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Libo Cao Ph.D., Analytical (Dr. P. B. Harrington) 19 th April, 2001

Peptide Sequencing by Matrix-assisted Laser Desorption-ionization Time-of-flight Mass Spectrometry (MALDI-TOF-MS) Combined with In-source Decay (ISD). Libo Cao Ph.D., Analytical (Dr. P. B. Harrington) 19 th April, 2001. Background. Matrix-assisted Laser Desorption-ionization (MALDI)

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Libo Cao Ph.D., Analytical (Dr. P. B. Harrington) 19 th April, 2001

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  1. Peptide Sequencing by Matrix-assisted Laser Desorption-ionization Time-of-flight Mass Spectrometry (MALDI-TOF-MS) Combined with In-source Decay (ISD) • Libo Cao • Ph.D., Analytical (Dr. P. B. Harrington) • 19th April, 2001

  2. Background • Matrix-assisted Laser Desorption-ionization (MALDI) • Time-of-flight MS (TOF-MS) • Delayed Pulsed Ion Extraction (DPIE) • In-source Decay (ISD)

  3. Components of a Mass Spectrometer • Skoog, D. A.; Holler, F. J.; Nieman, T. A. Principles of Instrumental Analysis, 5th Ed.; Saunders College Publishing: Philadelphia, 1998;pp 255 Inlet System Ion Source Mass Analyzer Detector Signal Processor Sample Vacuum System Readout

  4. MALDI • A sample introduction and ionization method • Matrix absorbs strongly at the wavelength of a pulsed laser beam • Analyte exhibits little absorbance at this wavelength Li, L.; Garden, R. W.; Sweedler, J. V. Trends in Biotech.2000, 18 (4), 151-160.

  5. Goals of MALDI • Vaporize and ionize the analytes without fragmentation • Very low background noise • Obtain accurate molecular weight information • Tolerate high level of impurities

  6. Matrices Most Frequently Used for MALDI Together with the Usable Wavelengths Matrix Wavelength (nm) • Nicotinic acid 266, 220-290 • Benzoic acid derivatives: • 2,5-Dihydroxybenzoic acid 266, 337, 355 • Vanillic acid 266 • 2-Amino-benzoic acid 266, 337, 355 Karas, M.; Bahr, U. Anal. Chem. 1990, 9, 322.

  7. Fundamental Equations for TOF-MS • KE =zV = ½ m1v12 = ½ m2v22 • L = v t • t = (m/2zV)1/2 L

  8. TOF Mass Analyzer • Cotter, R. J.Anal. Chem. News&Features,1999, 77, 445-450.

  9. Delayed Pulsed Ion Extraction (DPIE) in MALDI -200 V -100v Nitrogen Laser (337.1nm) -100v Brown, R. S.; Lennon, J. J. Anal. Chem. 1995, 67, 1998-2003. Sample Probe Tip Ion Guide (1) Signal XY Deflector Plates Ion Guide (2) -2 kV G1 G2 G1 Voltage = Pulse voltage (0—3 kv max) G2 Voltage = Bias Voltage (24 kv max)

  10. Benefits of DPIE • Kinetic energy focusing - Obtain same initial velocity for ions with the same mass to charge ratio (m/z)

  11. Ion-source Decay (ISD) • The decay of the precursor ions occur in the MALDI ion source with DPIE • Provide specific ions for certain sequencing information Li, L.; Garden, R. W.; Sweedler, J. V. Trends in Biotech.2000, 18 (4), 151-160

  12. a-, b-, c-, x-, y-, z- Series Ions y z x NH CHR CO NH CHR CO a c b

  13. MALDI Fast Metastable Ion Decay Spectrum of Porcine Insulin Obtained with DPIE • Brown, R. S.; Lennon, J. J. Anal. Chem.1995, 67, 3990-3999.

  14. MALDI Fast Metastable Ion Decay Spectrum of Cytochrome C Obtained with DPIE • Brown, R. S.; Lennon, J. J. Anal. Chem.1995, 67, 3990-3999.

  15. Metastable Ions (m*) • Broader peaks occurring at non-integer m/z values in MS • C6H6·+  C6H5·+ + H · m/z 78 m/z 77 • m* =m22/m1= 772/78 = 76.01 m/z

  16. Peptide Sequencing Information Brown, R. S.; Lennon, J. J. Anal. Chem.1995, 67, 3990-3999

  17. Conclusions • Mostly generate specific c-,y- ions • Sequencing of post-translational modifications • Do not need predigestion of proteins • Large peptides and small proteins (1,500 –18,000 Da.)

  18. Future Work • More suitable matrix materials need to be found • Simplify the instrumentation and operation • Potential applications need to be developed - for larger proteins - for mixture proteins

  19. Acknowledgements • Dr. Peter de B. Harrington • Tricia Buxton • Guoxiang Chen • Mingming Xu • Xueqing Liu • Erin Kolbrich • Jeannette Perr • Jennifer Cline • Yuka Minoshima

  20. ********************************

  21. G1 Voltage = Pulse voltage (0—3 kv max) G2 Voltage = Bias Voltage (24 kv max)

  22. Metastable ions (m*) m1v1 = m2v2 + (m1 – m2)v2 zV = ½ m1v2 m2 v2/r = Bzv m*/z= B2r2/2V

  23. Disadvantage * Edman degradation -----Tedious, indirect, labeling process complicated, need digested peptides

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