Tq xs

Cell extractions were prepared as described in [34 (link), 35 ]. Central carbon metabolites and nucleoside-phosphate were analyzed by capillary ion chromatography [34 (link)] coupled to tandem mass spectrometry, TQ-XS (Waters). Free amino acids were first derivatized by Edman’s reagent [36 (link)]. Derivatized samples were analyzed by UPLC (waters) coupled to TQ-XS as described in [37 ]. All metabolites measurements were corrected by isotopic dilution method as described in [37 ].
Determination of the NADH/NAD ratio was performed using an enzymatic assay kit, following the instruction supplied by the manufacturer (Sigma). Briefly, growing culture was centrifuged (3 min, 4500×g) and the supernatant was carefully discarded. Pellets were quickly frozen by liquid nitrogen for storage at −80 °C. Upon the measurements, the frozen cells were thawed on ice and resuspended in the extraction buffer supplied from the kit. An absorbance at 450 nm was measured every 20 min for 4 h using 96 well-plates in a plate-reader spark 20 M (Tecan).

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.