Post-translational modifications: A phosphate pile-upFunctional Glycomics (07 July 2011) | doi:10.1038/fg.2011.27
Mass spectrometry analyses reveal protein glycosyl phosphorylation sites.
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AP180, a protein that participates in clathrin assembly and synaptic vesicle endocytosis, is known to be regulated by phosphorylation and modified by O-GlcNAc. However, the timing, location and full functional consequences of these groups are not known. In their exploration of AP180 modifications, Graham et al. discovered a peptide sequence that was collected using a phosphoryl-specific column but showed mass spectra fragmentation patterns typical of a glycosylated sequence, leading the authors to suspect a tandem glycosylation-phosphorylation event. The authors were able to cleave the phosphate group from the peptide with alkaline phosphatase, ruling out a structure in which the phosphate linked the peptide and glycan. Instead, further mass spectrometry analysis, combined with the use of synthetic standards, demonstrated that the peptide was first modified by GlcNAc, and then the glycan itself was phosphorylated. Identification of a possible second 'protein glycosyl phosphorylation site', as termed by the authors, along with sequence comparison of the two sites, provided hints of a substrate motif for an unknown kinase. The authors note that current methods used to search for GlcNAc-modified proteins would not retain this unique modification, suggesting that additional doubly-functionalized species may await discovery.
Original research paper
- Graham, M.E. et al. A Novel Post-translational Modification in Nerve Terminals: O-Linked N-Acetylglucosamine Phosphorylation. J. Proteome Res. 10, 2725–2733 (2011). doi:10.1021/pr1011153. | Article