Enrichment of O-GlcNAc modified proteins by the periodate oxidation-hydrazide resin capture approach.
A chemical derivatization approach has been developed for the enrichment of O-GlcNAc modified proteins. The procedure is based on the isolation technique used for N-glycoproteins with appropriate modifications because of the differences in the two types of glycosylation: a prolonged periodate oxidation is followed by hydrazide resin capture, on-resin proteolytic digestion, and release of the modified peptides by hydroxylamine. This enrichment strategy offers a fringe benefit in mass spectrometry analysis. Upon collisional activation, the presence of the open carbohydrate ring leads to characteristic fragmentation facilitating both glycopeptide identification and site assignment. The enrichment protocol was applied to the Drosophila proteasome complex previously described as O-GlcNAc modified. The O-GlcNAc modification was located on proteasome interacting proteins, deubiquitinating enzyme Faf (CG1945) and a ubiquitin-like domain containing protein (CG7546). Three other proteins were also found GlcNAc modified, a HSP70 homologue (CG2918), scribbled (CG5462) and the 205 kDa microtubule-associated protein (CG1483). Interestingly, in the HSP70 homologue the GlcNAc modification is attached to an asparagine residue of a N-glycosylation motif.
Klement
Eva
E
Proteomics Research Group, Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary.
Lipinszki
Zoltán
Z
Kupihár
Zoltán
Z
Udvardy
Andor
A
Medzihradszky
Katalin F
KF
D000117:Acetylglucosamine / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D004330:Drosophila; D029721:Drosophila Proteins / Q000737:chemistry / Q000378:metabolism; D006020:Glycopeptides / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D006834:Hydrazines / Q000737:chemistry*; D010084:Oxidation-Reduction; D010091:Oximes / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D010504:Periodic Acid / Q000737:chemistry*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D029721:Drosophila Proteins; D006020:Glycopeptides; D006834:Hydrazines; D010091:Oximes; D010446:Peptide Fragments; D010504:Periodic Acid; C009288:metaperiodate; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D000117:Acetylglucosamine