Negative ion mass spectrometry of sialylated carbohydrates: discrimination of N-acetylneuraminic acid linkages by MALDI-TOF and ESI-TOF mass spectrometry

Negative ion MALDI and electrospray fragmentation spectra were recorded from 12 sialylated carbohydrates ranging from trisaccharides to biantennary N-linked glycans. D-Arabinosazone was found to be the most satisfactory MALDI matrix for these compounds. Fragmentation mechanisms were investigated with the aid of several synthesized analogues of the sugars labeled with 13C and 2H. The substitution position of the sialic acid (alpha2-->3 or alpha2-->6) was found to have a dramatic effect on the overall fragmentation pattern of these compounds, and several features of the spectra were identified that allowed the substitution pattern to be determined. In particular, the appearance of an ion at m/z 306 appeared to be diagnostic of the presence of an alpha2-->6-linked sialic acid. Selection and further fragmentation of the in-source (conevoltage) fragment ion corresponding to the trisaccharide Neu5Acalpha2-->3(or 6)Galbeta1-->4GlcNAc from larger, N-linked glycans, ionized by electrospray, gave fragmentation patterns identical to those of the reference trisaccharides, thus providing a method for confirming the sialic acid linkage.