Characterization of braun's lipoprotein and determination of its attachment sites to peptidoglycan by (252)Cf-PD and MALDI time-of-flight mass spectrometry.
A strategy for the characterization of bacterial lipoprotein-in this case Braun's lipoprotein (an outer membrane 7-ku lipoprotein) isolated from Escherichia coli-is described by time-of-flight mass spectrometric (TOF/MS) techniques [(252)Cf plasma desorption (PD) TOF/MS and matrix-assisted laser desorption-ionization (MALDI) TOF/MS]. Covalent linkage 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 class of proteins. Relative molecular mass determination of the major molecular species of Braun's lipoprotein was obtained by selection of an appropriate mixture of organic solvents compatible with matrix/support materials useful for the mass spectrometric techniques applied. Minor components of this lipoprotein that differ only in the fatty acid composition of the lipid anchor were detected by PD TOF/MS after enzymatic release of the extremely hydrophobic N-terminal amino acid followed by selective extraction with chloroform. Part of the primary sequence of this lipoprotein was confirmed based on peptide fragment ions observed 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 were enzymatically released, separated by reverse phase HPLC, and finally characterized by time-of-flight mass spectrometric techniques ((252)Cf-PD TOF/MS, MALDI TOF/MS). The results obtained with both techniques differed only in the better sensitivity obtained with MALDI TOF/MS, which consumed a factor of 100 to 1000 less material than with PD TOF/MS.
Institute for Analytical Chemistry, University of Vienna, Währinger Strasse 38, A-1090, Vienna, Austria.
E
E
Pittenauer
J C
JC
Quintela
E R
ER
Schmid
G
G
Allmaier
G
G
Paulus
M A
MA
de Pedro