Peptide Sequencing by Mass Spectrometry for Homology Searches and Cloning of Genes
It is now possible to obtain sequence information from gel-separated proteins by mass spectrometry at levels too low for conventional approaches. Usually this tandem mass spectrometric data are used for database searches with the aim of identifying the corresponding gene. Recently it has been shown that long and accurate amino acid sequences can be obtained which are sufficient for PCR-based strategies to clone the corresponding gene [Wilm et al. (1996), Nature379, 466–469]. More than eight proteins have now been cloned based on that method. In many more cases the sequence information identified homologous proteins. Issues involved in cloning by mass spectrometric sequence information are discussed, as are two case studies. These results clearly establish mass spectrometry as a viable tool not only for the database identification of proteins, but also for the de novo sequencing of gel-separated proteins at the low-picomole to femtomole level.
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- Eng, J. K., McCormack, A. L., and J. R. Yates, I. (1994). J. Am. Soc. Mass Spectrom. 5, 976–989.Google Scholar
- James, P., Quadroni, M., Carafoli, E., and Gonnet, G. (1993). Protein identification by mass profile fingerprinting, Biophys. Biochem. Res. Commun. 195, 58–64.Google Scholar
- King, T. B., Colby, S. M., and Reilly, J. P. (1995). High resolution MALDI-TOF mass spectra of three proteins obtained using space-velocity correlation focussing, Int. J. Mass Spectrom. Ion Processes 145, L1–L7.Google Scholar
- Mann, M. (1996). A shortcut to interesting human genes: Peptide sequence tags, ESTs and computers, Trends Biol. Sci., 21, 494–495.Google Scholar
- McNagny, K. M., Petterson, I., Rossi, F., Shevchenko, A., Mann, M., and Graf, T. (1997). MEP21: A podocalyxinrelated cell surface antigen expressed by primitive and definitive hematopoietic progenitors which modulates cell adhesion, submitted.Google Scholar
- Muzio, M., Chinnaiyan, A. M., Kischkel, F. C., Rouke, K. O., Shevchenko, A., Jian Ni, Scaffidi, C., Bretz, J. D., Zhang, M., Gentz, R., Mann, M., Krammer, P. H., Peter, M. E., and Dixit, V. M. (1996). FLICE, a novel FADD-homologous ICE/CED-3–like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex, Cell 85, 817–827.PubMedGoogle Scholar
- Shevchenko, A., Jensen, O. N., Podtelejnikov, A. V., Sagliocco, F., Wilm, M., Vorm, O., Mortensen, P., Shevchenko, A., Boucherie, H., and Mann, M. (1996a). Linking genome and proteome by mass spectrometry: Large scale identification of yeast proteins from two dimensional gels, Proc. Natl. Acad. Sci. USA, 93, 14440–14445.PubMedGoogle Scholar
- Thomson, B. A., Douglas, D. J., Corr, J. J., Hager, J. W., and Jolliffe, C. L. (1995). Improved collisionally activated dissociation efficiency and mass resolution on a triple quadrupole mass spectrometer system, Anal. Chem. 67, 1696–1704.Google Scholar
- Vestal, M. L., Juhasz, P., and Martin, S. A. (1995). Delayed extraction matrix-assisted laser desorption time-of-flight mass spectrometry, Rapid Commun. Mass Spectrom. 9, 1044–1050.Google Scholar
- Wilm, M. S., and Mann, M. (1994). Electrospray and Taylor-Cone theory, Dole's beam of macromolecules at last? Int. J. Mass Spectrom. Ion Processes 136, 167–180.Google Scholar