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Large-scale intact glycopeptide identification by Mascot database search
Ravi Chand Bollineni, Christian Jeffrey Koehler, Randi Elin Gislefoss, Jan Haug Anonsen & Bernd Thiede
Scientific Reports, volume 8, Article number: 2117 (2018)
The authors have developed a novel method for identifying intact glycopeptides in a high-throughput and batch-wise manner. They created a custom peptide database in which known glycan structures were linearized and then encoded using the one letter codes O (GlcNAc, GalNAc), J (Galactose, Mannose) and U (Neu5Ac). Additional monosaccharides could be encoded as variable modifications to these codes. By assigning the appropriate mass values and attaching the glycan sequence at the peptide N-terminus, Mascot was enabled to match glycosidic cleavage ions from both N-linked and O-linked glycans as pseudo y-ions.
Serum samples from controls and patients diagnosed with prostate cancer were analyzed to validate the approach. Samples were digested with trypsin and desalted with ZIC-HILIC SPE, enriched with TiO2 beads, followed by LC-MS analysis. The intact glycopeptide mass spectra were submitted to the Mascot search engine for identification and relative quantification with Mascot Distiller. The data was searched against a custom glycoprotein database.
A total of 257 glycoproteins were identified from the 24 serum samples. Within these 257 glycoproteins, a total of 970 unique glycosylation sites and 3447 non-redundant glycopeptide variants were identified.
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