13c6 isoleucine

S. aureus strain AH1263 was a USA300-014 genotype. [¹³C₆]Isoleucine, [d₃]leucine, and] [¹³C₅,¹⁵N]valine were from Cambridge Isotope Laboratories, Inc. [¹³C₅,¹⁵N]valine, rather than [¹³C₅]valine, was used because it was less expensive, and the presence of ¹⁵N has no impact on the experiment. [¹³C₂]C₂-CoA was from Millipore Sigma. Defined media components and other reagents were purchased from Millipore Sigma or Thermo Fisher Scientific. All MS reagents are of HPLC grade or better. The defined media used in this work consist of M9 salts supplemented with amino acids and other nutrients as outlined in Table S2. The growth characteristics of S. aureus strain AH1263 in defined media lacking each of the branched-chain amino acids are illustrated in Figure S4. Like other S. aureus strains, wildtype strain AH1263 requires extracellular Val as a supplement in defined media (47 , 51 (link)). Growth when Leu was removed was slower than in the other conditions as reported previously, but removal of either Ile or Ile + Leu did not affect growth (47 , 51 (link)) (Fig. S4). The Leu growth phenotype arises from the Ile repression of branched-chain amino acid synthesis by CodY (47 ).

All chemicals and reagents were reagent grade or better. [¹³C₆]Isoleucine, [d₃]leucine, [¹³C₅,¹⁵N]valine were purchased from Cambridge Isotope Laboratories, Inc. DSC-NH₂ SPE columns (Supelco, Sigma-Aldrich), Bee venom PLA₂ (P9297-Sigma), Orlistat (O4169-Sigma), [1-¹⁴C]oleate (specific activity 59 mCi/mmol) (Perkin Elmer).

Reference serum and study samples were thawed and metabolites were extracted using the combined extraction method (MTBE/methanol/water)^{56 (link)}. Briefly, 50 μL serum was mixed with 225 μL ice-cold methanol and 750 μL ice-cold methyl-tertbutyl ether (MTBE). The mixture was vortexed and incubated on ice for 1 h. The samples were then mixed with 188 μL MS-grade water containing internal standards (¹³C-lactate, ¹³C₃-pyruvate, ¹³C-methionine and ¹³C₆-isoleucine were purchased from Cambridge Isotope Laboratories) and vortexed. The mixture was then centrifuged at 15,000 × g at 4 °C for 10 min and the bottom phase was transferred to two tubes (125 μL/each tube) for positive and negative mode analysis of LC-MS. Finally, the samples were evaporated to dryness under a speed vacuum concentrator.