N-glycosylation site analysis of human platelet proteins by hydrazide affinity capturing and LC-MS/MS

A starting point for many glycosylation analysis pathways is marked by the determination of the respectivecarbohydrate attachment sites to the polypeptide backbone of proteins. Several methods have been reported for this purpose in the past, commonly divided into a three-step approach (1) affinity purification of glycoproteins/-peptides, (2) processing/trimming of the glycopeptides and (3) elucidation of the glycan attachment site by mass spectrometry. For N-glycosylation site analysis the last two steps are usually similar, while methods differ in the affinity purification step. Here, we describe the oxidative derivatisation of carbohydrate moieties and covalent trapping of glycopeptides on hydrazide functionalized beads. This method is suitable for large scale analysis of glycoproteins as well as isolated glycoproteins and can be applied readily to a number of different samples. In the described protocol, the elucidation of N-glycosylation sites of human platelet proteins is demonstrated as an example.