Analysis of carbohydrates on proteins by offline normal-phase liquid chromatography MALDI-TOF/TOF-MS/MS.

Glycoproteins typically exist as a diverse population of glycoforms, which can consist of a great number of different glycan structures (including structural isomers) that vary in their degree of occupancy at given glycosylation sites. Hence, the detailed characterization of such glycans can be a very demanding task. Liquid chromatography in combination with mass spectrometric techniques can provide a fast and sensitive tool for the analysis of these structurally complex molecules. Here we describe a sensitive method that employs capillary normal-phase HPLC coupled offline to MALDI-TOF/TOF-MS/MS for the detailed characterization of enzymatically released N-glycans. The normal-phase chromatography allows the separation of some isobaric glycan structures and analysis of the glycans by MALDI high-energy CID gives sequence, linkage and branching information.