Structural assignment of isomeric 2-aminopyridine-derivatized monosialylated biantennary N-linked oligosaccharides using negative-ion multistage tandem mass spectral matching

To investigate the possibility of structural assignment based on negative-ion tandem multistage (MSn) mass spectral matching, four isomers of 2-aminopyridine (PA)-derivatized monosialylated oligosaccharides (i.e., complex-type N-glycans with an alpha 2-3- or alpha 2-6-linked sialic acid on alpha 1-6 or alpha 1-3 antennae) were analyzed using high-performance liquid chromatography/electrospray ion trap time-of-flight mass spectrometry (HPLC/ESI-IT-TOFMS). The negative ion [M-2H](2-) is observed predominantly in the MS' spectra without the loss of a sialic acid. The MS2 spectra derived from it are sufficiently reproducible that MS2 spectral matching based on correlation coefficients can be applied to the assignment of these isomers. The isomers containing a sialic acid on alpha 1-6 or alpha 1-3 antennae can be distinguished by MS2 spectral matching, but the alpha 2-3 and alpha 2-6 linkage types of sialic acid cannot be distinguished by their MS2 spectra. However, MS3 spectra derived from fragment ions containing a sialic acid (i.e., C-4- and D-type ions) clearly differentiate the alpha 2-3 and alpha 2-6 linkage types of sialic acid in their MS3 spectral patterns. This difference might be rationalized in terms of a proton transfer from the reducing-end mannose to the negatively charged sialic acid. These two moieties are very close in the structural conformations of the precursor C-4-type fragment ions of alpha 2-6 linkage type, as predicted by molecular mechanics calculations. Thus, negative-ion MSn (n = 2, 3) spectral matching was demonstrated to be useful for the structural assignment of these four monosialylated PA N-glycan isomers. Copyright (c) 2005 John Wiley & Sons, Ltd.