High-performance targeted mass spectrometry with precision data-independent acquisition reveals site-specific glycosylation macroheterogeneity

K.Y. Benjamin Yeo, Panagiotis K. Chrysanthopoulos, Amanda S. Nouwens, Esteban Marcellin, Benjamin L. Schulz

Protein glycosylation is a critical post-translational modification that regulates the structure, stability, and function of many proteins. Mass spectrometry is currently the preferred method for qualitative and quantitative characterisation of glycosylation. However, the inherent heterogeneity of glycosylation makes its analysis difficult. Quantification of glycosylation occupancy, or macroheterogeneity, has proven especially challenging. Here, we used a variation of MRM(HR) or pseudo-MRM for targeted data independent acquisition which we term SWAT (Sequential Window Acquisition of Targeted fragment ions). We compared the analytical performance of SWATH, SWAT, and SRM using a suite of synthetic peptides spiked at various concentrations into a complex yeast tryptic digest sample. SWAT provided superior analytical performance than SWATH in a targeted approach. We then used SWAT to measure site-specific N-glycosylation occupancy in cell wall glycoproteins from yeast with defects in the glycosylation biosynthetic machinery. SWAT provided robust measurement of occupancy at more N-glycosylation sites, and with higher precision than SWATH, allowing identification of novel glycosylation sites dependent on the Ost3p and Ost6p regulatory subunits of oligosaccharyltransferase.