High-performance targeted mass spectrometry with precision data-independent acquisition reveals site-specific glycosylation macroheterogeneity.
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 characterization of glycosylation. However, the inherent heterogeneity of glycosylation makes its analysis difficult. Quantification of glycosylation occupancy, or macroheterogeneity, has proven to be especially challenging. Here, we used a variation of high-resolution multiple reaction monitoring (MRM(HR)) or pseudo-MRM for targeted data-independent acquisition that we term SWAT (sequential window acquisition of targeted fragment ions). We compared the analytical performance of SWATH (sequential window acquisition of all theoretical fragment ions), SWAT, and SRM (selected reaction monitoring) using a suite of synthetic peptides spiked at various concentrations into a complex yeast tryptic digest sample. SWAT provided superior analytical performance to 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.
School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland 4072, Australia.
K Y Benjamin
KYB
Yeo
Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, Brisbane, Queensland 4072, Australia.
Panagiotis K
PK
Chrysanthopoulos
School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland 4072, Australia; Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, Brisbane, Queensland 4072, Australia.
Amanda S
AS
Nouwens
Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, Brisbane, Queensland 4072, Australia. Electronic address: e.marcellin@uq.edu.au.
Esteban
E
Marcellin
School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland 4072, Australia. Electronic address: b.schulz@uq.edu.au.
Benjamin L
BL
Schulz
D006031:Glycosylation; D013058:Mass Spectrometry / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis* / Q000737:chemistry
D029701:Saccharomyces cerevisiae Proteins
Data-independent acquisition; Glycosylation; Macroheterogeneity; Mass spectrometry; Oligosaccharyltransferase