Rapid and sensitive analysis of N-glycans by MALDI-MS using permanent charge derivatization and methylamidation

Abstract Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has become an important technology for glycan analysis due to its ease of operation, short analysis time and impurity tolerance. However, the low ionization efficiency of N-glycans led to the difficulty in analyzing glycans of low abundance in complex biological samples due to the lack of basic site for protonation. Therefore, highly sensitive method for the glycans analysis is in urgent demand. Here we report a new strategy to introduce a permanent charge at the reducing end of N-linked glycans by a one pot reaction, where glycosylamines that were obtained by rapid deglycosylation within 5 min were labeled with N-succinimidyloxycarbonylmethyl tris (2,4,6- trimethoxyphenyl) phosphonium bromide (TMPP-Ac-OSu). With TMPP-Ac labeling, more than 50 fold enhancement in the sensitivity of method was achieved for neutral glycans from ribonuclease B (RNase B) in comparison to their native counterparts. In combination with methylamidation of sialic acid residues, this novel developed strategy could also be used for sialylated glycans analysis from sialoglycoproteins and complex serum sample. As a result, more than 50 glycans were detected with only 25 nL human serum sample.