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Gurcel C Vercoutter-Edouart AS Fonbonne C Mortuaire M Salvador A Michalski JC Lemoine J 《Analytical and bioanalytical chemistry》2008,390(8):2089-2097
The O-linked β-N-acetylglucosamine (O-GlcNAc) modification is an abundant post-translational modification in eukaryotic cells. This dynamic glycosylation plays
a fundamental role in the activity of many nuclear and cytoplasmic proteins and is associated with pathologies like type II
diabetes, Alzheimer’s disease or some cancers. However the exact link between O-GlcNAc-modified proteins and their function in cells is largely undefined for most cases. Here we report a strategy based
on the 1,3-dipolar cycloaddition, called click chemistry, between unnatural N-acetylglucosamine (GlcNAc) analogues (substituted with an azido or alkyne group) and the corresponding biotinylated probe
to specifically detect, enrich and identify O-GlcNAc-modified proteins. This bio-orthogonal conjugation confirms that only azido analogue of GlcNAc is metabolized by the
cell. Thanks to the biotin probe, affinity purification on streptavidin beads allowed us to identify 32 O-GlcNAc-azido-tagged proteins by LC-MS/MS analysis in an MCF-7 cellular model, 14 of which were previously unreported. This
work illustrates the use of the click-chemistry-based strategy combined with a proteomic approach to get further insight into
the pattern of O-GlcNAc-modified proteins and the biological significance of this post-translational modification.
Figure Detection of biotinylated O-GlcNAz proteins in MCF-7 cells
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
Caroline Gurcel and Anne-Sophie Vercoutter-Edouart contributed equally to this work. 相似文献