Transcription of Principles of MALDI-TOF mass spectrometry
1 Purdue-UAB Botanicals Center for Age-Related DiseasePrinciples of MALDIP rinciples of MALDI--TOF TOF mass spectrometrymass spectrometryStephen Barnes, Botanical Center Workshop 2002 Mass spectrometry Methods in Botanicals ResearchShining the light on samplesShining the light on samplesPurdue-UAB Botanicals Center for Age-Related Disease A focused laser beam, either in the UV or infra-red ranges, can evaporate compounds from the solid phase The resulting ions are injected into a tube (1 -2 m in length), accelerated and allowed to drift towards a detector. Their time-of-flightis proportional to their (MW)1/2 Matrix-Assisted Laser Desorption Ionization (MALDI)Accelerating pulsedetectorShort laserpulseFlight tube and drift region to measure the time-of-flight (TOF) MALDI-TOF ionizationN2laser pulse (337 nm)[M+H]+[M-H]-[M-H]-matrix ionsProtein/peptide/nucleotide/saccharid edeposited on crystal surfaceHow a TOF instrument worksLinear flight tube ( m)Oscilloscopeto time ionarrival (1 sec)SourceAcceleratingpulseOscilloscopet o time ionarrivalReflectronEffective flight tube ( m)Protein analysis 2002 WaterBath37oCdestainSpeed-VactrypsinIncu bateovernightEppendorftube1.
2 20 MALDI 1500 2000 2500 3000 Mass (m/z)Applications of timeApplications of time--ofof--flightflightPurdue-UAB Botanicals Center for Age-Related Disease Whole proteins -modified antibodies Peptide mixtures following trypsinolysis of spots or bands from 2D-gels Polyphenols Bile acids and many other biologically interesting molecules-Cytochrome Cytochrome C Modified by HNE C Modified by HNE MALDIMALDI--TOF Mass SpectrumTOF Mass SpectrumMass (m/z) [M+2H]2+[2M+H]+[M+H]+CONTROLPROTEIN + ONE 4 HNEPROTEIN + TWO 4 HNEPROTEIN +THREE 4 HNE% IntensityTrypsin Digest of Trypsin Digest of porinporin--P1; P1; VoltageVoltage--dependent aniondependent anion--selective channelselective channel0102030405060708090100% (m/z) MALDI-TOF Spectrum for Daidzein1001802603404205000020406080100% [M+H]+ + Na+Mass (m/z)MALDIMALDI--TOF of mouse bileTOF of mouse bileTDC [M-H] [M-H+Na] (m/z)544592496448400 Intensity204060801000640 CONHCH2CH2SO3-OHHHOTC [M-H] : -cyano-4-hydroxycinnamic acidN2laser 337 nm, 1:100 fold dilution of bile, 1 l spottedLC-MS analysis of mouse bileTauro-triOHTauro-diOHTotal ion currentMouse bile does not contain ANY glycine conjugates of bile acids, only taurine conjugates.
3 Several isomers are CE and LC to Connecting CE and LC to MALDI analysisMALDI analysisCE analysisnanoLC analysisParallel capture of effluents of 8 nanoLC separations on Mylar -can be scanned simultaneously by fast laserCreates 20 mm wide tracks thatcan be scanned by MALDI laserfor MS analysisPros/Cons of laying down LC or Pros/Cons of laying down LC or EC separations on matrix plateEC separations on matrix plate Allows off-line analysis both in real time and then in a retrospective mode MALDI-TOF analysis is very fast Can do TOF-TOF MS-MS analysis BUT what happens chemically on the acidic environment on the surface of the plate during storage Also, can the laser beam cause chemical changes?
