Journal
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS
Volume 1834, Issue 8, Pages 1474-1483Publisher
ELSEVIER
DOI: 10.1016/j.bbapap.2013.04.022
Keywords
Recombinant protein; Post-translational modification; Glycosylation; Mass spectrometry; Targeted proteomics
Categories
Funding
- Clarkson University
- Army Research Office through the Defense University Research Instrumentation Program (DURIP) [W911NF-11-1-0304]
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We used a targeted proteomics approach to investigate whether introduction of new N-linked glycosylation sites in a chimeric protein influence the glycosylation of the existing glycosylation sites. To accomplish our goals, we over-expressed and purified a chimeric construct that contained the Fc region of the IgG fused to the exons 7 & 8 of mouse ZP3 (IgG-Fc-ZP3E7 protein). Immunoglobulin heavy chain (IgG-HC protein) was used as control. We then analyzed the IgG-HC and IgG-Fc-ZP3E7 proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and by Western blotting (WB). We concluded that in control experiments, the glycosylation site was occupied as expected. However, in the IgG-Fc-ZP3E7 protein, we concluded that only one out of three NXS/T glycosylation sites is occupied by N-linked oligosaccharides. We also concluded that in the IgG-Fc-ZP3E7 protein, upon introduction of additional potential NXS/T glycosylation sites within its sequence, the original NST/S glycosylation site from the Pc region of the IgG-Fc-ZP3E7 protein is no longer glycosylated. The biomedical significance of our findings is discussed. (C) 2013 Elsevier B.V. All rights reserved.
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