Method for investigation of oligosaccharides using phenylhydrazine derivatization.

The reaction of carbohydrates with phenylhydrazine provides corresponding phenylhydrazone derivatives, yielding increased sensitivity for mass spectrometry (MS) and ultraviolet (UV) detection during high-performance liquid chromatography (HPLC) separation. After a simple derivatization procedure, 1 h of incubation at 37-70 degrees C, the samples can be analyzed by mass spectrometry techniques or with a combination of MS with HPLC using electrospray (ESI). Another useful aspect of this tagging is shown in the direct determination of glycosylation sites in proteins. Reaction conditions have enabled visualization of glycans next to their "mother peptides", that are otherwise usually not detectable because of suppression by abundant molecular ions of deglycosylated peptides.We have shown that oligosaccharides as phenylhydrazone derivatives under MALDI-tandem mass spectrometry (MS/MS) and post-source decay (PSD) conditions provide very useful data for the structural elucidation of saccharides, including assignment of dominant isomers. Sialylated oligosaccharides also produce B, C fragment ions and cross-ring cleavage fragments comprising sialic acid.