrix:analyte ratio Analyte does not need to absorb laserAnalyte does not need to absorb laser

A drop of this liquid is dried on a targetA drop of this liquid is dried on a target Analyte incorporated into matrix crystalsAnalyte incorporated into matrix crystals

Spot is irradiated by a laser pulseSpot is irradiated by a laser pulse Irradiated region sublimes, taking analyte with itIrradiated region sublimes, taking analyte with it Matrix is often promoted to the excited stateMatrix is often promoted to the excited state Charges exchange between matrix and analyte in the Charges exchange between matrix and analyte in the

plume (very fast <100 nsec)plume (very fast <100 nsec)

Ions are accelerated toward the detectorIons are accelerated toward the detector

MALDI DiagramMALDI Diagram

Image from http://www.noble.org/Plantbio/MS/iontech.maldi.html

MALDI AdvantagesMALDI Advantages

Relatively gentle ionization techniqueRelatively gentle ionization techniqueVery high MW species can be ionizedVery high MW species can be ionizedMolecule need not be volatileMolecule need not be volatileVery easy to get sub-picomole sensitivityVery easy to get sub-picomole sensitivityUsually 1-3 charge states, even for very Usually 1-3 charge states, even for very

high MW specieshigh MW speciesPositive or negative ions from same spotPositive or negative ions from same spotWide array of matrices availableWide array of matrices available

MALDI DisadvantagesMALDI DisadvantagesMALDI matrix cluster ions obscure low m/z MALDI matrix cluster ions obscure low m/z

(<600) range(<600) rangeAnalyte must have very low vapor pressureAnalyte must have very low vapor pressurePulsed nature of source limits compatibility Pulsed nature of source limits compatibility

with many mass analyzerswith many mass analyzersCoupling MALDI with chromatography can Coupling MALDI with chromatography can

be difficultbe difficultAnalytes that absorb the laser can be Analytes that absorb the laser can be

problematicproblematicFluorescein-labeled peptidesFluorescein-labeled peptides

MALDI ExampleMALDI Example

(Ubiq+H)+

(Ins+H)+

(Ub

iq+

2H

)2+

(ACTH 7-38+H)+

(AC

TH

18-

37

+H

)+

Types of Mass AnalyzersTypes of Mass Analyzers

Scanning: only one m/z ratio measured at Scanning: only one m/z ratio measured at a time (cf grating spectrophotometer)a time (cf grating spectrophotometer)Quadrupole mass filterQuadrupole mass filterMagnetic/electric sectorMagnetic/electric sector

Multiplexing: all m/z ratios analyzed Multiplexing: all m/z ratios analyzed simultaneously (cf FTIR or PDA)simultaneously (cf FTIR or PDA)Time-of-flightTime-of-flight Ion trapIon trapFourier transform ion cyclotron resonanceFourier transform ion cyclotron resonance

Time-of-Flight (TOF)Time-of-Flight (TOF)

All ions simultaneously accelerated All ions simultaneously accelerated through the same voltagethrough the same voltageExcellent choice for MALDIExcellent choice for MALDI

Ions drift through a field-free regionIons drift through a field-free regionLower m/z ions travel faster than higher Lower m/z ions travel faster than higher

m/z ionsm/z ionsKE = z*V = KE = z*V = ½½m*vm*v22 TOF TOF αα (m/z) (m/z)½½

MALDI-TOF DiagramMALDI-TOF Diagram

ReflectorDetector

LinearDetector

Lens

Target

ExtractionPlate

FlightTube

Entrance

Reflectron

337 nm Nitrogen laser

TOF AdvantagesTOF Advantages

All ions detected at once (multiplexing)All ions detected at once (multiplexing)High mass accuracy and resolving power High mass accuracy and resolving power

possiblepossibleReasonable performance for costReasonable performance for cost

<5 ppm mass accuracy and >20,000 resolving <5 ppm mass accuracy and >20,000 resolving power commercially available ($150k-$300k)power commercially available ($150k-$300k)

High mass, low charge ions not a problemHigh mass, low charge ions not a problemTheoretically unlimited mass rangeTheoretically unlimited mass range+1 Ion > 1,000,000 Th by MALDI-TOF+1 Ion > 1,000,000 Th by MALDI-TOF

TOF DisadvantagesTOF Disadvantages

High vacuum required for resolution and High vacuum required for resolution and accuracy (<10accuracy (<10-7-7 torr) torr)Complex vacuum system necessaryComplex vacuum system necessary

Must be recalibrated oftenMust be recalibrated oftenTemperature and voltage fluctuations alter Temperature and voltage fluctuations alter

flight timesflight timesFast detectors prone to saturationFast detectors prone to saturationLong flight tubes for high resolving power Long flight tubes for high resolving power

can make instruments largecan make instruments large

Quadrupole Mass Filter (QMF)Quadrupole Mass Filter (QMF)

QMF has radio frequency (RF) and DC QMF has radio frequency (RF) and DC field between 4 rodsfield between 4 rodsRods can be cylindrical or hyperbolicRods can be cylindrical or hyperbolic Ion motions governed by set of Mathieu Ion motions governed by set of Mathieu

equations (2equations (2ndnd order differential equations) order differential equations) A narrow range of m/z’s have stable A narrow range of m/z’s have stable

trajectories through the quadrupole trajectories through the quadrupole (usually 0.7 Th FWHM)(usually 0.7 Th FWHM)

Scanning the quadrupole generates the Scanning the quadrupole generates the mass spectrummass spectrum50.0, 50.2, 50.4, 50.6, 50.0, 50.2, 50.4, 50.6, 399.6, 399.8, 400.0 399.6, 399.8, 400.0

(repeat)(repeat)

Quadrupole DiagramQuadrupole Diagram

Movie URL: http://www.youtube.com/watch?v=8AQaFdI1Yow%20&%20mode=related%20&%20search=

QMF AdvantagesQMF Advantages

Very simple to implementVery simple to implementLow cost (<$100k)Low cost (<$100k)Moderate vacuum required (~10Moderate vacuum required (~10-5-5 torr) torr)Small sizeSmall sizeVery robustVery robustMost common MS in useMost common MS in use

QMF DisadvantagesQMF DisadvantagesLimited mass range (up to m/z 4,000)Limited mass range (up to m/z 4,000)Limited resolving power and mass Limited resolving power and mass

accuracyaccuracyUnit mass accuracy (+/- 0.2 Th for all ions)Unit mass accuracy (+/- 0.2 Th for all ions)Unit resolution (0.5 Th wide) peakUnit resolution (0.5 Th wide) peak

Cannot resolve isotopes on multiply charged ionsCannot resolve isotopes on multiply charged ionsHigh resolving power, less sensitivityHigh resolving power, less sensitivity

Scanning limits sensitivity and speedScanning limits sensitivity and speedQuad can rapidly jump between select m/z Quad can rapidly jump between select m/z

ratios for increased speed & sensitivityratios for increased speed & sensitivity

Walk-up Instruments in the MSFWalk-up Instruments in the MSF

Agilent 6890n/5973i GC-MSAgilent 6890n/5973i GC-MS EI QMF instrumentEI QMF instrument 10-800 m/z range10-800 m/z range All analytes MUST pass through GC columnAll analytes MUST pass through GC column

Waters LCT Classic (2 in lab)Waters LCT Classic (2 in lab) ESI-TOF instrumentESI-TOF instrument One is set up for flow injection analysis of small One is set up for flow injection analysis of small

molecules (no LC column)molecules (no LC column) The other is set up for LC-MS of biomoleculesThe other is set up for LC-MS of biomolecules

Bruker Biflex IIIBruker Biflex III MALDI-TOF instrumentMALDI-TOF instrument

Upcoming Application SeminarsUpcoming Application Seminarsin Ballantine Hall 006in Ballantine Hall 006

Analyzing small molecules by ESI-TOFAnalyzing small molecules by ESI-TOF Monday July 28 @ 1:30 noonMonday July 28 @ 1:30 noon

Analyzing proteins/peptides by MALDI-TOFAnalyzing proteins/peptides by MALDI-TOF Tuesday July 29 @ 1:30 noonTuesday July 29 @ 1:30 noon

Analyzing semi-volatiles by GC-MSAnalyzing semi-volatiles by GC-MS Thursday July 31 @ 1:30 noonThursday July 31 @ 1:30 noon

Analyzing proteins/peptides by ESI-TOFAnalyzing proteins/peptides by ESI-TOF Monday Aug. 4 @ 1:30 noon)Monday Aug. 4 @ 1:30 noon)

Please indicate which ones you want to attend Please indicate which ones you want to attend on the sign-up sheeton the sign-up sheet