Methionine

Isotope labeling of proteins was achieved by expressing protein in 2×M9 minimal media (28 (link)) with the relevant isotopes supplemented to the media (H₂O supplemented with (¹⁵NH₄)Cl for [U-¹⁵N]-labeled protein or with both (¹⁵NH₄)Cl and d-(¹³C)-glucose for uniform double labeling [U-¹³C,¹⁵N], or D₂O supplemented with (¹⁵NH₄)Cl yielding [U-²H,¹⁵N]-labeled proteins or D-(²H,¹³C)-glucose for uniform triple labeling [U-²H,¹³C,¹⁵N]). For specific methyl group labeling, Met, Ala, Leu, Val and Ile (MALVI^proS)-labeled DinB sample was produced in Bioexpress rich (2%) D₂O-based 2× M9 media supplemented with (¹⁵NH₄)Cl, d-(²H, ¹²C)-glucose as well as 50 mg/l 2-Ketobutyric acid-4–¹³C,3,3-d₂ sodium salt hydrate (Isoleucine), 4 vials/l DLAM-LV^proS-kit (2-(¹³C)-methyl-4-(D₃)-acetolactate (valine/leucine proS methyl group only), 50 mg/l [U-²H, ¹³CH₃] methionine, and 50 mg/l 2-[²H], 3-[¹³C] L-alanine (alanine) precursors added 1 h prior to induction (29–31 (link)). Bioexpress, methionine, and alanine were purchased from Cambridge Isotopes Laboratories and the DLAM-LV^proS precursors from NMR-Bio. All other isotopes were purchased from Merck.

Non-polar extracts were reconstituted in 75 µl of IPA containing isotopically labeled lipid standards (a detailed list of standards is included in the SI) and analyzed by LC-MS using a ThermoFisher Scientific Accucore C30 150 × 2.1mm, 2.6 µm column paired with a Thermo Fisher Orbitrap ID-X in positive and negative polarity. Polar (80:20 CH₃OH:H₂O) extracts were reconstituted in 75 µl of 80:20 CH₃OH:H₂O containing isotopically labeled arginine, hypoxanthine, hippuric acid, and methionine (Cambridge Isotope Laboratories, Inc.) and analyzed by LC-MS using a Waters BEH Amide 150 × 2.1 mm, 1.7 µm column paired with a Thermo Fisher Orbitrap ID-X in positive and negative polarity. LC-MS/MS data for each mode of analysis was collected using three rounds of iterative DDA (Thermo Scientific AcquireX) performed on pooled test samples.
Data for each sample were collected in full MS1 with a resolution of 240,000 FWHM (full-width half-maximum) and MS/MS spectra of pooled samples were collected at a resolution of 30,000 FWHM using a 0.8 Da isolation window and stepped HCD collision energies of 15, 30, and 45.

Amino acid standards (alanine, arginine, aspartic acid, cysteine,
glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine,
phenylalanine, proline, serine, threonine, tyrosine, valine, glutamine,
asparagine, GABA, citrulline, ornithine, taurine, tryptophan, 5-HTP,
norvaline, and kynurenine) were purchased from Sigma (St. Louis, MO).
Isotopically labeled amino acid standards (alanine (¹³C₃,¹⁵N), arginine (¹³C₆, ¹⁵N₄), aspartic acid (¹³C₄, ¹⁵N), cystine (¹³C₆, ¹⁵N₂), glutamic acid (¹³C₅, ¹⁵N), glycine (¹³C₂, ¹⁵N), histidine
(¹³C₆, ¹⁵N₃), isoleucine
(¹³C₆, ¹⁵N), leucine (¹³C₆, ¹⁵N), lysine (¹³C₆, ¹⁵N₂), methionine (¹³C₅, ¹⁵N), phenylalanine (¹³C₉, ¹⁵N), proline (¹³C₅, ¹⁵N),
serine (¹³C₃, ¹⁵N), threonine (¹³C₄, ¹⁵N), tyrosine (¹³C₉, ¹⁵N), valine (¹³C₅, ¹⁵N), citrulline (d4), GABA (¹³C₄), glutamine
(¹³C₅), asparagine(¹³C₄), ornithine (d6), tryptophan (d8), kynurenine (d4)) were obtained
from Cambridge Isotope Laboratories (Andover, MA). Stock solutions
of each compound were prepared at a concentration of 500 ng/μL
and stored at −80 °C. A 10-point calibration curve (0.01–100
pmol/μL) was prepared by serial dilutions and spiked with internal
standards at a final concentration of 5 pmol/μL. Mass spectrometry-grade
formic acid was purchased from Sigma-Aldrich (St Louis, MO), and HPLC-grade
acetonitrile was purchased from Fisher Scientific (Fair Lawn, NJ).