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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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

N-glycosylation is one of the most frequently occurring protein post-translational modifications (PTMs) with broad cellular, physiological and pathological relevance. Mass spectrometry-based N-glycomics has become the state-of-the-art instrumental analytical pipeline for sensitive, high-throughput and comprehensive characterization of N-glycans and N-glycomes. Improvement and new development of methods in N-glycan release, enrichment, derivatization, isotopic labeling, separation, ionization, MS, tandem MS and informatics accompany side-by-side wider and deeper application. This review provides a comprehensive update of mass spectrometry-based qualitative and quantitative N-glycomics in the years of 2017-2018., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Phosphorylation is one of the most important and widely studied protein post-translational modifications. Tandem mass spectrometry using higher-energy collisional dissociation has evolved into a state-of-the-art analytical platform for both phosphorylation identification and site localization. Tens of thousands of phosphopeptides can now be routinely identified from a single shotgun proteomics study; site localization, however, is much more complicated and many challenges still exist. Here, we report our development of P-bracket using direct experimental evidence of phospho-containing site-determining product ions for accurate site localization without the need for additional FLR control. A P-bracket is defined as a complementary product ion pair that forms a bracket to confine a phosphorylation event to a unique site. P-bracket has been successfully benchmarked with a set of six synthetic phosphopeptides with a single phosphorylation event, a set of 96 synthetic peptides and phosphopeptide reference libraries, and two HeLa phosphopeptide LC-MS/MS (HCD) datasets; Accurate phosphosite localization by P-bracket will greatly enhance identification confidence of phosphopeptides and contribute to structural and functional studies of phosphoproteins. [ABSTRACT FROM AUTHOR]