PEPTIDE REFOLDING DEFINED BY ENZYMATIC DIGESTION

  • Backlund, Peter S. (PI)
  • Cowburn, David (PI)
  • Roeder, Robert G. (PI)
  • Darst, Seth A. (PI)
  • Merrifield, Robert B. (PI)

Project: Research project

Project Details

Description

We have synthesized a cysteine-rich domain in h-factor IX. This EFG-like domain contains 45 amino acids and three disulfide bonds. An extremely difficult point in this work is the definition of the peptide disulfide bond refolding pattern. In order to characterize this peptide and deduce the correct and formation of native disulfide bonds, a series of enzymatic digestions are carried out (under non-reducing conditions) and the disulfide connectivity is established using MS. Typically, V8-protease and trypsin are used to produce the needed proteolytic peptides. During these studies, we have found that matrix-assisted laser desorption ion trap mass spectrometry is particularly useful for this application to S-S mapping (J. Qin & B. T. Chait "Rapid, Accurate Identification and Characterization of Posttranslational Modifications of Proteins by MALDI Ion Trap Mass Spectrometry" Anal. Chem. 69 (1997) 4002 - 4009).
StatusFinished
Effective start/end date10/1/919/30/01

Funding

  • National Center for Research Resources

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  • Natl Resource for Mass Spectrom of Biolog Macromolecules

    Chait, B. T., Abreu, I. A., Adzuma, K., Aebersold, R. H., Aitchison, J. D., Allis, C. D., Andren, P. E., Backlund, P. S., Beavis, R. C., Bern, M. W., Beyreuther, K., Birken, S., Blobel, G., Boeke, J. D., Bradley, C. C., Bradshaw, R. A., Breslow, N. J., Breslow, J. L., Burley, S. K., Burley, S., Buxbaum, J. D., Cadene, M., Campbell, E. A., Canfield, R. E., Canfield, R., Cardozo, C., Cerami, A., Chang, E. J., Chang, E. Y., Chen, E., Christea, I. M., Chua, N., Cohen, S. L., Cohen, S., Cowburn, D., Cross, G. A. M., Cross, F. R., Darnell, J. E., Darst, S. A., de Lange, T., Degrasse, J., Demmers, J., Deshaies, R. J., Dodd, J., Duff, K. E., Dutzler, R., Engelberg-Kulka, H., Eriksson, J., Fenyo, D., Fenyo, J., Field, F. H., Field, M., Fischetti, V. A., Friedman, J. M., Funabiki, H., Gadsby, D. C., Gandy, S. E., Gautier, J., Gilman, A. G., Giriat, I., Glavy, J. S., Goddard, N. L., Gotschlich, E. C., Greene, L. J., Greengard, P., Grendell, J. H., Gu, W., Hajjar, K. A., Haley, J. D., Hall, W. W., Heintz, N., Hingorani, M. M., Ho, D. D., Holford, M. N., Hudspeth, A. J., Hugli, T. E., Jacobs, E. Y., Jeruzalmi, D., Jiang, Y., Jin, M., Jin, S. V., Jordan, B., Jordan, W. B., Kaiser, N. E., Kalkum, M., Kapoor, T. M., Kato, H., Keeney, S., Kent, S., Kim, Y. I., Kipper, J., Kreek, M. J., Kretzschmar, M. D., Krutchinsky, A. N., Kufe, D. W., Kuriyan, J., Lander, H. M., Lazarow, P. B., Leonard, J., Lima, C. D., Lindsey, J., Lindsey, J., Lipton, S. A., Lloyd, N. K., Logothetis, D. E., Luo, Y., MacKinnon, R., Makriyannis, A., Malamud, D., Manning, J. M., Marian Orlowski, N., Matunis, M. J., Mclachlin, D. T., Merrifield, R. B., Merrifield, B., Merrifield, N. R., Miller, D. L., Miller, C. M., Minakhin, L., Mirza, U. A., Mirza, U., Molloy, K., Muesing, M. A., Muir, T., Muir, T. W., Müller, M., Myung, S., Nairn, N. A., Nairn, A. C., Nakanishi, K., Neubert, T. A., Niu, S., Nogoceke, E., Nui, S., Nussenzweig, M. C., Nussenzweig, R. S., O'donnell, M. E., Oakley, M. G., Oda, Y., Oelgeschlager, T., Orlowski, M., Padovan, J. C., Padovan, J., Peterson, C. L., Pochapsky, T. C., Prusiner, S. B., Qin, J., Raleigh, D. P., Ravetch, J. V., Rice, C. M., Riggs, A. F., Roeder, R. G., Romanelli, A., Rout, M. P., Sakmar, T. P., Sali, A., Sechi, S., Sehayek, E., Seibert, C., Sekedat, M., Severinov, N. K., Severinov, K. V., Shenk, T. E., Smith, A. B., Speigel, A., Spiegel, A. M., Stebbins, C. E., Steinman, R. M., Steller, H., Stoffel, M., Stuart, A., Tackett, A. J., Tam, J. P., Tang, N. H., Tarakhovsky, A., Tischer, E., Tjernberg, A., Tomasz, N. A., Trester, M., Ueberheide, B. M., Upmacis, R. K., Vallee, R. B., Varmus, H. E., Wang, Q., Wang, R., Wang, Z., Weiss, E., Wendel, H., Werner, M. H., Wlodawer, A., Woo, E., Woolford, J. L., Wu, X., Yanagisawa, K., Young, R., Yue, Z., Zhang, J. J., Zhang, J., Zhang, W., Zhao, Y., Zimniak, P., Buku, A., Chait, B. T., Goodman, S., Kappas, A., Lin Yao-zhong Lin, N., Marchal, G., Severinov, K., Tang, H. & Unson, C.

    12/1/843/31/12

    Project: Research project