A Perspective on the Confident Comparison of Glycoprotein Site-Specific Glycosylation in Sample Cohorts

Protein glycosylation, resulting from glycosyl transferase reactions under complex control in the secretory pathway, consists of a distribution of related glycoforms at each glycosylation site. Because the biosynthetic substrate concentration and transport rates depend on architecture and other aspects of cellular phenotypes, site-specific glycosylation cannot be predicted accurately from genomic, transcriptomic, or proteomic information. Rather, it is necessary to quantify glycosylation at each protein site and how this changes among a sample cohort to provide information about disease mechanisms. At present, mature mass spectrometry-based methods allow for qualitative assignment of the glycan composition and glycosylation site of singly glycosylated proteolytic peptides. To make such quantitative comparisons, it is necessary to sample the glycosylation distribution with sufficient coverage and accuracy for confident assessment of the glycosylation changes that occur in the biological cohort. In this Perspective, we discuss the unmet needs for mass spectrometry acquisition methods and bioinformatics for the confident comparison of protein site-specific glycosylation among sample cohorts.