Abstract
O-linked β-N-acetylglucosamine (O-GlcNAc) is an important post-translational modification (PTM) consisting of a single N-acetylglucosamine moiety attached via an O-β-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, β-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.
Highlights
O-linked β-N-acetylglucosamine (O-GlcNAc) is a ubiquitous modification with a single N-acetylglucosamine attachment to hydroxyl groups of Ser and/or Thr residues of target proteins, which occurs in all metazoas
Since we have found the existence of OGlcNAc in multiple mitochondrial proteins by western blot, we further explored what these O-GlcNAcylated proteins were and where the O-GlcNAcylation sites located by mass spectrometry
All of the 11 identified O-GlcNAcylated mitochondrial proteins are enzymes involved in a wide variety of biological processes
Summary
O-linked β-N-acetylglucosamine (O-GlcNAc) is a ubiquitous modification with a single N-acetylglucosamine attachment to hydroxyl groups of Ser and/or Thr residues of target proteins, which occurs in all metazoas. The definite proteins/peptides and exact amino acids sites with O-GlcNAc modification were seldom known [14], because the evidence of mitochondrial protein O-GlcNAcylation in these studies was almost got from immunological methods, such as immunoprecipitation and western blot with antibody RL2 or lectin WGA. Immunoaffinity/lectin chromatography is the simplest approach to purify OGlcNAcylated proteins This method prefers to enrich high abundance proteins or those with multiple clustered OGlcNAc residues instead of low abundance proteins with single or widely separated O-GlcNAcylation sites [1]. Chemoenzymatic approach [39], can greatly enrich O-GlcNAcylated proteins/peptides and can be combined with ETD/MS/MS analysis for sites mapping, it is still not suitable for sites mapping for highthrough put by direct CID/CAD MS/MS for its labile and large mass of tag attached to the O-GlcNAc [1]. In our study, we took advantage of BEMAD method and combined tandem mass spectrometry and immunological methods to identify and validate the O-GlcNAcylated proteins in rat liver mitochondria, and tried to explore the role of such modification in mitochondria
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