c knockout cells can be used as a model system for identification of deacetylase substrates as 
illustrated by identification of several known substrates of Sirt3. We show that it is feasible to identify substrates of deacetylases by manipulating their expression levels by ectopic protein expression or gene knockdown by siRNA-based approaches. It is plausible that ectopic expression of deacetylases may result in deacetylation of non-native substrates, and may complicate the interpretation of results. 5 Identification of Endogenous Substrates of Sirt3 6 Identification of Endogenous Substrates of Sirt3 Nevertheless, this approach can be useful for identification of putative substrates of lysine deacetylases or their validation in different model organisms, as we have shown here. This strategy is also applicable for discovering substrates of deacetylases that are important for cellular viability, and for which it is not possible to obtain complete knockout cells. In summary, our proteomics screen identified a large number of endogenous substrates of Sirt3 in mammalian cells. Our 481-53-8 web results show that Sirt3 deacetylates a large number of enzymes involved in key metabolic pathways implying a broader regulatory role of Sirt3 in mitochondrial processes. These results exemplify the usefulness of combining genetic tools with quantitative MS screens to identify endogenous substrates of lysine deacetylases. The Sirt3 targets identified here may provide a starting point to analyze their functional relevance in cellular metabolism. using 5% skimmed milk powder in PBS tween-20. PDHX was detected using an anti-PDHX antibody followed by incubation with Protein-G conjugated to horseradish peroxidase. Sirt3-expression was analyzed by subjecting total cell lysate to SDS-PAGE and immunodetected using an anti-Sirt3 antibody. As a loading control, an antibody against Vinculin or Acetyl-a-Tubulin were used. The secondary antibodies coupled to horseradish peroxidase were used for immunodetection. The detection was performed with Novex ECL Chemiluminescent Substrate Reagent Kit. MS sample preparation Sirt3 knockout and wild-type control cells were cultured in SILAC media. Also, SILAC-labeled Sirt3 knockdown or overexpressing cells, U2OS cells expressing exogenous Sirt3 or U2OS expressing Sirt3 shRNA were cultured for 48 hours in the presence of 1 mg/ml doxycycline prior to lysis. Cells were washed twice with 16phosphate buffered saline and lysed in RIPA-buffer containing 1% NP-40, 1 mM sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 50 mM Tris, and protease inhibitor. Lysates were incubated for 15 minutes on ice, and were cleared by centrifugation. Protein concentration of the cleared lysates was measured with BCA Protein Assay Reagent and proteins from two SILAC populations were mixed 1:1. Proteins from cell lysates were acetone precipitated by adding 4-fold excess acetone, and storing overnight at 220uC. The precipitated proteins were re-dissolved in 6M urea/2M thiourea/10 mM HEPES, reduced with 1 mM dithiothreitol, alkylated with 5.5 mM chloroacetamide and subsequently digested with endoproteinase Lys-C and modified sequencing grade trypsin as described previously. Protease digestion was stopped by adding trifluoroacetic acid to a final concentration of 1%, and precipitation was cleared by centrifugation. Peptides were purified using reversed-phase Sep-Pak C18 cartridges and peptides were eluted in 50 percent acetonitrile, 0.1% TFA. The peptides were re-dissolved in