MassIVE MSV000080705

Imported Reanalysis Dataset Public PXD002591

GNPS - A novel quantitative mass spectrometry platform for determining site-specific protein O-GlcNAcylation dynamics

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Description

Over the past decades, protein O-GlcNAcylation has been found to play a fundamental role in cell cycle control, metabolism, transcriptional regulation, and cellular signaling. Nevertheless, quantitative approaches to determine in vivo GlcNAc dynamics at a large-scale are still not readily available. Here, we have developed an approach to isotopically label O-GlcNAc modifications on proteins by producing 13C-labeled UDP-GlcNAc from 13C6-glucose via the hexosamine biosynthetic pathway. This metabolic labeling was combined with quantitative mass spectrometry-based proteomics to determine site-specific protein O-GlcNAcylation turnover rates. First, an efficient enrichment method for O-GlcNAc peptides was developed with the use of phenylboronic acid solid-phase extraction and anhydrous DMSO. The near stoichiometry reaction between the diol of GlcNAc and boronic acid dramatically improved the enrichment efficiency. Additionally, our kinetic model for turnover rates integrates both metabolomic and proteomic data, which increase the accuracy of the turnover rate estimation. Other advantages of this metabolic labeling method include in vivo application, direct labeling of the O-GlcNAc sites and higher confidence for site identification. Concentrating only on nuclear localized GlcNAc modified proteins, we are able to identify 159 O-GlcNAc sites on 74 proteins and determine turnover rates of 24 O-GlcNAc peptides from 21 proteins extracted from HeLa nuclei. In general, we found O-GlcNAcylation turnover rates are slower than those published for phosphorylation or acetylation. Nevertheless, the rates widely varied depending on both the protein and the residue modified. We believe this methodology can be broadly applied to reveal turnovers/dynamics of protein O-GlcNAcylation from different biological states and will provide more information on the significance of site-specific O-GlcNAcylation, enabling us to study the temporal dynamics of this critical modification in a site-specific manner for the first time. [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: mass spectrometry method determines protein O-GlcNAcylation rates

Contact

Principal Investigators:
(in alphabetical order) 		Benjamin A. Garcia, Department of Biochemistry and Biophysics, University of Pennsylvania, N/A
Submitting User: ccms

Publications

Wang X, Yuan ZF, Fan J, Karch KR, Ball LE, Denu JM, Garcia BA.
A Novel Quantitative Mass Spectrometry Platform for Determining Protein O-GlcNAcylation Dynamics.
Mol. Cell Proteomics. 2016 Jul;15(7):2462-75. Epub 2016 Apr 25.

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GNPS content goes here (MSV000080705 [task=af4652a1fd7a45d7a4ab8120537c4c03])
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Distinct protein accessions are counted across all files submitted in the "Statistical Analysis of Quantified Analytes" category having a "Protein" column in this dataset.

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