Purification of the O-glycosylated protein p135 and identification as O-GlcNAc transferase

We have previously shown that rat pancreatic islets contain a predominant 1 35 kDa O-glycosylated protein (p135) that is recognized by immunoprecipitat ion and Western blotting with anti-O-GlcNAc antibody. In this paper, we sho w that p135 is also detectable in other rat tissues including brain, heart, liver, spleen, and lung, but not kidney. To identify p135, the protein was purified from rat brain using a multistep procedure including selective ab sorption with anti-O-GlcNAc antibody. After electrophoresis, and Coomassie staining, the protein was excised from the gel for tryptic digestion. Next, O-methylisourea was used to convert lysine residues to homoarginine to inc rease the sequence coverage, and MALDI-TOF mass spectrometry detection was performed. MALDI-TOF identified p135 as rat 0-GlcNAc transferase (OGT), an identity confirmed by LC/MS of individual peptides. The identification of p 135 as 0GT is consistent with previous reports of the tissue distribution o f OGT, as well as reports that OGT is itself O-glyeosylated. (C) 2001 Acade mic Press.