67 3 Responses of alveolar-like macrophages to lysine deacetylase inhibition and chloroform were stored at -20°C until the moment of use. Methanol/chloroformcontaining cell extracts were first shaken for 30 minutes at 4°C and then centrifuged for 10 minutes at 16100 g at 4°C. The upper aqueous phase was then transferred to a new tube and evaporated under nitrogen. The lower non-polar fraction was also transferred to a new tube and the solvent was evaporated under nitrogen. The interphase was washed with methanol and centrifuged for 10 minutes at 16000 g at 4°C. The supernatant was removed and a small volume of fresh methanol was added to the pellet and samples were stored at -80°C for further protein analysis (see the paragraph “Targeted proteomics of metabolic enzymes by QconCATs”). The dried metabolites present in the upper aqueous phase were dissolved in 2% methoxyamine HCL in pyridine and incubated for 90 minutes at 37°C. The samples were then silylated for 1 hour at 55°C by adding N-tert-butyldimethysilylN-methyltrifluoroacetamide with 1% tert-butyldimethylchlorosilane. The derivates were transferred to a GC/MS vial with a micro-insert and analyzed by GC/MS. GC/MS measurements were carried out on a 7890A GC (Agilent) coupled to a 5975C Quadrupole MS (Agilent), equipped with a CTC Analytics PAL autosampler (CTC Analytics AG, Switzerland). Quantification of the amino acids and TCA cycle metabolites was performed based on 6-point internal standard corrected calibration curves in water, treated like the cell samples to follow the complete extraction method. Amino acid quantification by HPLC Amino acids were extracted by washing cells three times with ice-cold PBS and then quenched with methanol while being placed on ice. Millipore water was then added and the cells were scraped off the plastic. The cell extract was transferred to an Eppendorf tube containing chloroform. Methanol and chloroform were stored at -20°C until use. Cells were first shaken for 20 minutes at 1400 rpm, at 4°C, and then centrifuged for 5 minutes at of 16100 g at 4°C. The upper aqueous phase was then transferred to a new tube and dried overnight. Sample preparation and amino acid measurements were performed as described by Hernandez-Valdes et al. (29). Briefly, samples were first resuspended in water-acetonitrile (1:4), aliquoted, dried again, and then resuspended in water containing three internal standards (citrulline, norvaline and sarcosine). Standard amino acids and samples were derivatized automatically in an HPLC autosampler with o-phtalaldehyde and 9-fluorenylmethyloxycarbonyl reagent solutions. HPLC amino acid analysis was performed on an Agilent 1100 HPLC binary system (Agilent, Santa Clara, USA) equipped with an 1100 Fluorescence detector (FLD) and a Zorbax Eclipse C18 column (Ø3 × L250 mm, 3 μm particle size, Agilent, Santa Clara, USA) at 40°C. A 10mM
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