Acquity pda

Tissue samples harvested from sacrificed animals were weighed before homogenization, frozen, and stored at −80°C. Tissues were homogenized in PBS + protease inhibitor buffer using a TissueLyser (Qiagen). Oligomycin B (Cat #11343, Cayman Chemical) was used as an internal standard at a final concentration of 1 μg/mL. Homogenates were vortexed for 30 s, left for 5 min, then extracted using an excess (1 mL) of acetonitrile, vortexed for 3 min and placed on a shaker for an additional 15 min. All samples were then centrifuged at 10,000 x g for 10 min and the supernatants were transferred to a clean vial and evaporated to dryness, followed by reconstitution with 50 μL of 75% acetonitrile (LC-MS grade). Samples were run on an UPLC-MS system using an Acquity UPLC BEH C18 column (50 mm x 2.1 mm) and a mobile phase of 75% acetonitrile in water was applied under isocratic conditions at a flow rate of 0.5 mL/min. A UPLC-MS system equipped with a single quadrupole (Acquity QDA, Waters) coupled to a UPLC system (Acquity PDA, Waters) was used in multiple reaction monitoring (MRM) mode. The ESI-MS of Oligomycin A [C₄₅H₇₃O₁₁Na]⁺ was calculated as 813.51 and was found as 813.79, whereas Oligomycin B [C₄₅H₇₁O₁₂Na]⁺ was calculated as 827.49 and was found as 827.67. The retention times for Oligomycin A and Oligomycin B were 1.09 min and 0.77 min, respectively.

Peptides were incubated in RKO cells (ATCC, #CRL-2577) as follows: Experiment 1: RKO cells were incubated in 25 µM compound for 90 min and 24 h. Experiment 2: RKO cells were incubated in 25 µM for 30 min or 72 h. Following peptide incubation, the cells were washed twice with PBS, trypsinized and pelleted. The pellets were lysed in RIPA buffer (Perkin Elmer) for total lysis, or CelLytic NuCLEAR Extraction Kit (SIGMA) for subcellular fractionation. Total protein quantification BCA (Pierce) was performed prior to MS analysis to normalize sample data. Total and fractionated cell lysates were analysed by quantitative UPLC-MS utilizing a Waters Xevo TQ-XS (WBA0259) and an Acquity UPLC system from Waters consisting of Sample Manager (M16UFL953M), Acquity PDA (F17UPD457A), Column Oven (E17CMP703G) and Binary Solvent Manager (E17BUR621G). The Waters TQ-XS was operated in +ve ion Electrospray (ESI) mode with the optimized transitions for peptides 4, 4E, 7, and TAT. The chromatograms at each transition were extracted, smoothed, and integrated to give the standard curve. Chromatograms of the samples were treated similarly, and by linear regression (1/x) an in-cell concentration was established in the re-suspended cell lysate using the Waters MassLynx TargetLynx^TM product.