N-glycosylation proteome of endoplasmic reticulum in mouse liver by ConA affinity chromatography coupled with LTQ-FT mass spectrometry

Abstract

Glycosylation is the most versatile and one of the most significant protein post-translational modifications. It is generally classified into three categories according to the amino acid to which the glycan is attached: N-glycosylation, O-glycosylation and C-glycosylation. Synthesis of N-glycoproteins occurs in the rough endoplasmic reticulum (rER), and all N-glycoproteins synthesized in rER have uniform glycan endings with mannose (Man) and glucose (Glc). A systematic strategy was developed to comprehensively profile N-glycoproteins in complex biological samples. The lectin Concanvalin A (ConA), which has a high affinity for glycans ending with Man, was used to extract the N-glycoproteins synthesized or located in the rER, and identified the N-glycoproteins and their glycosylation sites by LTQ-FT MS. The analysis was repeated three times at both the biological sample and mass spectrometry levels. In total, 323 glycosylation sites on 212 N-glycoproteins were identified in the mouse liver. Of these, 131 were known N-glycoproteins in the Swissprot database. The identified N-glycoproteins were classified according to their cell location by the Swissprot database and GO software. The identified N-glycoproteins in this study would serve as a good complement to the N-glycoprotein database for the mouse liver. © Science China Press and Springer-Verlag Berlin Heidelberg 2010.