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Absolute Quantitation of Glycosylation Site Occupancy Using Isotopically Labeled Standards and LC-MS
Zhu, Zhikai ; Go, Eden P. ; Desaire, Heather
Zhu, Zhikai
Go, Eden P.
Desaire, Heather
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Abstract
N-linked glycans are required to maintain appropriate biological functions on proteins. Underglycosylation leads to many diseases in plants and animals; therefore, characterizing the extent of glycosylation on proteins is an important step in understanding, diagnosing, and treating diseases. To determine the glycosylation site occupancy, protein N-glycosidase F (PNGase F) is typically used to detach the glycan from the protein, during which the formerly glycosylated asparagine undergoes deamidation to become an aspartic acid. By comparing the abundance of the resulting peptide containing aspartic acid against the one containing non-glycosylated asparagine, the glycosylation site occupancy can be evaluated. However, this approach can give inaccurate results when spontaneous chemical deamidation of the non-glycosylated asparagine occurs. To overcome this limitation, we developed a new method to measure the glycosylation site occupancy that does not rely on converting glycosylated peptides to their deglycosylated forms. Specifically, the overall protein concentration and the non-glycosylated portion of the protein are quantified simultaneously by using heavy isotope-labeled internal standards coupled with LC-MS analysis, and the extent of site occupancy is accurately determined. The efficacy of the method was demonstrated by quantifying the occupancy of a glycosylation site on bovine fetuin. The developed method is the first work that measures the glycosylation site occupancy without using PNGase F, and it can be done in parallel with glycopeptide analysis because the glycan remains intact throughout the workflow.
Description
This document is the Accepted Manuscript version of a Published Work that appeared in final form in the Journal of the American Chemical Society, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://doi.org/10.1007/s13361-014-0859-2.
Date
2014-06
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American Chemical Society
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Keywords
N-linked glycosylation, Site occupancy, PNGase F, Liquid chromatography/mass spectrometry, Chemical deamidation
Citation
Zhu, Z., Go, E. P., & Desaire, H. (2014). Absolute Quantitation of Glycosylation Site Occupancy Using Isotopically Labeled Standards and LC-MS. Journal of the American Society for Mass Spectrometry, 25(6), 1012–1017. http://doi.org/10.1007/s13361-014-0859-2