Characterization of mannooligosaccharide caps in mycobacterial lipoarabinomannan by capillary electrophoresis/electrospray mass spectrometry

A new analytical approach based on capillary electrophoresis-electrospray m ass spectrometry (CE/ESI-MS) has provided new insight into the characteriza tion of mannooligosaccharide caps from lipoarabinomannans (LAMs), which are key molecules in the immunopathogenesis of tuberculosis. This analytical a pproach requires oligosaccharide labeling with the fluorophore 1-aminopyren e-3,6,8-trisulfonate (APTS) by reductive amination at the reducing termini. Optimization of the separation and ionization conditions, such as the choi ce of capillary electrophoresis (CE) electrolyte buffers, is presented and discussed. Anionic separation of the mono and oligosaccharide APTS derivati ves was finally achieved with aqueous triethylammonium formate buffer. It w as found that in contrast to the triethylammonium phosphate buffer, the tri ethylammonium formate buffer was appropriate for CE/ESI-MS coupling analysi s of APTS-carbohydrate derivatives. In this case, negative ESI-mass spectra of APTS-carbohydrate adducts showed mainly (M-2H)(2-) pseudomolecular ions and some sequence fragment ions allowing their non-ambiguous structural ch aracterization at the picomolar level. This analytical approach was success fully applied to more complex mixtures of carbohydrates released by mild ac id hydrolysis of the lipoarabinomannans from Mycobacterium bovis BCG. The A PTS-mannooligosaccharide cap adducts were separated by CE and their structu ral characterization achieved by CE/ESI-MS analyses. Mannooligosaccharide c aps were routinely analyzed by capillary electrophoresis-laser induced fluo rescence (CE-LIF) from 50 fmol of lipoarabinomannans with mannosyl capping (ManLAMs) but sensitivity was about 50 times lower using ESI-MS detection.