A R T I C L E S GGAAACCCCGGGAATCTAACCCGGCTGAACGGATTTAG- AG, which is speciﬁc for MbtDNACPyUlA. The hybridized probe was detected with North2South chemiluminescent hybridization and detection kit (Pierce). The number of cells was used to control the total amount of RNA loaded. Phenotyping Yeast Cells. Phenotyping was performed as described in Chin et al..8 Brieﬂy, S. cereVisiae MaV203 (Invitrogen) was transformed by the lithium acetate method with the pGADGAL4- (2TAG) reporter, pMbPylRS and tDNAPyl CUA constructs. Overnight cultures were serially diluted and replica plated onto selective media in the presence or absence of 2 mM Nε-[(2-propynyloxy)carbonyl]- L-lysine (1). X-Gal assays were performed using the agarose overlay method. Protein Expression, Puriﬁcation, western blot Analysis, and Mass Spectrometry. Appropriate selective medium ( unnatural amino acid was inoculated with a stationary phase culture to give an OD600 ≈ 0.2. Cultures were grown at 30°C for 24-48 h. Proteins were extracted from yeast cells using Y-PER reagent (Thermo Scientiﬁc) containing complete, EDTA-free inhibitor cocktail (Roche). Clariﬁed supernatants were separated by SDS-PAGE, and western blots were performed using anti-His6 (Qiagen). Human superoxide dismutase was puriﬁed using Ni2+-NTA resin (Qiagen) as previously described.26 For expressions with Nε-acetyl-L-lysine Hancock et al. (2), 20 mM nicotinamide was added to the cultures and to lysis buffers; for expressions with Nε-triﬂuoroacetyl-L-lysine (3), 10 mM sodium butyrate was added to the cultures and to lysis buffers. Protein concentration was determined using the Biorad Protein Assay in comparison to IgG standard. Total mass analysis was performed on a LCT time-of-ﬂight mass spectrometer with elec- trospray ionization (Micromass) with protein solutions in 20 mM ammonium bicarbonate and mixed 1:1 with 1% formic acid in 50% MeOH. Samples were injected at 10 µ L · min-1, and calibration was performed in positive ion mode using horse heart myoglobin. MS/ MS analysis was performed on a LTQ-Orbitrap mass spectrometer on protein samples that were in-gel digested with Glu-C (Roche). Acknowledgment. We are grateful to Farida Begum and Sew Peak-Chew (MRC-LMB) for mass spectrometry, and Heinz Neu- mann and Sebastian Greiss for assistance. This work was supported by the Medical Research Council (MRC) UK and a European Research Council (ERC) StG to J.W.C. Supporting Information Available: Supplementary ﬁgures, details of plasmid construction, and construct sequences. This material is available free of charge via the Internet at http:// pubs.acs.org. (26) Chen, S.; Schultz, P. G.; Brock, A. J. Mol. Biol. 2007, 371, 112. JA104609M 14824 J. AM. CHEM. SOC. 9 VOL. 132, NO. 42, 2010