Workflows capable of determining glycopeptides in large-scale are missing in the field of glycoproteomics. We present an approach for automated annotation of intact glycopeptide mass spectra. The steps in adopting the Mascot search engine for intact glycopeptide analysis included: (i) assigning one letter codes for monosaccharides, (ii) linearizing glycan sequences and (iii) preparing custom glycoprotein databases. Automated annotation of both N- and O-linked glycopeptides was proven using standard glycoproteins. In a large-scale study, a total of 257 glycoproteins containing 970 unique glycosylation sites and 3447 non-redundant N-linked glycopeptide variants were identified in 24 serum samples. Thus, a single tool was developed that collectively allows the (i) elucidation of N- and O-linked glycopeptide spectra, (ii) matching glycopeptides to known protein sequences, and (iii) high-throughput, batch-wise analysis of large-scale glycoproteomics data sets.
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