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 OGlcNAc. 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 OGlcNAc-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 OGlcNAc-6-phosphate is a general post-translation modification of mammalian proteins with a variety of possible cellular functions. Molecular & Cellular

Advances in purification and separation of posttranslationally modified proteins

“…In recent years approaches for analyzing protein PTMs have rapidly developed, but large proportions of peaks in MS spectra of protein samples are still typically not assigned, and may originate from hidden PTMs. This hypothesis has been recently corroborated by the detection of a signature fragment of a novel Oacetylglucosamine-6-phosphate PTM in a re-analysis of a large-scale proteomics dataset [260]. Thus, we are still clearly far from an optimal procedure for PTM analyses, and further advances are required not only in protein isolation and MS sensitivity, but also in bioinformatics.…”

Mass Informatics: From Mass Spectrometry Peaks to Biological Pathways

“…In some rare cases this has already happened, culminating in republication of insight from previously acquired data. [42,43] However, as stated by the authors of these republication efforts and others, there is currently no easy way to study large collections of raw spectral data. This is because the fields of proteomics, metabolomics and small-molecule mass spectrometry do not have a universally accepted and heavily used curated repository of raw data.…”

Functional decorations: post-translational modifications and heart disease delineated by targeted proteomics

“…Superficial elevation of O -GlcNAcylation with the addition of multiple O-GlcNAcase inhibitors in mouse embryonic fibroblasts resulted in reciprocal regulation of phosphorylation at over 400 sites (280 of which showed reduced phosphorylation), providing evidence for crosstalk or competition between protein kinases and O -GlcNAc transferase [136]. Adding a further level of complexity to the relationship between O -GlcNAc and phosphate is the recent discovery of a single O -GlcNAc-6-phosphate modification [137] that can be attached to multiple proteins [138]. …”