Expanding N-glycopeptide identifications by modeling fragmentation, elution, and glycome connectivity

Accurate glycopeptide identification in mass spectrometry-based glycoproteomics is a challenging problem at scale. Recent innovation has been made in increasing the scope and accuracy of glycopeptide identifications, with more precise uncertainty estimates for each part of the structure. We present a dynamically adapting relative retention time model for detecting and correcting ambiguous glycan assignments that are difficult to detect from fragmentation alone, a layered approach to glycopeptide fragmentation modeling that improves N-glycopeptide identification in samples without compromising identification quality, and a site-specific method to increase the depth of the glycoproteome confidently identifiable even further. We demonstrate our techniques on a set of previously published datasets, showing the performance gains at each stage of optimization. These techniques are provided in the open-source glycomics and glycoproteomics platform GlycReSoft available at https://github.com/mobiusklein/glycresoft .