Direct microextraction and analysis of rough-type lipopolysaccharides by combined thin-layer chromatography and MALDI mass spectrometry

A rapid method for the microscale extraction of lipopolysaccharides (endoto xins, LPSs) from rough-type Gram-negative bacteria was developed using thin -layer chromatography (TLC) combined with improved conditions for LPS analy sis by mass spectrometry. TLC of intact bacteria on silica gel plates in an appropriate solvent selectively extracted and separated their LPS componen ts. The bands of molecular species were scraped from the plates after nonde structive visualization, directly mixed with matrix, and analyzed by laser desorption time-of-flight mass spectrometry. Lipids A and Re-type LPSs were analyzed after transfer to a membrane. Adding citric acid to the matrix ga ve greatly improved mass spectra. The system allows characterization of bac terial LPS at the microscale level and is equally well applicable to hetero geneous LPS and lipid A preparations (Escherichia coli lipid A and Bordetel la lipopolysaccharides were used). The technique provides a rapid determina tion of the heterogeneity of unmodified preparations and the determination of the molecular weight of each separated component.