A novel two-step protease digestion and glycopeptide capture approach has been developed. It is different from traditional tryptic digestion, glycopeptide enriching and identification approach in glycoproteomics. Here, proteins were first digested by Lys-C into relatively large peptides. Glycopeptides among them were selectively captured by hydrazide resin through oxidized glycans. After thorough washing steps, trypsin was used as a second protease to in situ release non-glycosylated part (named as LT-peptides) from glycopeptides. Subsequently, the remaining part of glycopeptides on resin was de-glycosylated by peptide-N-glycosidase F, and collected as DG-peptides. Finally, both LT- and DG-peptides could be analyzed by mass spectrometer, achieving glycoprotein and glycosite identification. The approach was applied to cell lysate after positive validation by a model glycoprotein: 143 N-glycoproteins identified from DG- and LT-fraction both. In those glycoproteins, 189 DG-peptide-revealed N-glycosites got further confirmation by neighboring LT-peptides, which, in the meantime, made 109 glycoproteins get improved sequence coverage with increase even up to 350% (averagely 79.4%). Through controllable release, separate identification and combined interpretation of non-glycopeptides (newly introduced LT-peptides here) and traditional de-glycopeptides, the approach could not only achieve routine N-glycosite identification, but also provide further proofs of N-glycosites and increase glycoprotein sequence coverage.
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