First comprehensive identification of cardiac proteins with putative increased O-GlcNAc levels during pressure overload hypertrophy
- Seattle Children’s Research Institute, Seattle, WA (United States)
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Environmental Molecular Sciences Division
- Seattle Children’s Research Institute, Seattle, WA (United States); Univ. of Washington, Seattle, WA (United States)
Protein posttranslational modifications (PTMs) by O-GlcNAc globally rise during pressure-overload hypertrophy (POH). However, a major knowledge gap exists on the specific proteins undergoing changes in O-GlcNAc levels during POH primarily because this PTM is low abundance and easily lost during standard mass spectrometry (MS) conditions used for protein identification. Methodologies have emerged to enrich samples for O-GlcNAcylated proteins prior to MS analysis. Accordingly, our goal was to identify the specific proteins undergoing changes in O-GlcNAc levels during POH. We used C57/Bl6 mice subjected to Sham or transverse aortic constriction (TAC) to create POH. From the hearts, we labelled the O-GlcNAc moiety with tetramethylrhodamine azide (TAMRA) before sample enrichment by TAMRA immunoprecipitation (IP). We used LC-MS/MS to identify and quantify the captured putative O-GlcNAcylated proteins. We identified a total of 700 putative O-GlcNAcylated proteins in Sham and POH. Two hundred thirty-three of these proteins had significantly increased enrichment in POH over Sham suggesting higher O-GlcNAc levels whereas no proteins were significantly decreased by POH. We examined two MS identified metabolic enzymes, CPT1B and the PDH complex, to validate by immunoprecipitation. We corroborated increased O-GlcNAc levels during POH for CPT1B and the PDH complex. Enzyme activity assays suggests higher O-GlcNAcylation increases CPT1 activity and decreases PDH activity during POH. In summary, we generated the first comprehensive list of proteins with putative changes in O-GlcNAc levels during POH. Our results demonstrate the large number of potential proteins and cellular processes affected by O-GlcNAc and serve as a guide for testing specific O-GlcNAc-regulated mechanisms during POH.
- Research Organization:
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
- Sponsoring Organization:
- USDOE Office of Science (SC), Biological and Environmental Research (BER)
- Grant/Contract Number:
- AC05-76RL01830
- OSTI ID:
- 1900156
- Report Number(s):
- PNNL-SA-170854
- Journal Information:
- PLoS ONE, Vol. 17, Issue 10; ISSN 1932-6203
- Publisher:
- Public Library of ScienceCopyright Statement
- Country of Publication:
- United States
- Language:
- English
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