Abstract
A combination of techniques is presented allowing gel-purified protein identification in the femtomole range using matrix-assisted-laser-desorption-ionization mass spectrometry. The proteins are detected in the primary gel by a sensitive negative staining procedure, transferred, and concentrated in a secondary gel matrix. There, they are digested in the presence of H2 18O and their sequences are predicted (1) by peptide mass fingerprinting, (2) by comparing the post-source-decay (PSD) spectra with theoretical spectra of candidate isobaric peptides using a computer algorithm called MassFrag, and (3) by a manual readout of the 18O/16O-labeled fragmentation ions in the PSD spectra.
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REFERENCES
Aebersold, R., Teplow, D. B., Hood, L. E., and Kent, S. B. (1986). J. Biol. Chem. 261, 4229–4239.
Aebersold, R. H., Leavitt, J., Saavedra, R. A., Hood, L. E., and Kent, S. B. H. (1987). Proc. Natl. Acad. Sci. USA 84, 6970–6974.
Bauw, G., De Loose, D., Inze, M., Van Montagu, M., and Vandekerckhove, J. (1987). Proc. Natl. Acad. Sci. USA 84, 4806–4810.
Biemann, K. (1990). In Methods in Enzymology, Vol. 193 (McCloskey, J. A., ed.), Academic Press, San Diego, pp. 455–480.
Eckerskorn, C., Mewes, W., Goretzki, H., and Lottspeich, F. (1988). Eur. J. Biochem. 176, 509–515.
Fernandez-Patron, C., Calero, M., Collazo, P. R., Garcia, J. R., Madrazo, J., Musacchio, A., Soriano, F., Estrada, R., Frank, R., Castellanos-Serra, L. R., and Mendez, E. (1995). Anal. Biochem. 224, 203–211.
Gevaert, K., Rider, M. H., Puype, M., Van Damme, J., De Boeck, S., and Vandekerckhove, J. (1995). In Methods in Protein Structure Analysis (Atassi, M. Z. and Appella, E., eds.), Plenum Press, New York, pp. 15–26.
Gevaert, K., Verschelde, J.-L., Puype, M., Van Damme, J., Goethals, M., De Boeck, S., and Vandekerckhove, J. (1996). Electrophoresis 17, 918–924.
Henzel, W. J., Billecci, T. M., Stults, J. T., Wong, S. C., Grimley, C., and Watanabe, C. (1993). Proc. Natl. Acid. Sci. USA 90, 5011–5015.
Hess, D., Covey, T. C., Winz, R., Brownsey, R., and Aebersold, R. (1993). Protein Sci. 2, 1342–1351.
Kaufmann, R., Spengler, B., and Lutzenkirchen, F. (1993). Rapid Commun. Mass Spectrom. 7, 902–910.
Matsudaira, P. J. (1987). J. Biol. Chem. 262, 10035–10038.
Matsudaira, P. J., and Burgess, D. R. (1978). Anal. Biochem. 87, 386–396.
O'Farrell, P. H. (1975). J. Biol. Chem. 250, 4007–4021.
Pappin, D. J., Højrup, P., and Bleasby, A. J. (1993). Curr. Biol. 3, 327–332.
Rider, M. H., Puype, M., Van Damme, J., Gevaert, K., De Boeck, S., D'Alayer, J., Rasmussen, H.-H., Celis, J. E., and Vandekerckhove, J. (1995). Eur. J. Biochem. 230, 258–265.
Rosenfeld, J., Capdevielle, J., Guillemot, J. C., and Ferrara, P. (1992). Anal. Biochem. 203, 173–179.
Schnölzer, M., Jedrzejewski, P., and Lehmann, W. D. (1996). Electrophoresis 17, 945–953.
Takao, T., Gonzalez, J., Yoshidome, K., Sato, K., Asada, T., Kammei, Y., and Shimonishi, Y. (1993). Anal. Chem. 65, 2394–2399.
Yates, J. R., Eng, J. K., McCormack, A. L., and Schieltz, D. (1995). Anal. Chem. 67, 1426–1436.
Vandekerckhove, J., Bauw, G., Puype, M., Van Damme, J., and Van Montagu, M. (1985). Eur. J. Biochem. 152, 9–19.
Weiner, A. M., Platt, T., and Weber, K. (1972). J. Biol. Chem. 247, 3242–3251.
Wilm, M., Shevchenko, A., Houthaeve, T., Breit, S., Schweigerer, L., Fotsis, T., and Mann, M. (1996). Nature 379, 466–469.
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Gevaert, K., De Mol, H., Verschelde, JL. et al. Novel Techniques for Identification and Characterization of Proteins Loaded on Gels in Femtomole Amounts. J Protein Chem 16, 335–342 (1997). https://doi.org/10.1023/A:1026320318489
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DOI: https://doi.org/10.1023/A:1026320318489