MOLECULAR CHARACTERIZATION OF SURFACE-TOPOLOGY IN PROTEIN TERTIARY STRUCTURES BY AMINO-ACYLATION AND MASS-SPECTROMETRIC PEPTIDE-MAPPING

Amino-acetylation and -succinylation reactions in combination with mas s spectrometric peptide mapping of tryptic peptide mixtures have been employed for surface topology-probing of lysine residues in bovine rib onuclease A, lysozyme, and horse heart myoglobin as model proteins of different surface structures. Direct molecular weight determinations i dentifying the precise number of acyl groups in partially modified pro teins were obtained by electrospray and Cf-252-plasma desorption mass spectrometry. Electrospray mass spectra of multiply protonated molecul ar ions and deuterium exchange experiments provided a relative conform ational characterization of protein derivatives and enabled the direct determinations of intact, partially acylated heme-myoglobin derivativ es. Tryptic peptide mapping analysis, using plasma desorption and fast atom bombardment mass spectrometry, ascertained by mass spectrometric characterization of HPLC-separated modified peptides, yielded the exa ct identification of acylation sites. Relative reactivities of the ami no acylation were derived from the peptide mapping data and from quant itative estimations of modified peptides upon acetylation/trideuteroac etylation and provided direct correlations with the relative surface a ccessibilities of lysine-epsilon-amino groups taken from X-ray crystal lographic structure data of the proteins. The reactive lysine-41 resid ue in ribonuclease A which is part of the substrate binding site was d irectly identified from the mass spectrometric data. These results ind icate tertiary structure-selective acylation combined with mass spectr ometric peptide mapping as an efficient approach for the molecular cha racterization of surface topology and reactive fundamental lysine resi dues in proteins.