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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. [Display omitted] • One-hour N -glycoproteomics with 1-h gradient is developed for fast analysis. • 85 % more IDs was achieved per elution time than previous 4-h gradient. • Fast N -glycoproteomics may find wide application of large-cohort study. [ABSTRACT FROM AUTHOR]

Rationale: We previously isolated antibodies binding to glycopeptide neoantigen epitopes centering the GSTA sequence of the highly glycosylated tandem repeat region of MUC1. Epitopes centering the GSTA sequence are also predicted by NetMHC programs to bind to MHC molecules. Detecting MUC1 glycopeptide epitopes remains a challenge since antigenic epitopes are often shorter than 10 amino acids. Methods: In this study, we used pronase from Streptomyces griseus, which has no amino acid sequence preference for enzymatic cleavage sites, to digest synthetic glycopeptides RPAPGST (Tn)APPAHG and RPAPGS (Tn)TAPPAHG, and analyzed the digests by liquid chromatography/mass spectrometry (LC/MS) using electron transfer dissociation (ETD) and higher‐energy collisional dissociation (HCD) methods with an Orbitrap Fusion Lumos Tribid mass spectrometer. Results: We found that short glycopeptides containing 8 to 11 amino acids could be efficiently generated by pronase digestion. Such glycopeptides of minimal epitope lengths were clearly distinguished by characteristic MS/MS ion patterns and LC elution profiles. A glycopeptide library was generated which may serve as a standard for measuring neoantigen epitopes centering the GSTA sequence. Conclusions: Our data established the LC/MS/MS identities of a clinically relevant MUC1 glycopeptide neoantigen epitope centering the GSTA motif. A library of short MUC1 glycopeptides centered on the GSTA motif was created, which is a critical step for analysis of such antigen epitopes in real biological samples. [ABSTRACT FROM AUTHOR]