Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry of lipopolysaccharide species separated by slab-polyacrylamide gel electrophoresis: high-resolution separation and molecular weight determination of lipooligosaccharides from Vibrio fischeri strain HMK.
We recently demonstrated that the combined use of lipopolysaccharide (LPS) reverse staining and high-efficiency passive elution techniques can be successfully used as a suitable interface between LPS slab-gel separation and electrospray ionization-mass spectrometry (ESI-MS) of LPS-derived oligosaccharides. Here, we extend our micropurification strategy for the analysis of O-deacylated LPS forms from Vibrio fischeri HMK after recovery from single reverse-stained LPS bands using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The quantities (30-40 microg) obtained from the two gel-resolved LPS bands were sufficient to allow MALDI-TOF-MS detection of O-deacylated LPS glycoforms at m/z 3767.1, 3890.1 for the high-molecular-weight or at m/z 2522.5, 2645.4, 2725.7, and 2848.7 for the low-molecular-weight LPS band. These LPS band heterogeneities resulted not only from variations in the oligosaccharide region of the LPS but also from two phosphorylation states of the lipid A (diphosphoryl and diphosphoryl plus a single phosphoethanolamine substitution). On the other hand, MALDI-TOF mass spectra of the separated LPS bands displayed reduced heterogeneity and increased signal-to-noise ratios as compared to spectra of the unpurified LPS. Furthermore, micropurification of LPS bands prior MALDI-TOF-MS led to a higher sensitivity of detection of less abundant low-molecular-weight LPS glycoforms. Taken together, this and our previous study on gel-micropurified LPS using ESI definitively show how one can unambiguously determine the different molecular species contained within each gel-separated LPS band, their relative abundance and oligosaccharide sequences.
Pupo
Elder
E
Center for Genetic Engineering and Biotechnology, Havana, Cuba.
Phillips
Nancy J
NJ
Gibson
Bradford W
BW
Apicella
Michael A
MA
Hardy
Eugenio
E
D048908:Aliivibrio fischeri / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D008070:Lipopolysaccharides / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D008070:Lipopolysaccharides