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PXD002591

PXD002591 is an original dataset announced via ProteomeXchange.

Dataset Summary
TitleA novel quantitative mass spectrometry platform for determining site-specific protein O-GlcNAcylation dynamics
DescriptionOver 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.
HostingRepositoryPRIDE
AnnounceDate2016-07-19
AnnouncementXMLSubmission_2016-07-19_05:01:50.xml
DigitalObjectIdentifierhttp://dx.doi.org/10.6019/PXD002591
ReviewLevelPeer-reviewed dataset
DatasetOriginOriginal dataset
RepositorySupportSupported dataset by repository
PrimarySubmitterXiaoshi Wang
SpeciesList scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationListdeamidated residue; monohydroxylated residue; iodoacetamide derivatized residue; N-acetylaminoglucosylated
InstrumentQ Exactive
Dataset History
RevisionDatetimeStatusChangeLog Entry
02015-07-23 00:39:06ID requested
12016-07-19 05:01:52announced
Publication List
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, 15(7):2462-75(2016) [pubmed]
Keyword List
curator keyword: Biological, Technical
submitter keyword: mass spectrometry method determines protein O-GlcNAcylation rates
Contact List
Benjamin A. Garcia
contact affiliationDepartment of Biochemistry and Biophysics, University of Pennsylvania
contact emailbgarci@mail.med.upenn.edu
lab head
Xiaoshi Wang
contact affiliationUniversity of Pennsylvania
contact emailchemwangxs@gmail.com
dataset submitter
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