Journal
PLOS ONE
Volume 8, Issue 10, Pages -Publisher
PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pone.0076399
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Funding
- National Key Scientific Program of China [2008ZX10208, 2008ZX10207]
- National Science and Technology Key Project of China [2011CB910600]
- National Natural Science Foundation of China [31100590, 20975024]
- Shanghai Municipal Natural Science Foundation [11ZR1403000]
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O-linked beta-N-acetylglucosamine (O-GlcNAc) is an important post-translational modification (PTM) consisting of a single N-acetylglucosamine moiety attached via an O-beta-glycosidic linkage to serine and threonine residues. Glycosylation with O-GlcNAc occurs on myriad nuclear and cytosolic proteins from almost all functional classes. However, with respect to O-GlcNAcylated proteins special in mitochondria, little attention has been paid. In this study, we combined mass spectrometry and immunological methods to perform global exploration of O-GlcNAcylated proteins specific in mitochondria of rat liver. First, highly purified mitochondrial proteins were obviously shown to be O-GlcNAcylated by immunoblot profiling. Then, beta-elimination followed by Michael Addition with Dithiothreitol (BEMAD) treatment and LC-MS/MS were performed to enrich and identify O-GlcNAcylated mitochondrial proteins, resulting in an unambiguous assignment of 14 O-GlcNAcylation sites, mapping to 11 O-GlcNAcylated proteins. Furthermore, the identified O-GlcNAcylated mitochondrial proteins were fully validated by both electron transfer dissociation tandem mass spectrometry (ETD/MS/MS) and western blot. Thus, for the first time, our study definitely not only identified but also validated that some mitochondrial proteins in rat liver are O-GlcNAcylated. Interestingly, all of these O-GlcNAcylated mitochondrial proteins are enzymes, the majority of which are involved in a wide variety of biological processes, such as urea cycle, tricarboxylic acid cycle and lipid metabolism, indicating a role for protein O-GlcNAcylation in mitochondrial function.
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