6120 quadrupole

Bacterially produced Ch-S was obtained by culturing P. merdae ATCC 43184 (WT) and P. merdae Δsult in the presence of 500 μM Ch for 168 hours alongside media controls (n = 3) and P. merdae WT with no additional Ch. A 1.25 mL sample was removed from each culture, acidified to pH 1 with HCl (Sigma), and extracted twice with ethyl acetate (Sigma). The organic phase was collected and evaporated to dryness using a turbovap (Biotage). Dried extracts were resuspended in 100 μL of HPLC-grade methanol (Sigma) and quantified by running 1:10 dilutions on the UPLC-MS (Agilent Technologies 1290 Infinity II UPLC system coupled online to an Agilent Technologies 6120 Quadrupole LC/MS spectrometry in negative electrospray mode) alongside a standard curve according to the above method. Quantified samples were used for immune cell migration assays (see below).

Peptides were synthesized on solid support using Fmoc chemistry. Rink amide MBHA resin (25 μmol) was deprotected using 25% piperidine in 1-methyl-2-pyrrolidinone (NMP) for 25 min. All subsequent deprotections were performed under the same conditions. Couplings were performed for 45 min using 0.5 M Fmoc-protected amino acid (0.5 mL, 250 μmol, 10 equiv.), 0.5 M HCTU (0.495 mL, 247.5 μmol, 9.9 equiv.), and diisopropylethylamine (87 μL, 500 μmol, 20 equiv.) in NMP. After the final deprotection the peptides were then cleaved from the resin in a solution containing 95% TFA, 2.5% triisopropylsilane, and 2.5% water for 4 hours. The resin was removed by filtration through glass wool, and the peptides were precipitated in ice cold tert-butyl methyl ether. The precipitate was pelleted by centrifugation and the supernatant was discarded. The dry peptide was dissolved in methanol and characterized by LC/MS using a Zorbax SB-C18 5 μm column with an Agilent 1200 series HPLC coupled to an Agilent 6120 quadrupole LC/MS. Peptides were purified by reverse-phase HPLC using a gradient of 100% acetonitrile in water containing 0.1% TFA. Neurochondrin peptide I molecular weight = 2418.0 (expected = 2418.7); neurochondrin peptide II = 2229.9 (expected = 2230.6).

Samples were acidified to a pH of 1 with HCl (Sigma) with GCA (Steraloids) as the internal standard and extracted twice with ethyl acetate (Sigma). The organic phase was collected and dried down using a turbovap (Biotage). Dried extracts were resuspended in 75% HPLC-grade methanol in dH₂O and analyzed by UPLC-MS (Agilent Technologies 1290 Infinity II UPLC system coupled online to an Agilent Technologies 6120 Quadrupole LC/MS spectrometry in negative electrospray mode) using a published method^{69 (link),75} with modifications outlined as follows. Extracted molecule solutions were injected onto a Phenomenex 1.7 μm, C18 100 Å, 100 × 21 mm LC column with a flow rate of 0.350 mL/min using 0.05% formic acid in water as mobile phase A and acetone as mobile phase B. The following gradient was applied: 0–1 min: 25–60% B, 1–5 min: 60–70% B, 5–6 min: 70–100% B, 6–7 min: 100% B isocratic, 7–8 min: 100–25% B, 8–10 min: 25% isocratic. Extracted ion chromatograms and areas under the curve were generated using Agilent ChemStation C.01.07 SR3. Exported data were analyzed in Microsoft Excel and GraphPad Prism V9.

Streptomycete cultures were pelleted by centrifugation (4,000 x g for 15 min), and the spent media were extracted 1:1 by volume with ethyl acetate. Ethyl acetate was removed by rotary evaporation, and the dry extract was dissolved in 300 μL of methanol for liquid chromatography-mass spectrometry (LC-MS) analysis by 10 μL injection onto an Agilent Technologies 6120 Quadrupole LC-MS instrument with an Agilent Eclipse Plus C18 column (4.6 × 100 mm). For high resolution MS (HRMS) analysis, the extract in methanol could be diluted by a factor of 5 for LC-HRMS and HRMS/MS analysis by 10 μL injection onto an Agilent Technologies 6545 Q-TOF LC-MS instrument with the same column. Linear gradients of 5-95% acetonitrile (vol/vol) in water with 0.1% formic acid (vol/vol) at 0.5 mL/min were used.