Structural assignment of disialylated biantennary N‐glycan isomers derivatized with 2‐aminopyridine using negative‐ion multistage tandem mass spectral matching

To investigate the possibility of structural assignment based on negative‐ion multistage tandem mass (MS n ) spectral matching, four isomers of disialylated biantennary N‐glycans ( α 2‐6 and/or α 2‐3 linked sialic acid on α 1‐6 and α 1‐3 antennae) derivatized with 2‐aminopyridine (PA) were analyzed by employing high‐performance liquid chromatography/electrospray ionization linear ion trap time‐of‐flight mass spectrometry (HPLC/ESI‐LIT‐TOFMS), which uses helium gas for ion trapping and collision‐induced dissociation (CID). It is shown that the MS 2 spectra derived from each precursor ion [M−2H] 2− are reproducible and useful for distinguishing the four isomers. Thus, they can be assigned by negative‐ion MS 2 spectral matching based on correlation coefficients. In addition, MS 3 spectra derived from D‐type fragment ions clearly differentiate the α 2‐3‐ or α 2‐6‐linked sialic acid on the α 1‐6 antenna due to their characteristic spectral patterns. The C 4 ‐type fragment ions, which are produced from both the α 1‐6 and α 1‐3 antennae, show the characteristic MS 3 spectra reflecting α 2‐3‐ or α 2‐6‐ linkage type or a mixture of both types. Thus, the differentiation and assignment of these disialylated biantennary N‐glycan isomers can also be supported with the MS 3 spectra of C 4 ‐ and D‐type ions. Copyright © 2006 John Wiley & Sons, Ltd.