Anal. Chem. 1995, 67, 2864-2869

Electrospray Mass Spectrometric Study of Melittin Trypsinolysis by a Kinetic Approach

Ekatherina P. Mirgorodskaya, Olga A. Mirgorodskaya, and Sergey V. Dobretsov
Institute for Cytology of the Russian Academy of Sciences, 4 Tikhoretsky Avenue,
194064 St. Petersburg, Russia

Andrei A. Shevchenko
Institute for Analytical Instrumentation of the Russian Academy of Sciences,
198103 St, Petersburg, Russia

Alexander F. Dodonov, Vyacheslav I. Kozlovskiy, and Valeri V. Raznikov
Institute of Energy Problems of Chemical Physics, Russian Academy of Sciences,
142432 Chemogolovka, Moscow Region, Russia

Analytical Chemisfry, Vol. 67, No. 17, September 1, 1995, pp. 2864–2869.

Received for review July 11, 1994. Accepted March 30, 1995

   The kinetics of tryptic digestion of melittin was studied by combined electrospray ionization time-of-flight mass-spectrometry and high-performance liquid chromatography. The ratios of the kinetic constants for cleavage of the peptide bonds that are susceptible to trypsin action were determined. It is shown that trypsin does not manifest affinity for the hydrolysis of the peptide bonds inside the Arg, Lys cluster series as efficiently as it cleaves the peptide at the separately localized Lys residue. This feature demonstrates clearly the advantage of the kinetic approach to tryptic mapping of proteins. The kinetic approach allows the determination of not only discrete structural segments in protein structure but also their relative locations and their amino acid sequences. Using the melittin digests and some artificially prepared amino acids and dipeptides mixtures as models, it is shown that the presence and nature of basic amino acids predetermines the charge states of the molecules analyzed by electrospray but not the yields of their ions. The aliphatic parts of the molecules seem to be more important in determining the actual ion yields.

© 1995 American Chemical Society