Identification of new O-GlcNAc modified proteins using a click-chemistry-based tagging.
The O-linked beta-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: see text]
Gurcel
Caroline
C
UMR CNRS 5180, Sciences Analytiques, Université de Lyon, Lyon 1, 43 Bld du 11 novembre 1918, 69622, Villeurbanne cedex, France.
Vercoutter-Edouart
Anne-Sophie
AS
Fonbonne
Catherine
C
Mortuaire
Marlène
M
Salvador
Arnaud
A
Michalski
Jean-Claude
JC
Lemoine
Jérôme
J
D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000032:analysis* / Q000378:metabolism; D001710:Biotin / Q000737:chemistry; D019921:Biotinylation / Q000379:methods; D045744:Cell Line, Tumor; D005260:Female; D006031:Glycosylation; D006801:Humans; D015336:Molecular Probe Techniques; D015335:Molecular Probes / Q000737:chemistry*; D015394:Molecular Structure; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D019809:Streptavidin / Q000737:chemistry; D013997:Time Factors
D015335:Molecular Probes; D011506:Proteins; D001710:Biotin; D019809:Streptavidin; D000117:Acetylglucosamine