68 Chapter 3 Na2HPO4/Na2B4O7 pH 8.2 buffer was used as solvent A and a mixture of 45:45:10 acetonitrile/methanol/water as solvent B at a flow-rate of 0.5 ml/min and a gradient from 2-57% B from 0.5-20 min after the injection followed by respectively a 4 min 100% cleaning and 9 min 2% B reconditioning period. The fluorescence detector was set to λEx = 340 nm, λEm = 450 nm for all 2-O-phthaldialdehyde (OPA) derivatives and λEx = 266, λEm = 305 nm for the 9-fluorenylmethyl chloroformate (FMOC) derivatives at 18 min, eluting at the end of the chromatogram. Quantification of amino acids was performed based on 5-point internal standard corrected calibration curves in water ranging from 2-250 µM (r2>0.99). Targeted proteomics of metabolic enzymes in the interphase by quantification concatemers (QconCATs) For proteome analysis, interphases obtained during metabolite extractions as described in the section “TCA cycle metabolites measurement by GC-MS” were used. Samples stored at -80°C were dried and resuspended in 8M Urea, 100 mM ammonium bicarbonate to be further diluted with 100 mM AmBiCa to 2M urea. Samples were then sonicated (Vibra-Cell, Sonics, Newton, United States) for 3×10 seconds, 30% without pulse. Protein concentrations were determined with a microplate BCA protein assay following the manufacturer’s instructions (Pierce™ Microplate BCA Protein Assay Kit, Thermo Fisher Scientific). For insolution digestion, 10 µg of sample was diluted in 2M Urea, 100 mM ammonium bicarbonate (AmBiCa) to a final volume of 300 µL. Proteins were reduced in 10mM dithiothreitol for 30 minutes at 37°C, alkylated with 55 mM iodoacetamide, and incubated in the dark at room temperature for 60 minutes. To further dilute the urea concentration, 300 µL of 100 mM AmBiCa was added. Samples were then digested with 100 ng of trypsin (sequencing grade modified trypsin V5111, Promega) at 37 °C for 16 hours and the trypsin was deactivated with formic acid before cleanup using C18 SPE columns (Gracepure TM SPE C18-Aq, 50 mg/1mL). Targeted proteome analyses were performed by injecting 1 µg total protein starting material plus 2 ng high abundant proteins or 0.1 ng low abundant proteins of predigested QconCAT standards (quantification concatemers; designed to target a set of proteins, details about the assay development and applications for quantification have been described elsewhere previously) (30,31). Untargeted proteomics of the interphase For proteome analysis, the same digested samples from the interphases obtained during metabolite extractions as described in the section “TCA cycle metabolites measurement by GC-MS” and processed as described in the section “Targeted
RkJQdWJsaXNoZXIy MTk4NDMw