title: Discovery of O-GlcNAc-6-phosphate modified proteins in large-scale phosphoproteomics data.
abstract: Phosphorylated O-GlcNAc is a novel post-translational modification that has so far only been found on the neuronal protein AP180 from the rat (Graham et al., J. Proteome Res. 2011, 10, 2725-2733). Upon collision induced dissociation, the modification generates a highly mass deficient fragment ion (m/z 284.0530) that can be used as a reporter for the identification of phosphorylated O-GlcNAc. Using a publically available mouse brain phosphoproteome data set, we employed our recently developed Oscore software to re-evaluate high resolution/high accuracy tandem mass spectra and discovered the modification on 23 peptides corresponding to 11 mouse proteins. The systematic analysis of 220 candidate phosphoGlcNAc tandem mass spectra as well as a synthetic standard enabled the dissection of the major phosphoGlcNAc fragmentation pathways, suggesting that the modification is O-GlcNAc-6-phosphate. We find that the classical O-GlcNAc modification often exists on the same peptides indicating that O-GlcNAc-6-phosphate may biosynthetically arise in two steps involving the O-GlcNAc transferase and a currently unknown kinase. Many of the identified proteins are involved in synaptic transmission and for Ca(2+)/calmodulin kinase IV, the O-GlcNAc-6-phosphate modification was found in the vicinity of two autophosphorylation sites required for full activation of the kinase suggesting a potential regulatory role for O-GlcNAc-6-phosphate. By re-analyzing mass spectrometric data from human embryonic and induced pluripotent stem cells, our study also identified Zinc finger protein 462 (ZNF462) as the first human O-GlcNAc-6-phosphate modified protein. Collectively, the data suggests that O-GlcNAc-6-phosphate is a general post-translation modification of mammalian proteins with a variety of possible cellular functions.
authors: Center of Life and Food Sciences Weihenstephan, Technische Universität München, Emil-Erlenmeyer-Forum 5, 85354 Freising, Germany.
authors: Hannes
authors: H
authors: Hahne
authors: Bernhard
authors: B
authors: Kuster
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4 / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000032:analysis; D010766:Phosphorylation; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D051381:Rats; D012984:Software; D009435:Synaptic Transmission / Q000235:genetics; D053719:Tandem Mass Spectrometry; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D016335:Zinc Fingers / Q000235:genetics
chemical_list: D004268:DNA-Binding Proteins; D009419:Nerve Tissue Proteins; D010455:Peptides; D014157:Transcription Factors; C578243:ZNF462 protein, human; D017351:N-Acetylglucosaminyltransferases; C449880:O-GlcNAc transferase; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; D000117:Acetylglucosamine