High-throughput N-glycoproteomics with fast liquid chromatographic separation.

N-glycosylation is a common protein post translation modification, which has tremendous structure diversity and wide yet delicate regulation of protein structures and functions. Mass spectrometry-based N-glycoproteomics has become a state-of-the-art pipeline for both qualitative and quantitative characterization of N-glycosylation at the intact N-glycopeptide level, providing comprehensive information of peptide backbones, N-glycosites, monosaccharide compositions, sequence and linkage structures. For high-throughput analysis of large-cohort clinic samples, fast and high-performance separation is indispensable. Here we report our development of 1-h liquid chromatography gradient N-glycoproteomics method and accordingly optimized MS parameters. In the benchmark analysis of cancer and paracancerous tissue of hepatocellular carcinoma, 5,218 intact N-glycopeptides were identified, where 422 site- and structure-specific differential N-glycosylation on 145 N-glycoproteins was observed. The method, representing substantial increase of throughput, can be adopted for fast and efficient analysis of N-glycoproteomes at large scale.