6130 single quadrupole lc ms

Analysis was performed on an Agilent 6130 Single Quadrupole LC/MS instrument. Chromatographic separation was achieved using a Kinetex Hilic column (100 × 2.1 mm, 100 Å, 2.6-μm particles; Phenomenex, Torrance, CA, USA). 0.1% (v/v) formic acid in water was used as mobile phase A and 0.1% (v/v) formic acid in acetonitrile was used as mobile phase B. The mobile phase flow rate was 500 μL min⁻¹. The injection volume was 5 μL. The gradient elution method was: 90% B to 10% B from 0 to 10 min, held at 10% B from 10 to 13 min, 10% B to 90% B from 13 to 14 min.

Synthesis of CoA esters was performed as described (63 (link)) unless specified otherwise. β-Methylmalyl-CoA was synthesized from propionyl-CoA and a 12-fold molar excess of glyoxylate (in buffer: 100 mM MOPS/KOH pH 7.5, 5 mM MgCl₂) using heterologously expressed and purified Rhodobacter sphaeroides Mcl-1 (accession number ACI22682) as catalyst. The resulting esters were purified via preparative HPLC-MS with a 1260 Infinity II LC System (Agilent, USA) in combination with a 6130 Single Quadrupole LC/MS (Agilent, USA). Lyophilized CoA esters were stored at −80°C and dissolved in ddH₂O before use. The concentrations of the respective CoA esters were determined photometrically using a Carry 60 UV-Vis spectrophotometer (Agilent, USA) and calculated from the absorbance at 260 nm using defined extinction coefficients (22,300 M⁻¹cm⁻¹ for α-β-unsaturated fatty acid CoA thioesters and 16,400 M⁻¹cm⁻¹ for saturated ones). The amount of free sulfhydryl groups (indicating free CoA) present in the samples was quantified by treatment with Ellman’s reagent (DNTB) in EDTA-HEPES/KOH buffer (15 mM EDTA, 200 mM HEPES-KOH pH 7.8) and subsequent measurement of the absorbance at 412 nm (extinction coefficient: 14,000 M⁻¹cm⁻¹).