Abstract
A quadrupole mass spectrometer with an ionspray interface was used to measure the molecular weight (MW) of proteins up to 80,000 u . With the improvements in instrument cahbration by a statistical averaging method and in data analysis by a gaussian curve-fitting method, precision of MW determination as high as 12 ppm was achieved with equine myoglobin (MW 16,950.4 ± 0.2 u). Exact MW determination of three components in cerato-ulmin revealed that the two minor ones had lost amino acid residues Ser and Ser-Asp, respectively, from the major component (MW 7618.4 ± 0.2 u). MW classifIcation of eight components in the Fab fragment of a monoclonal antibody revealed that one set of four had MW − 47,540 u and the other − 47,640 u. The MW difference of 100.2 ± 0.6 u between fragment 1 and 2, attributed to inhomogeneous cleavage at the Fab C-terminus, was probably due to one additional Thr in 1. The MW of bovine serum albumin (BSA) was found to be 66,431.5 ± 1.3 u, − 164 u higher than the calculated sequence MW, most probably because of the incorrectness in the previously reported BSA amino acid sequence. The MW of human serum transferrin (79,556.8 ± 1.7 u) was shown to be 4414 u higher than the sequence MW, pointing to a glycosylation of 22.7 sugar units in this protein. The greater complexity in bovine serum transferrin (MW 78,030.5 ± 1.8 and 78,326 ± 3.3 u for the two major components) was correlated with the heterogeneity in the glycosylation.
Article PDF
Similar content being viewed by others
References
Karas, M.; Bahr, U.; lngendoh, A.; Hillenkamp, F. Agnew. Chem., Int. Ed. Engl. 1989, 28, 760–761.
Beavis, R. C.; Chait, B. T. Anal. Chem. 1990, 62, 1836–1840.
Spengler, B.; Cotter, R. J. Anal. Chem. 1990, 62, 793–796.
Fenn, J.B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64–71.
Smith, R. D.; Loo, J. A.; Edmonds, C. G.; Barinaga, C. J.; Udseth, H. R. Anal. Chem. 1990, 62, 882–899.
Covey, T. R.; Bonner, R. F.; Shushan, B. I.; Henion, J. Rapid Cornrnun. Mass Spectrom. 1988, 2, 249–256.
Macfarlane, R. D.; Sundqvist, B. U. R. Mass Spectrom. Rev. 1985, 4, 421–460.
Jonsson, G.; Hedin, A.; Hakansson, P.; Sundqvist, B. U. R.; Bennich, H.; Roepstorff, P. Rapid Commun. Mass Spectrom, 1989, 3, 190–191.
Hillenkamp, F. Proceedings of the 37th ASMS Conference on Mass Spectrometry and Allied Topics, Miami Beach, FL, May 21–26, 1989, pp 1168–1169.
Loo, J. A.; Udseth, H. R.; Smith, R. D. Anal. Biochem. 1989, 179, 404–412.
Penn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Mass Spectrom. Rev. 1990, 9, 37–70.
Chowdhury, S. K.; Katta, V.; Chait, B. T. Rapid Commun. Mass Specirom. 1990, 4, 81–87.
Henry, K. D.; Williams, E. R.; Wang, B. H.; McLafferty, F. W.; Shabanowitz, J.; Hunt, D. F. Proc. Natl. Acad. Sci. USA 1989, 86, 9075–9078.
Berkel, G. J. V.; Glish, G. L.; McLuckey, S. A. Anal. Chem. 1990, 62, 1284–1295.
Bruins, A. P.; Covey, T. R.; Henion, J. D. Anal. Chem. 1987, 59, 2642–2646.
Miller, I.; Freund, J. E. Probability and Statistics for Engineers, 3rd ed.; Prentice-Hall: Englewood Cliffs, NT, 1985.
Mann, M.; Meng, C. K.; Fenn, J. B. Anal. Chem. 1989, 61, 1702–1708.
Yergey, J.; Heller, D.; Hansen, G.; Cotler, R. J.; Fenselau, C. Anal. Chem. 1983, 55, 353–356.
Hsu, C. S. et al. Private communications.
Wang, B. H.; Williams, E. R.; Henry, K. D.; McLafferty, F. W. Proceedings of the 37th ASMS Conference on Mass Spectrometry and Allied Topics, Miami Beach, FL, May 21–26, 1989, pp 236–237.
Yaguchi, M. et at. Private communications.
Bradley, C. V.; Williams, D. H.; Hanley, M. R. Biochem. Biophys. Res. Commun. 1982, 104, 1223–1230.
Klarskov, K.; Breddam, K.; Roepstorff, P. Anal. Biochem. 1989, 180, 28–37.
Cygler, M. et al. Manuscript in preparation.
Lehninger, A. L. Biochemistry, 2nd ed.; Worth Publishers: New York, 1975; p 1002.
Walker, J. M.; Cox, M. The Language of Biotechnology; American Chemical Society: Washington, DC, 1988, p 127.
Siegel, M. M.; Tsao, R.; Doelling, V. W.; Hollander, I. J. Anal. Chem. 1990, 62, 1536–1542.
Berkel, G. J. V.; Mcl.uckey, S. A.; Glish, G. L. Proceedings of the 38th ASMS Conference on Mass Spectrometry and Allied Topics; Tucson, AZ, June 3–8, 1990, pp 12–13.
Chowdhury, S. K; Kalla, V.; Chait, B. T. Proceedings of the 38th ASMS Conference on Mass Spectrometry and Allied Topics, Tucson, AZ, June 3–8,1990, pp 138–139.
Feng, R; Konishi, Y.; Bell, A. Proceedings of the 38th ASMS Conference on Mass Spectrometry and Allied Topics, Tucson, AZ, June 3–8, 1990, pp 275–276.
Henry, K. D.; Wang, B. H.; Williams, E. R.; Alexander, A. J.; Quinn, J. P.; McLafferty, F. W. Proceedings of the 38th ASMS Conference on Mass Spectrometry and Allied Topics, Tucson, AZ, June 3–8, 1990, pp 433–434.
Hirayama, K.; Akashi, S.; Furuya, M.; Fukuhara, K. Biochem. Biophy. Res. Commun. 1990, 173, 639–646.
Alsen, P.; Listowsky, I. Ann. Rev. Biochem. 1980, 49, 357–393.
Brock, J. H. In Metalloproteins, Part 2; Harrison, P. M., Ed.; Verlag Chemie: Weinhein, 1985; p 183.
MacGillivray, R. T. A.; Mendez, E.; Shewale, J. G.; Sinha, S. K.; Lineback-Zins, J.; Brew, K. J. Biol. Chem. 1983, 258, 3543–3553.
Dorland, L.; Haverkamp, J.; Schut, B. L.; Vliegenthart, J. F. G.; Spik, G.; Strecker, G.; Fournet, B.; Montreuil, J. FEBS Lett. 1977, 77, 15–20.
Stryer, L. Biochemistry, 3rd ed.: W. H. Freeman: New York, 1988, p 344.
Loa, J. A.; Edmonds, C. G.; Udseth, H. R.; Smith, R. D. Anal. Chem. 1990, 62, 693–698.
Feng, R; Bell, A.; Dumas, F.; Konishi, Y. Proceedings of the 38th ASMS Conference on Mass Spectrometr and Allied Topics, Tucson, AZ, June 3–8, 1990, pp 273–274.
Feng, R.; Konishi, Y. Proceedings of the 39th ASMS Conference on Mass Spectrometry and Allied Topics, Nashville, TN, May 19–24, 1991, in press.
Chowdhury, S. K.; Katta, V.; Chait, B. T. J. Am. Chem. Soc. 1990, 112, 9012–9013.
Maeda, K.; McKenzie, H. A.; Shaw, D. C. Anim. Blood Grps. Biochem. Genet. 1980, 11, 63–75.
Edmonds, C. G.; Loo, J. A.; Barinaga, C. J.; Udseth, H. R.; Smith, R. D. J. Chromatog. 1989, 474, 21–37.
Richardson, N. E.; Buttress, N.; Feinstein, A.; Stratil, A.; Spooner, R. L. Biochem. J. 1973, 135, 87–92.
Stratil, A.; Spooner, R. L. Biochem. Genet. 1971, 5, 347–365.
Chowdhury, S. K.; Katta, V.; Beavis, R. C.; Chait, B. T. J. Am. Soc. Mass Spectram. 1990, 1, 382–388.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Feng, R., Konishi, Y. & Bell, A.W. High Accuracy Molecular Weight Determination and Variation Characterization of Proteins Up To 80 ku by Ionspray Mass Spectrometry. J Am Soc Mass Spectrom 2, 387–401 (1991). https://doi.org/10.1016/1044-0305(91)85005-Q
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1016/1044-0305(91)85005-Q