ABSTRACT
With the advancement in chromatographic separation and MS detection, high-throughput and deep site- and structure-specific characterization of glycosylation can be and can only be achieved at the intact glycopeptide level. For an intact N-glycopeptide, site-specific information of amino acid sequence and glycosite can be achieved with selective fragmentation of the peptide backbone; and structure-specific information of the N-glycan can be achieved with the corresponding selective fragmentation. Every LC-MS/MS analysis of intact glycopeptides from any complex sample yield a raw dataset containing tens of thousands of MS and MS/MS spectra. For high-throughput interpretation and database search of raw LC-MS/MS datasets, lots of powerful dedicated intact glycopeptide search engines and auxiliary databases have been developed. This chapter provides a general overview of current analytical software and databases in N-glycoproteomics; for each software, the search algorithm and procedure, method of (false discovery rate) FDR control as well as recent applications are mainly covered. Early search engines (including Byonic, GPQuest, etc.) report site-specific information of amino acid sequences, N-glycosites as well as monosaccharide compositions of intact N-glycopeptides; whereas latest GPSeeker reports additional structure-specific information of monosaccharide sequences, part of which are confirmed with structure-diagnostic fragment ions. Additionally, search engines that support O-glycosylation identification are also briefly introduced.
