Abstract
The rapid accumulation of sequence data generated by the various genome sequencingprojects and the generation of expressed sequence tag databases has resulted in the need forthe development of fast and sensitive methods for the identification and characterisation oflarge numbers of gel electrophoretically separated proteins to translate the sequence data intobiological function. To achieve this goal it has been necessary to devise new approaches toprotein analysis: matrix-assisted laser desorption and electrospray mass spectrometry havebecome important protein analytical tools which are both fast and sensitive. When combinedwith a robotic system for the in-gel digestion of electrophoretically separated proteins, itbecomes possible to rapidly identify many proteins by searching databases with MS data. Thepower of this combination of techniques is demonstrated by an analysis of the proteins presentin the myofibrillar lattice of the indirect flight muscle of Drosophila melanogaster. Theproteins were separated by SDS-PAGE and in-gel proteolysis was performed bothautomatically and manually. All 16 major proteins could quickly be identified by massspectrometry. Although most of the protein components were known to be present in theflight muscle, two new components were also identified. The combination of methodsdescribed offers a means for the rapid identification of large numbers of gel separatedproteins.
Similar content being viewed by others
REFERENCES
Gaasterland, T. and Sensen, C.W., Automated Microbial Genome Analysis, Tutorial given at the 4th International Conference on Intelligent Systems for Molecular Biology, St. Louis, MO, U.S.A., June 12, 1996.
Boguski, M.S., Lowe, T.M. and Tolstoshev, C.M., Nat. Genet., 4 (1993) 332.
Swinbanks, D., Nature, 378 (1995) 653.
Kahn, P., Science, 270 (1995) 368.
Wilkins, M.R., Pasquali, C., Appel, R.D., Ou, K., Golaz, O., Sanchez, J.C., Yan, J.X., Gooley, A.A., Hughes, G., Humphery-Smith, I., Williams, K.L. and Hochstrasser, D.F., Biotechnology, 14 (1996) 61.
Shevchenko, A., Wilm, M., Vorm, O. and Mann, M., Anal. Chem., 68 (1996) 850.
Eckerskorn, C. and Lottspeich, F., Chromatographia, 28 (1989) 92.
Houthaeve, T., Gausepohl, H., Mann, M. and Ashman, K., FEBS Lett., 376 (1995) 91.
Houthaeve, T., Gausepohl, H., Ashman, K., Nillson, T. and Mann, M., J. Protein Chem., in press.
Mann, M. and Talbo, G., Curr. Opin. Biotechnol., 7 (1996) 11.
Mann, M. and Wilm, M., Trends Biochem. Sci., 20 (1995) 219.
Jensen, O.N., Podtelejnikov, A. and Mann, M., Rapid Commun. Mass Spectrom., 10 (1996) 1371.
Mann, M., In Kellner, R., Lottspeich, F. and Meyer, H.E. (Eds.) Microcharacterisation of Proteins, VCH, Weinheim, Germany, 1994, pp. 223–244.
Wilm, M., Shevchenko, A., Houthaeve, T., Breit, S., Schweigerer, L., Fotsis, T. and Mann, M., Nature, 379 (1996) 466.
Peckham, M., Molloy, J.E., Sparrow, J.C. and White, D.C.S., J. Muscle Res. Cell Motil., 11 (1990) 203.
Cripps, R.M. and Sparrow, J.C., Biochem. Genet., 30 (1992) 159.
Vorm, O., Roepstorff, P. and Mann, M., Anal. Chem., 66 (1994) 3281.
Shevchenko, A., Jensen, O.N., Podtelejnikov, A.V., Sagliocco, F., Wilm, M., Vorm, O., Mortensen, P., Shevchenko, An., Boucherie, H. and Mann, M., Proc. Natl. Acad. Sci. USA, 93 (1996) 14440.
Ball, E., Karlik, C.C., Beall, C.J., Saville, D.L., Sparrow, J.C., Bullard, B. and Fyrberg, E.A., Cell, 51 (1987) 221.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Ashman, K., Houthaeve, T., Clayton, J. et al. The application of robotics and mass spectrometry to the characterisation of the Drosophila melanogaster indirect flight muscle proteome. Letters in Peptide Science 4, 57–65 (1997). https://doi.org/10.1023/A:1008827212347
Issue Date:
DOI: https://doi.org/10.1023/A:1008827212347