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

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Abstract
Derivatization procedures using 1‐phenyl‐3‐methyl‐5‐pyrazolone (PMP) and 2‐aminonaphthalene trisulfone (ANTS) were selected among a number of well known methods for labelling carbohydrates. PMP derivatives were selected owing to our laboratory's previous high‐performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI‐MS) experience with these, whereas the ANTS‐labelled compounds were prepared for fluorophore‐assisted carbohydrate electrophoresis (FACE) separation. ANTS‐oligosaccharide standards were characterized to study their ionization patterns. Reversed‐phase and normal‐phase HPLC systems were coupled on‐line with ESI‐MS. Each necessitated its own mobile phase system which, in turn, imposed some important changes in the ionization conditions used and/or on the ionization patterns and spectra obtained. Following characterization of the intact glycoprotein ovalbumin with ESI‐MS, its glycans were detached using the enzyme PNGase‐F. The glycans were subjected to PMP and ANTS derivatization. It was very difficult to separate ANTS derivatives by reversed‐phase HPLC owing to lack of retention, and normal‐phase HPLC offered reasonable retention with limited separation. PMP compounds overall yielded better normal‐ and reversed‐phase separations and improved sensitivity over the ANTS‐labelled sugars, for which negative mode ESI had to be used. The combination of ESI of intact ovalbumin 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 © 2001 John Wiley & Sons, Ltd.

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