Diet-induced perturbation of the rat liver mitochondrial acetylome studied by quantitative (iTRAQ) LC-MS/MS

Introduction
Post-translational modifications (PTMs) regulate protein networks in the cell. -N-acetylation of lysine residues is a reversible and highly regulated PTM, which is controlled by networks of lysine-acetyl transferases (KATs) and lysine-deacetylases (KDACs). Lysine-acetylation of mitochondrial proteins has emerged as a key regulator of cellular metabolism. The acetylated proteome includes enzymes involved in glycolysis, gluconeogenesis, the tricarboxylic acid (TCA) cycle, the urea cycle, fatty acid metabolism and glycogen metabolism.
A mammal’s inability to handle excess energy intake leads to metabolic abnormalities. Thus, the discovery of dietary supplements that can control lipid metabolism is of great significance. For example, fish oil and tetradecylthioacetic acid (TTA) have induced beneficial effects in in vivo experiments by lowering plasma cholesterol and triacylglycerol levels1, 2.
Methods
A rat liver mitochondria-enriched sample was used to establish an iTRAQ-based LC-MS/MS strategy for determination of acetylated proteins. Immuno-affinity enrichment of acetylated peptides was carried out with anti-acetyl Lysine antibody (Immune Pharmaceuticals), as previously described3. Quantitation by MS/MS was achieved by using iTRAQ 8-Plex reagents (Life Technologies). LC-MS/MS analyses were performed using an Easy-nLC (Proxeon/ThermoFisher) fitted with an in-house made 17 cm C18 column that was interfaced to an LTQ-OrbiTrap XL instrument (ThermoFisher). Data analysis was performed by Proteome Discoverer (ThermoFisher) and Mascot (Matrix Science).
Preliminary data
Acetylated proteins were recovered from 200 ug of mitochondrial protein sample. A total of 321 e-N-acetyl-lysine sites in 115 proteins were identified. Sequence analysis revealed a KY Lys-acetylation motif in mitochondrial proteins, and a completely novel motif. A total of 60 proteins contained multiple e-N-acetyl-lysine residuesx. Acetylated protein variants were detected in the respiratory chain complex 5 proteins. We identified regulated acetyl-Lys residues, independent of changes at the protein expression level. For example, fish oil and TTA induced opposite effects on the abundance of Lys-acetylation and the abundance of the trifunctional enzyme beta subunit, which is involved in lipid metabolism. We also identified proteins in which the increase of protein expression is followed by the increase of their Lys-acetylation levels. Thus, it suggests that some lysine acetylation sites might be necessary for the some proteins in order to keep, for instance, protein conformation or enzymatic activity.

Novel aspect
iTRAQ-based quantitative LC-MS/MS workflow for determination of the acetylome and its diet-induced regulations. Novel sequence motif was identified.
References
1. Connor, W. E., Importance of n-3 fatty acids in health and disease. Am J Clin Nutr 2000, 71, (1 Suppl), 171S-5S.
2. Vigerust, N. F.; Cacabelos, D.; Burri, L.; Berge, K.; Wergedahl, H.; Christensen, B.; Portero-Otin, M.; Viste, A.; Pamplona, R.; Berge, R. K.; Bjorndal, B., Fish oil and 3-thia fatty acid have additive effects on lipid metabolism but antagonistic effects on oxidative damage when fed to rats for 50 weeks. J Nutr Biochem.
3. Choudhary, C.; Kumar, C.; Gnad, F.; Nielsen, M. L.; Rehman, M.; Walther, T. C.; Olsen, J. V.; Mann, M., Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 2009, 325, (5942), 834-40.