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Profiling of Endogenous Intact N-linked and O-linked Glycopeptides from Human Serum by Using an Integrated Platform.

Endogenous glycopeptides in serum are an invaluable resource for biomarker discovery. Due to the low abundance and the poor fragmentation in tandem mass spectrometry, the identification of endogenous intact glycopeptides still faces many challenges. Herein, an integrated platform is fabricated for the identification of N- and O-linked endogenous intact glycopeptides. In this platform, the high-temperature acid denaturation, ultrafiltration, and HILIC steps are combined together to highly efficient extract the endogenous intact glycopeptides from a small amount of serum. Additionally, twin spectra scheme and in silico deglycosylation strategy were applied for the identification of N- and O-linked endogenous glycopeptides, respectively. In total, 223 intact N-glycopeptides and 51 intact O-glycopeptides are identified from only 40 L human serum sample. This is the first study reporting the identification of endogenous intact N- and O-linked glycopeptide and is also the largest dataset of endogenous intact glycopeptides reported so far. The distributions of glycans among peptides and proteins and cleavage sites on peptides are further analyzed to seek the regulation of endogenous glycosylation for disease mechanism. The developed strategy provides a novel platform for disease biomarker discovery.