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.

Fig. 1.

HCD spectrum of an O-GlcNAc-phosphate peptide corresponding to the sequence SPEVMSTVSGTR of the protein 2 Shank2. Diagnostic fragment ions (*) and a low Oscore indicate that this peptide is O-GlcNAc-phosphate modified. The exact site of modification can however not be deduced with certainty.

Fig. 2.

Major fragmentation routes of GlcNAc-phosphate. A, Upper spectrum: Low m/z region of a GlcNAc-phosphate peptide with the proposed structures and fragmentation of diagnostic ions. Lower spectrum: HCD spectrum of a synthetic GlcNAc-6-phosphate standard. The two fragmentation spectra are virtually identical supporting the assignment of GlcNAc-6-phosphate. B, Average ion counts for the most abundant diagnostic fragment ions from 220 experimental GlcNAc-phosphate spectra. The intensity distribution of the fragment ions is consistent with the two proposed fragmentation pathways of GlcNAc-6-phosphate and intensity distribution of the synthetic standard.

Fig. 3.

Schematic representation of the post-translational modifications and protein domains of CaMKIV. The different post-translational modifications are denoted as follows: a: acetylation; g: O-GlcNAc; p: phosphorylation; u: ubiquitination. O-GlcNAc-6-phosphate is located at a serine or threonine residue between Ser-5 and Ser-33 where also several phosphorylation sites are localized.