Liquid Chromatography-Mass Spectrometry (LC-MS)
Reagents and chemicals
The following reagents were purchased: water, acetonitrile, isopropanol,
and methanol from Optima grade (Fisher Scientific); high purity formic
acid (Thermo-Scientific); 3-HPMA-d6 and Isobutyril-Coenzyme A were
purchased from Cayman chemicals.
Preparation of standards and internal standard stock solutions
CoA was resuspended in HEPES buffer pH 7.5 to generate a 3.83 mg
ml-1 stock solution (stock A). A 1 mg
mL-1 solution (stock B) of Isobutyryl-Coenzyme A
(IB-CoA) was prepared by dissolving IB-CoA in HEPES buffer pH 7.5. The
stock A and B were diluted in extraction buffer (3-HPMA-d6 was dissolved
in methanol to achieve a 1 mg mL-1 stock solution, and
subsequently diluted to a final concentration 500 ng
mL-1 of IS) to generate stock C (20 µg
ml-1 of CoA and 10 µg ml-1 IB-CoA).
Stock C was then serially diluted to generate calibration curve
standards ranging from 20 ng ml-1 to 10 µg
ml-1 (IB-CoA) and 40 ng ml-1 to 20
µg ml-1 (CoA).
Sample preparation
MbcS acyl-CoA activity was assessed via LC-MS. The reaction mixture
consisted of 50 mM HEPES pH 7.5, 2.5 mM Mg-ATP, 493 μM CoA, 73 μM
isobutyric acid, and 60 nM MbcS. MbcS was denatured through incubation
at 85°C for 10 minutes and used as a negative control. The reactions
were initiated by adding isobutyric acid and incubated at 37°C for 2
hours, followed by placing the samples on ice. For IB-CoA measurements,
to the 40 µl of samples was added 360 µl of extraction buffer and
vortexed. For CoA measurements, the samples prepared for IB-CoA
measurements were diluted 10-fold using extraction buffer. The samples
were transferred to the MS vials for data acquisition.
LC-MS methods
This method was designed to measure CoA and Isobutyryl-CoA by UPLC-MS.
The samples were resolved on a HSS T3, 1.8 µm, 2.1 x 100 mm column
online with a triple quadrupole mass spectrometer (Xevo-TQ-S, Waters
Corporation) operating in the multiple reaction monitoring (MRM) mode.
The LC gradient method started with 98% of mobile phase A (10 mM
ammonium formate in water) that involved a gradient change from 2% B
(ACN:water [95:5] with 10 mM ammonium formate) to 98% phase B in
2.5 minutes after an initial lag phase of 0.5 minutes with flow rate 0.6
ml/min. The column was maintained at 25 °C and injection volume was kept
at 5 µL. The autosampler was maintained at 5 °C. The column eluent was
introduced directly into the TQS mass spectrometer by electrospray
operating in positive mode at a capillary voltage of 3.0 kV and a
sampling cone voltage of 83 V. The desolvation gas flow was set to 1000
l/h and the desolvation temperature was set to 500 °C. The cone gas flow
was 150 l/h and the source temperature was set to 150 °C.
The sample cone voltage and collision energies were optimized for the
analyte to obtain maximum ion intensity for parent and daughter ions
using “IntelliStart” feature of MassLynx software (Waters
Corporation). The instrument parameters were optimized to gain maximum
specificity and sensitivity of ionization for the parent and daughter
ions. Signal intensities from the MRM Q1>Q3 ion pairs for
the CoA (768.1>261.1), IB-CoA (838.1>331.1)
and 3-HPMA-d6 (228>169) were ranked to ensure selection of
the most intense precursor and fragment ion pair for MRM-based
quantitation. This approach resulted in selection of cone voltages and
collision energies that maximized the generation of each fragment ion
species. The details for the calibration curves for standards are
included in Supplementary Materials (Table S4) . CoA was
detectable as low as 5.0 ng ml-1 (limit of detection
[LOD]) and showed 250 ng ml-1 as the lowest limit
of quantification (LLOQ). While IB-CoA showed the LOD and LLOQ as 25 ng
ml-1 and 50 ng ml-1, respectively.
The quantification range (QR) for CoA and IB-CoA were 250 ng
ml-1 to 20 µg ml-1 and 50 ng
ml-1 to 10 µg ml-1, respectively. As
ascertained by blanks run on either side of sample sets, no
sample-to-sample carryover was observed. Each experiment was conducted
in triplicate. Blanks (solvent alone) were injected on either side of
test samples to assess sample carryover. Data was processed using Target
Lynx 4.1. The relative quantification values of analytes were determined
by calculating the ratio of peak areas of transitions of samples
normalized to the peak area of the internal standard.