Abstract
The molecular scanner combines protein separation using gel electrophoresis with peptide mass fingerprinting (PMF) techniques to identify proteins in a highly automated manner. Proteins separated in a 2-dimensional polyacrylamide gel (2-D PAGE) are digested in parallel and transferred onto a membrane keeping their relative positions. The membrane is then sprayed with a matrix and inserted into a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer, which measures a peptide mass fingerprint at each site on the scanned grid. First, visualization of PMF data allows surveying all fingerprints at once and provides very useful information on the presence of chemical noise. Chemical noise is shown to be a potential source for erroneous identifications and is therefore purged from the mass fingerprints. Then, the correlation between neighboring spectra is used to recalibrate the peptide masses. Finally, a method that clusters peptide masses according to the similarity of the spatial distributions of their signal intensities is presented. This method allows discarding many of the false positives that usually go along with PMF identifications and allows identifying many weakly expressed proteins present in the gel.
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Godovac-Zimmermann, J.; Brown, L. R. Perspectives for Mass Spectrometry and Functional Proteomics. Mass Spectrom. Rev. 2001, 20(1), 1–57.
Bienvenut, W. V.; Müller, M.; Palagi, P.; Gasteiger, E.; Heller, M.; Jung, E.; Giron, M.; Gras, R.; Gay, S.; Binz, P. A.; Hughes, G. J.; Sanchez, J. C.; Appel, R. D.; Hochstrasser, D. Proteomics and Mass Spectrometry: Some Aspects and Developments. In Mass Spectrometry and Genomic Analysis. Kluwer: The Netherlands, 2001; 1st ed.; pp 1–53.
Bjellqvist, B.; Ek, K.; Righetti, P. G.; Gianazza, E.; Gorg, A.; Westermeier, R.; Postel, W. Isoelectric Focusing in Immobilized pH Gradients: Principle, Methodology, and Some Applications. J. Biochem. Biophys. Methods 1982, 6(4), 317–339.
Karas, M.; Hillenkamp, F. Laser Desorption Ionization of Proteins With Molecular Masses Exceeding 10,000 Daltons. Anal. Chem. 1988, 60(20), 2299–2301.
Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T. Protein and Polymer Analysis up to m/z 100,000 by Laser Ionization Time-of-Flight Mass Spectrometry. Rapid Commun. Mass Spectrom. 1988, 2, 151–153.
Henzel, W. J.; Billeci, T. M.; Stults, J. T.; Wong, S. C.; Grimley, C.; Watanabe, C. Identifying Proteins from Two-Dimensional Gels by Molecular Mass Searching of Peptide Fragments in Protein Sequence Databases. Proc. Natl. Acad. Sci. U.S.A. 1993, 90(11), 5011–5015.
James, P.; Quadroni, M.; Carafoli, E.; Gonnet, G. Protein Identification by Mass Profile Fingerprinting. Biochem. Biophys. Res. Commun. 1993, 195(1), 58–64.
Mann, M.; Hojrup, P.; Roepstorff, P. Use of Mass Spectrometric Molecular Weight Information to Identify Proteins in Sequence Databases. Biol. Mass Spectrom. 1993, 22(6), 338–345.
Pappin, D. J. C.; Hojrup, P.; Bleasby, A. J. Rapid Identification of Proteins by Peptide-Mass Fingerprinting. Curr. Biol. 1993, 3, 327–345.
Yates, J. R. I.; Speicher, S.; Griffin, P. R.; Hunkapiller, T. Peptide Mass Maps: A Highly Informative Approach to Protein Identification. Anal. Biochem. 2001, 214, 397–408.
Gras, R.; Müller, M.; Gasteiger, E.; Gay, S.; Binz, P. A.; Bienvenut, W.; Hoogland, C.; Sanchez, J. C.; Bairoch, A.; Hochstrasser, D. F.; Appel, R. D. Improving Protein Identification from Peptide Mass Fingerprinting Through a Parameterized Multi-Level Scoring Algorithm and an Optimized Peak Detection. Electrophoresis 1999, 20(18), 3535–3550.
Eriksson, J.; Chait, B. T.; Fenyo, D. A Statistical Basis for Testing the Significance of Mass Spectrometric Protein Identification Results. Anal. Chem. 2000, 72(5), 999–1005.
Christian, N. P.; Arnold, R. J.; Reilly, J. P. Improved Calibration of Time-of-Flight Mass Spectra by Simplex Optimization of Electrostatic Ion Calculations. Anal. Chem. 2000, 72(14), 3327–3337.
Juhasz, P.; Vestal, M. L.; Martin, S. A. On the Initial Velocity of Ions Generated by Matrix-Assisted Laser Desorption Ionization and Its Effect on the Calibration of Delayed Extraction Time-of-Flight Mass Spectra. J. Am. Soc. Mass Spectrom. 1997, 8, 209–217.
Vestal, M. L.; Juhasz, P. Resolution and Mass Accuracy in Matrix-Assisted Laser Desorption Ionization Time-of-Flight. J. Am. Soc. Mass Spectrom. 1998, 9, 892–911.
Lopez, M. F. Better Approaches to Finding the Needle in a Haystack: Optimizing Proteome Analysis Through Automation. Electrophoresis 2000, 21(6), 1082–1093.
Traini, M.; Gooley, A. A.; Ou, K.; Wilkins, M. R.; Tonella, L.; Sanchez, J. C.; Hochstrasser, D. F.; Williams, K. L. Towards an Automated Approach for Protein Identification in Proteome Projects. Electrophoresis 1998, 19(11), 1941–1949.
Binz, P. A.; Müller, M.; Walther, D.; Bienvenut, W. V.; Gras, R.; Hoogland, C.; Bouchet, G.; Gasteiger, E.; Fabbretti, R.; Gay, S.; Palagi, P.; Wilkins, M. R.; Rouge, V.; Tonella, L.; Paesano, S.; Rossellat, G.; Karmime, A.; Bairoch, A.; Sanchez, J. C.; Appel, R. D.; Hochstrasser, D. F. A Molecular Scanner to Automate Proteomic Research and to Display Proteome Images. Anal. Chem. 1999, 71(21), 4981–4988.
Bienvenut, W. V.; Sanchez, J. C.; Karmime, A.; Rouge, V.; Rose, K.; Binz, P. A.; Hochstrasser, D. F. Toward a Cinical Molecular Scanner for Proteome Research: Parallel Protein Chemical Processing Before and During Western Blot. Anal. Chem. 1999, 71(21), 4800–4807.
Pacholski, M. L.; Winograd, N. Imaging with Mass Spectrometry. Chem. Rev. 1999, 99, 2977–3005.
Stoeckli, M.; Chaurand, P.; Hallahan, D. E.; Caprioli, R. M. Imaging Mass Spectrometry: A New Technology for the Analysis of Protein Expression in Mammalian Tissues. Nat. Med. 2001, 7(4), 493–496.
Toffoli, T.; Margolus, N. Cellular Automata Machines. MIT Press: Cambridge, 1987.
Hoogland, C.; Sanchez, J. C.; Tonella, L.; Binz, P. A.; Bairoch, A.; Hochstrasser, D. F.; Appel, R. D. The 1999 SWISS-2-D PAGE Database Update. Nucleic Acids Res. 2000, 28(1), 286–288.
Keller, B. O.; Li, L. Discerning Matrix-Cluster Peaks in Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectra of Dilute Peptide Mixtures. J. Am. Soc. Mass Spectrom. 2000, 11(1), 88–93.
Land, C. M.; Kinsel, G. R. The Mechanism of Matrix to Analyte Proton Transfer in Clusters of 2,5-Dihydroxybenzoic Acid and the Tripeptide VPL. J. Am. Soc. Mass Spectrom. 2001, 12(6), 726–731.
Egelhofer, V.; Bussow, K.; Luebbert, C.; Lehrach, H.; Nordhoff, E. Improvements in Protein Identification by MALDI-TOF-MS Peptide Mapping. Anal. Chem. 2000, 72(13), 2741–2750.
Press, W. H.; Teukolsky, S. A.; Vetterling, W. T.; Flannery, B. P. Numerical Recipes in C. Cambridge University Press: Cambridge, 1995, 699–706.
Gusev, A. I.; Wilkinson, W. R.; Proctor, A.; Hercules, D. M. Improvement of Signal Reproducibility and Matrix/Comatrix Effects in MALDI Analysis. Anal. Chem. 1995, 67(6), 1034–1041.
Duncan, M. W.; Matanovic, G.; Cerpa-Poljak, A. Quantitative Analysis of Low Molecular Weight Compounds of Biological Interest by Matrix-Assisted Laser Desorption Ionization. Rapid Commun. Mass Spectrom. 1993, 7(12), 1090–1094.
Gobom, J.; Kraeuter, K. O.; Persson, R.; Steen, H.; Roepstorff, P.; Ekman, R. Detection and Quantification of Neurotensin in Human Brain Tissue by Matrix-Assisted Laser Desorption/ Ionization Time-of-Flight Mass Spectrometry. Anal. Chem. 2000, 72(14), 3320–3326.
Gusev, A. I.; Wilkinson, W. R.; Proctor, A.; Hercules, D. M. Direct Quantitative Analysis of Peptides Using Matrix Assisted Desorption Ionization. Fresenius J. Anal. Chem. 1996, 354, 455–463.
Kratzer, R.; Eckerskorn, C.; Karas, M.; Lottspeich, F. Suppression Effects in Enzymatic Peptide Ladder Sequencing Using Ultraviolet-Matrix Assisted Laser Desorption/Ionization-Mass Spectrometry. Electrophoresis 1998, 19(11), 1910–1919.
Han, J.; Kamber, M. Data Mining. Academic Press: San Diego, 2001, 354–362.
Medzihradszky, K. F.; Campbell, J. M.; Baldwin, M. A.; Falick, A. M.; Juhasz, P.; Vestal, M. L.; Burlingame, A. L. The Characteristics of Peptide Collision-Induced Dissociation Using a High-Performance MALDI-TOF/TOF Tandem Mass Spectrometer. Anal. Chem. 2000, 72(3), 552–558.
Loboda, A. V.; Krutchinsky, A. N.; Bromirski, M.; Ens, W.; Standing, K. G. A Tandem Quadrupole/Time-of-Flight Mass Spectrometer With a Matrix-Assisted Laser Desorption/Ionization Source: Design and Performance. Rapid Commun. Mass Spectrom. 2000, 14(12), 1047–1057.
Wei, J.; Buriak, J. M.; Siuzdak, G. Desorption-Ionization Mass Spectrometry on Porous Silicon. Nature 1999, 399(6733), 243–246.
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Müller, M., Gras, R., Appel, R.D. et al. Visualization and analysis of molecular scanner peptide mass spectra. J Am Soc Mass Spectrom 13, 221–231 (2002). https://doi.org/10.1016/S1044-0305(01)00358-0
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DOI: https://doi.org/10.1016/S1044-0305(01)00358-0