Use of a glycosylation site database to improve glycopeptide identification from complex mixtures

New mass spectrometry instrumentation, particularly those with electron transfer dissociation fragmentation, has made the analysis of complex glycopeptide mixtures accessible. However, software tools need to be optimized for interpretation of this type of data. Glycopeptide identification is challenging due to the number of different peptide and sugar moieties that can be combined, leading to a large number of potential compositions to consider. In this manuscript, different strategies for reducing the number of peptides and glycopeptides considered in database searching are compared. Adaptation of the software Protein Prospector to support the use of a reference modification site database doubled the number of glycopeptide IDs. The potential of this as an improved analysis strategy is discussed. Graphical abstract This manuscript compares the use of a restricted protein database based on a list of accession numbers of identified proteins to the use of a modification site database for intact glycopeptide analysis. It was found that the modification database is more effective for glycopeptide identification, particularly for larger glycopeptides.