Amino acid stocks were prepared at 10 mM concentration or their maximum solubility (always >1 mM) and were stored at –20°C. For translation assays the pH of the stocks was adjusted with KOH to 7–7.5, and the stocks were filtered. Supplier information for the amino acids has been described previously 
. The α-hydroxy acids were purchased from Fluka (OF, OM, OS, OV), Sigma/Aldrich (OA, OP, OG, OH, OI, OL, OW, OY) or City Chemical (OE).
AARSs and translation factors were prepared as His-tagged constructs as previously described 
Quantitative AARS Assays Each assay contained 40 mM HEPES-KOH pH 7.4, 17 mM MgCl2, 45 mM KCl, 3.4 mM BME, 6 mM ATP, 6% glycerol, 0.02 units/mL yeast inorganic pyrophosphatase, 50–350 µM E. coli total tRNA (Roche), 0.09 mg/mL BSA, and various AARS, natural amino acid and analog concentrations (). For each analog inhibition experiment three different assays were performed (A, B, and C). These assays were then mixed together (Mixing protocols 1–3) to determine differences between the ratios of the natural AA [M]+ and [M+15]+ peaks in the presence of the analog.
AARS and AA concentrations in competition assays
: Only the natural amino acid was included in this charging assay at the indicated concentration (). The assay was initiated by the addition of the appropriate AARS and was incubated for 30 min at 37°C. A solution of 3 M NaOAc pH 5.2 (0.1 vol., 2.5 µL) was added and the assay solution extracted with unbuffered phenol:CHCl3
:isoamyl alcohol (25:24:1), and then CHCl3
. A portion (45 µL) of the final aqueous layer was removed and precipitated with EtOH (3 vol.) and 3 M NaOAc pH 5.2 (0.1 vol.) at –20°C. The resulting pellet was washed twice with 70% EtOH (250 µL each), allowed to dry at room temperature (~5 min), and dissolved in 200 mM NaOAc pH 5.0 (12.5 µL). Half of the solution was frozen at –20°C, and the remaining half was added to water (3.75 µL), a freshly prepared solution of 4-formylphenoxypropyl triphenylphosphonium bromide 
(each 69 mM in MeOH, 12.5 µL), and freshly prepared NaCNBH3
(200 mM in 50 mM NaOAc pH 5.0, 2.5 µL). The solution was placed in a tumbler at 37°C (occasionally additional MeOH (1–2 µL) was required for complete dissolution). After 2 hours, 4.4 M NH4
OAc pH 5.0 (0.2 vol., 5 µL) was added to quench and the assay was mixed as described below (Mixing protocol).
: Assay B followed the identical procedure, except the deuterated aldehyde 4-formylphenoxypropyl triphenylphosphonium-d15
was added in place of the non-deuterated aldehyde.
Assay C: Assay C included both the natural amino acid and the analog at the specified concentrations () and used the deuterated aldehyde 4-formylphenoxypropyl triphenylphosphonium-d15 bromide. The contents of the three assays are summarized in .
Mixing protocol #1 (used to determine the ratio of the [M]+ and [M+15]+ peaks when no unnatural AA competitor was present). After the NH4OAc quenching of the reductive amination step, 25 µL of Assay A and 25 µL of Assay B were mixed together and the combined assays were precipitated with EtOH (3 vol.). The pellet was washed with 70% EtOH (2X) and 100% EtOH (2X) and was allowed to dry at room temperature. The dry pellet was then dissolved in 200 mM NH4OAc pH 5.0 (2.25 µL) and 1 U/µL Nuclease P1 in 200 mM NH4OAc pH 5.0 (~0.25 µL) was added. After 20 min at room temperature, an aliquot (1 µL) was removed and added to a saturated solution of CHCA (α-cyano-4-hydroxycinnamic acid) matrix in 1:1 MeCN:1% TFA (9 µL). An aliquot (1 µL) of the resulting suspension was added to a MALDI plate and analyzed.
Mixing protocol #2 (used to determine the ratio of [M]+ and [M+15]+ that would be found if complete, 100%, inhibition by the analog was observed). For this protocol, assay A was used alone without mixing and treated exactly as described for Mixing protocol #1.
Mixing protocol #3 (used to determine the ratio of [M]+ and [M+15]+ when the analog was present). For this protocol, Assays A and C (25 µL each) were mixed and treated exactly as in Mixing protocol #1.
After software baseline subtraction, the ratio of peak heights for derivatized natural AA-AMP and d15
-AA-AMP was determined for the assay mixtures described above. To get the final % inhibition, the following equation was used % inh
(ratio A/C mix – ratio A mix)/(ratio A/B mix – ratio A mix)*100. For MetRS, ValRS, IleRS, GlnRS, GluRS, and AspRS analogs the % inhibition value was instead calculated with a calibration curve determined by mixing known ratios of d15
-AA-AMP and H15
Translation reactions (50 µL) were carried out as previously described 
. The mRNAs used for the translation reactions are described in the supporting information (Figure S1
). For a particular template only the required natural or analog amino acids (400 µM each) and AARSs (0.1 µM MetRS, 0.3 µM LeuRS, 0.6 µM GluRS, 0.2 µM ProRS, 1.0 µM GlnRS, 1.0 µM HisRS, 0.3 µM PheRS or 0.25 µM PheRS A294G, 1.5 µM TrpRS, 0.2 µM SerRS, 0.2 µM IleRS, 0.4 µM ThrRS, 0.6 µM AsnRS, 0.6 µM AspRS, 0.5 µM TyrRS, 0.5 µM LysRS, 0.4 µM ArgRS, 0.2 µM ValRS, 0.2 µM AlaRS, 0.5 µM CysRS, 0.06 µM GlyRS) were added.
Multiple Analog template translation Peptide synthesis was carried out with the following optimized amino acid concentrations: N1 (800 µM), A3 (1600 µM), C6 (800 µM), A15 (1600 µM), A10 (400 µM), P5 (800 µM), N12 (3200 µM), A18 (800 µM), C7 (400 µM), A24 (800 µM), N23 (800 µM), C10 (400 µM), N18 (1600 µM), Ala (400 µM), Gln (400 µM).
α-hydroxy methionine peptide cleavage assay
Translation reactions (100 µL) were performed as described 
using mRNA template f
. After elution from the Ni-NTA resin (Qiagen) into 0.2% TFA (50 µL), the peptides were treated with 1 M NaOH (15 µL) for 60 min at 37°C. The assays were quenched with 10% TFA (6 µL) and 1 M HOAc (5 µL) and were purified by Zip-Tip C18
chromatography as described above. The products were analyzed by MALDI-TOF MS.