Software Design and Analytical Challenges for Confident Glycopeptide Identification With Data-Independent Acquisition

Glycosylation is an abundant post-translational modification that impacts a wide variety of functions, including protein regulation, cell adhesion, and structural integrity. The application of proteomics methods to glycopeptide assignment faces unique challenges due to high heterogeneity, which results in complex populations with low overall abundance per glycopeptide. In addition, glycans dissociate at a lower collision energy compared to their attached peptide component. The resulting mass spectral data require specialized assignment software, which has caused glycoproteomics to lag traditional proteomics. Existing software primarily focuses on data-dependent acquisition (DDA), but manual validation is frequently required, and experiments are necessarily limited by the stochastic nature of DDA ion-selection. Data-independent acquisition (DIA) allows for a more complete and robust analysis of glycopeptide samples, but analysis software is still sparse. In this review, we discuss the current state of DDA analysis software, the limitations, and how it can inform our forays into DIA glycoproteomics.