Structural characterization of Mycobacterium tuberculosis lipoarabinomannans by the combination of capillary electrophoresis and matrix-assisted laser desorption/ionization time of-flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometr y (MALDI-TOF-MS) associated with capillary electrophoresis (CE) has been us ed for structural characterization of mannooligosaccharide caps from Mycoba cterium tuberculosis H37rv mannosylated lipoarabinomannans (ManLAMs), The m annooligosaccharide caps were released by mild acid hydrolysis, labeled wit h 1-aminopyrene-3,6,8-trisulfonate (APTS) prior to being separated by CE, c ollected, and analyzed by MALDI-TOFMS and post-source decay experiments. Th is approach was optimized using standard APTS-labeled oligosaccharides. Wit h the selected (9:1) mixture of 2,5-dihydroxybenzoic acid (DHB) and 5-metho xysalicylic acid (MSA) as matrix and the on-probe sample cleanup procedure with cation-exchange resin, standard APTS-maltotriose was successfully dete cted down to 50 fmol using linear-mode negative MALDI-TOF-MS. Moreover, usi ng extraction delay time, only 100 and 500 fmol of this standard were requi red, respectively, to obtain accurate reflectron mass measurements and sequ ence determination through post-source decay experiments. Applied to only 5 mug (294 pmol) of M, tuberculosis ManLAMs, this analytical approach allowe d successful mass characterization of the mannooligosaccharide cap structur es from the deprotonated molecular ions [M - H](-) and the y-type ion fragm ents obtained in post-source decay experiments. This powerful analytical ap proach opens new insights into both the characterization of oligosaccharide s and the capping motifs displayed by ManLAMs purified from mycobacteria is olated from tubercular patients without in vitro culturing.