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Mass Spectrometry for O-GlcNAcylation.

ABSTRACT: O-linked β-N-acetylglucosamine modification (O-GlcNAcylation) at proteins with low-abundance expression level and species diversity, shows important roles in plenty of biological processes. O-GlcNAcylations with abnormal expression levels are associated with many diseases. Systematically profiling of O-GlcNAcylation at qualitative or quantitative level is vital for their function understanding. Recently, the combination of affinity enrichment, metabolic labeling or chemical tagging with mass spectrometry (MS) have made significant contributions to structure-function mechanism elucidating of O-GlcNAcylations in organisms. Herein, this review provides a comprehensive update of MS-based methodologies for quali-quantitative characterization of O-GlcNAcylation.

SUBMITTER: Yin R

PROVIDER: S-EPMC8685217 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project 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 (13)C-labeled UDP-GlcNAc from (13)C6-glucose via the hexosamine biosynthetic pathway. This metabolic labeling was combined with quantitative mass spectrometry-based proteomics to determine 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 105 O-GlcNAc peptides on 42 proteins and determine turnover rates of 20 O-GlcNAc peptides from 14 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 O-GlcNAcylation, enabling us to study the temporal dynamics of this critical modification for the first time.

Dataset Information

Mass Spectrometry for O-GlcNAcylation.

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