Initially, mitochondrial and cytosolic fractions were separated using a commercially available kit (ProteoExtract Cytosol/Mitochondria Fractionation Kit, Merck, USA) according to manufacturer’s instructions. Mito-Esc was quantified in the mitochondrial and cytosolic fractions obtained from HAEC and aortas of ApoE−/− mice of different treatment groups as mentioned in the Animal Experiments section. Electrospray ionization (ESI)-mass spectrometry (MS) measurements (positive mode) were performed using a quadrupole time-of-flight mass spectrometer (QSTAR XL, Applied Biosystems/MDS Sciex, Foster City, CA). The data acquisition was under the control of Analyst QS software (Applied Biosystems). For the CID (collision-induced dissociation) experiments, the precursor ions were selected using the quadrupole analyzer and the product ions were analyzed using the TOF analyzer47 (link).
The mitochondrial/cytosolic extracts (50 μl) were diluted with 50 μl of methanol and introduced into the ESI source (injection volume 20 μl) using methanol: water (80:20, v/v) as a mobile phase gradient with flow rate 600 μl/min. Stock solution (0.5 mM) of Mito-Esc was made in methanol: water (50:50, v/v). For spiking experiments, appropriate volumes of standard solutions (1–50 μM) were added to the mitochondrial/cytosolic extracts of untreated samples.
The mitochondrial/cytosolic extracts (50 μl) were diluted with 50 μl of methanol and introduced into the ESI source (injection volume 20 μl) using methanol: water (80:20, v/v) as a mobile phase gradient with flow rate 600 μl/min. Stock solution (0.5 mM) of Mito-Esc was made in methanol: water (50:50, v/v). For spiking experiments, appropriate volumes of standard solutions (1–50 μM) were added to the mitochondrial/cytosolic extracts of untreated samples.
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