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 mass spectrometric peptide mapping of tryptic peptide mixtures have been employed for surface topology-probing of lysine residues in bovine ribonuclease A, lysozyme, and horse heart myoglobin as model proteins of different surface st...
Saved in:
Published in | Bioconjugate chemistry Vol. 5; no. 6; pp. 583 - 590 |
---|---|
Main Authors | , , , , |
Format | Journal Article |
Language | English |
Published |
United States
American Chemical Society
01.11.1994
|
Subjects | |
Online Access | Get full text |
Cover
Loading…
Summary: | Amino-acetylation and -succinylation reactions in combination with mass spectrometric peptide mapping of tryptic peptide mixtures have been employed for surface topology-probing of lysine residues in bovine ribonuclease A, lysozyme, and horse heart myoglobin as model proteins of different surface structures. Direct molecular weight determinations identifying the precise number of acyl groups in partially modified proteins were obtained by electrospray and 252Cf-plasma desorption mass spectrometry. Electrospray mass spectra of multiply protonated molecular ions and deuterium exchange experiments provided a relative conformational characterization of protein derivatives and enabled the direct determinations of intact, partially acylated heme-myoglobin derivatives. 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 exact identification of acylation sites. Relative reactivities of the amino acylation were derived from the peptide mapping data and from quantitative estimations of modified peptides upon acetylation/trideuteroacetylation and provided direct correlations with the relative surface accessibilities of lysine-epsilon-amino groups taken from X-ray crystallographic structure data of the proteins. The reactive lysine-41 residue in ribonuclease A which is part of the substrate binding site was directly identified from the mass spectrometric data. These results indicate tertiary structure-selective acylation combined with mass spectrometric peptide mapping as an efficient approach for the molecular characterization of surface topology and reactive fundamental lysine residues in proteins. |
---|---|
Bibliography: | istex:0B6ED50C414B7231DAE0052F990F5E6C85BAEF04 ark:/67375/TPS-129QVJQC-P ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 1043-1802 1520-4812 |
DOI: | 10.1021/bc00030a014 |