CHARACTERIZATION OF BRAUN LIPOPROTEIN AND DETERMINATION OF ITS ATTACHMENT SITES TO PEPTIDOGLYCAN BY CF-252-PD AND MALDI TIME-OF-FLIGHT MASS-SPECTROMETRY
E. Pittenauer et al., CHARACTERIZATION OF BRAUN LIPOPROTEIN AND DETERMINATION OF ITS ATTACHMENT SITES TO PEPTIDOGLYCAN BY CF-252-PD AND MALDI TIME-OF-FLIGHT MASS-SPECTROMETRY, Journal of the American Society for Mass Spectrometry, 6(10), 1995, pp. 892-905
A strategy for the characterization of bacterial lipoprotein-in this c
ase Braun's lipoprotein (an outer membrane 7-ku Lipoprotein) isolated
from Escherichia coli-is described by time-of-flight mass spectrometri
c (TOF/MS) techniques [Cf-252 plasma desorption (PD) TOF/MS and matrix
-assisted laser desorption-ionization (MALDI) TOF/MS]. Covalent linkag
e of lipid at the N-terminal cysteine (posttranslationally modified to
a S-[2,3-bis(acyloxy)-propyl]-N-acylcysteine) and, therefore, strict
insolubility in aqueous solution constitute common features for this c
lass of proteins. Relative molecular mass determination of the major m
olecular species of Braun's Lipoprotein was obtained by selection of a
n appropriate mixture of organic solvents compatible with matrix/suppo
rt materials useful for the mass spectrometric techniques applied. Min
or components of this lipoprotein that differ only in the fatty acid c
omposition of the lipid anchor were detected by PD TOF/MS after enzyma
tic release of the extremely hydrophobic N-terminal amino acid followe
d by selective extraction with chloroform Part of the primary sequence
of this lipoprotein was confirmed based on peptide fragment ions obse
rved in the positive ion PD mass spectra of cyanogen bromide-generated
peptide fragments that had been isolated previously by reverse phase
high-performance liquid chromatography (HPLC). Peptidoglycan fragments
that represent the attachment sites of Lipoprotein to peptidoglycan w
ere enzymatically released, separated by reverse phase HPLC, and final
ly characterized by time-of-flight mass spectrometric techniques (Cf-2
52-PD TOF/MS, MALDI TOF/MS). The results obtained with both techniques
differed only in the better sensitivity obtained with MALDI TOF/MS, w
hich consumed a factor of 100 to 1000 less material than with PD TOF/M
S.