Investigation of different combinations of derivatization, separation methods and electrospray ionization mass spectrometry for standard oligosaccharides and glycans from ovalbumin

Derivatization procedures using 1-phenyl-3-methyl-5-pyrazolone (PMP) and 2- aminonaphthalene trisulfone (ANTS) were selected among a number of well kno wn methods for labelling carbohydrates. PMP derivatives were selected owing to our laboratory's previous high-performance liquid chromatography/electr ospray ionization mass spectrometry (HPLC/ESI-MS) experience with these, wh ereas the ANTS-labelled compounds were prepared for fluorophore-assisted ca rbohydrate electrophoresis (FACE) separation. ANTS-oligosaccharide standard s were characterized to study their ionization patterns. Reversed-phase and normal-phase HPLC systems were coupled on-line with ESI-MS. Each necessita ted its own mobile phase system which, in turn, imposed some important chan ges in the ionization conditions used and/or on the ionization patterns and spectra obtained. Following characterization of the intact glycoprotein ov albumin with ESI-MS, its glycans were detached using the enzyme PNGase-F. T he glycans were subjected to PMP and ANTS derivatization. It was very diffi cult to separate ANTS derivatives by reversed-phase HPLC owing to lack of r etention, and normal-phase HPLC offered reasonable retention with limited s eparation. PMP compounds overall yielded better normal- and reversed-phase separations and improved sensitivity over the ANTS-labelled sugars, for whi ch negative mode ESI had to be used. The combination of ESI of intact ovalb umin and ESI of PMP-glycans gave rise to the detection of over 20 different glycoforms, excluding the possible presence of structural isomers for each sugar composition detected. Copyright (C) 2001 John Wiley & Sons, Ltd.