Abstract
MOLECULAR analysis of complex biological structures and processes increasingly requires sensitive methods for protein sequencing. Electrospray mass spectrometry1 has been applied to the high-sensitivity sequencing of short peptides2, but technical difficulties have prevented similar success with gel-isolated proteins. Here we report a simple and robust technique for the sequencing of proteins isolated by polyacrylamide gel electro-phoresis, using nano-electrospray3,4 tandem mass spectrometry5,6. As little as 5ng protein starting material on Coomassie- or silver-stained gels can be sequenced. Multiple-sequence stretches of up to 16 amino acids are obtained, which identify the protein unambiguously if already present in databases or provide information to clone the corresponding gene. We have applied this method to the sequencing and cloning of a protein which inhibits the proliferation of capillary endothelial cells in vitroand thus may have potential antiangiogenic effects on solid tumours.
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References
Fenn, J. B., Mann, M., Meng, C. K., Wong, S. F. & Whitehouse, C. M. Science 246, 64–71 (1989).
Hunt, D. F. et al. Science 255, 1261–1263 (1992).
Wilm, M. S. & Mann, M. Int. J. Mass Spectrom. Ion Process. 136, 167–180 (1994).
Wilm, M. & Mann, M. Analyt. Chem. 68, 1–8 (1996).
Biemann, K. & Scoble, H. A. Science 237, 992–998 (1987).
Hunt, D. F., Yates, J. R., Shabanowitz, J., Winston, S. & Hauer, C. R. Proc. natn. Acad. Sci. U.S.A. 83, 6233–6237 (1986).
Mann, M. & Wilm, M. S. Analyt. Chem. 66, 4390–4399 (1994).
Fotsis, T. et al. Proc. natn. Acad. Sci. U.S.A. 90, 2690–2694 (1993).
Fotsis, T. et al. Nature 368, 237–239 (1994).
Walker, J. E. et al. J. molec. Biol. 226, 1051–1072 (1992).
Hunkapiller, M. et al. Nature 310, 105–111 (1984).
Erdjument-Bromage, H., Lui, M., Sabatini, D. M., Snyder, S. H. & Tempst, P. Protein Sci. 3, 2435–2446 (1994).
Matsudaira, P. (ed.) A Practical Guide to Protein and Peptide Purification for Microsequencing (Academic, San Diego, CA, 1993).
Roepstorff, P. & Fohlman, J. Biomed. Mass Spectrom. 11, 601 (1984).
Jenö, P., Mini, T., Hintermann, E. & Horst, M. Analyt. Biochem. 224, 451–455 (1995).
Rosenfeld, J., Capdevielle, J., Guillemot, J. C. & Ferrara, P. Analyt. Biochem. 203, 173–179 (1992).
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Wilm, M., Shevchenko, A., Houthaeve, T. et al. Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry. Nature 379, 466–469 (1996). https://doi.org/10.1038/379466a0
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DOI: https://doi.org/10.1038/379466a0
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