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

Yeo, K. Y. Benjamin, Chrysanthopoulos, Panangiotis K., Nouwens, Amanda S., Marcellin, Esteban and Schulz, Benjamin L. (2016) High performance targeted mass spectrometry with precision data independent acquisition reveals site-specific glycosylation macroheterogeneity. Analytical Biochemistry, 510 106-113. doi:10.1016/j.ab.2016.06.009


Author Yeo, K. Y. Benjamin
Chrysanthopoulos, Panangiotis K.
Nouwens, Amanda S.
Marcellin, Esteban
Schulz, Benjamin L.
Title High performance targeted mass spectrometry with precision data independent acquisition reveals site-specific glycosylation macroheterogeneity
Journal name Analytical Biochemistry   Check publisher's open access policy
ISSN 0003-2697
1096-0309
Publication date 2016-10-01
Year available 2016
Sub-type Article (original research)
DOI 10.1016/j.ab.2016.06.009
Open Access Status Not yet assessed
Volume 510
Start page 106
End page 113
Total pages 8
Place of publication Philadelphia, PA United States
Publisher Elsevier
Collection year 2017
Language eng
Formatted abstract
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 MRMHR 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.
Keyword Glycosylation
Oligosaccharyltransferase
Macroheterogeneity
Mass spectrometry
Data independent acquisition
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status UQ

 
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Created: Tue, 28 Jun 2016, 11:39:28 EST by Mrs Louise Nimwegen on behalf of School of Chemistry & Molecular Biosciences