025 in a nitrogen-free synthetic medium containing the following components: 5 g.L-1 glucose, 3.5 g.L-1 fructose, 10 g.L-1 D,L- malic acid, 0.6 g.L-1 KH2PO4,
0.45 g.L-1 KCl, 0.13 g.L-1 CaCl2, 2H2O, 0.13 g.L-1 MgSO4, 7H2O, 3 mg.L-1 MnSO4, H2O, and 1 mL.L-1 Tween 80, at pH 5. Amino acids were added one by one as nitrogen sources according to Terrade et al. [53]. This medium corresponds to the first culture condition where amino acids are free and contains 1.6 mM of tyrosine. Otherwise, in a second condition, tyrosine was replaced by 1.6 mM of a mix of synthetic peptides containing tyrosine: Gly-Gly-Tyr-Arg, Tyr-Ala and Gly-Leu-Tyr purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). Selleck Autophagy Compound Library Aliquots of 50 mL of PCI-34051 in vivo culture were harvested after various times of the growth and centrifuged for 10 min at 6,000 rpm. The pellets were stocked at −20°C until RNA Crenolanib extraction. A 1 mL sample of each supernatant
was derivatized and analyzed by HPLC to assay biogenic amines and amino acids. The rest of the supernatant was stored at −20°C. Amino acid and biogenic amine analysis by HPLC Free AA and BA were analyzed by HPLC using the method described by Gomez-Alonso et al. [47]. The derivatization reaction was performed by adding 1.75 mL of borate buffer pH 9, 1 mL of methanol, 40 μL of internal standard (2,4,6-trimethylphenethylamine hydrochloride, 2 mg.mL-1), and 30 μL of DEEMM (diethyl ethoxymethylenemalonate) to 1 mL of target sample. The samples were placed for 30 min in an ultrasound bath, then heated to 70°C for 2 h to allow complete degradation of excess DEEMM and reagent byproducts. The analyses were performed on a Varian HPLC (Varian Inc., Walnut Creek, CA) using an Alltech (Grace, Templemars, France) HPLC column (C18-HL), particle size 5 μm (250 mm × 4.6 mm), maintained at 16°C, with a binary gradient. Phase A was modified with 10 mM ammonium acetate pH 5.8 Branched chain aminotransferase to allow the identification of AA and BA by mass spectrometry. The mobile phase, phase B, was 80:20 mixture of acetonitrile and methanol and the flow rate a constant
0.9 mL.min-1. HPLC-MS conditions LC-MS/MS analyses were performed on a ThermoFinnigan TSQ Quantum triple quadrupole mass spectrometer equipped with a standard electrospray ionization source fitted with a 100 μm i.d. H-ESI needle. HPLC was performed using an Accela™ LC pump from ThermoFinnigan (San Jose, CA, USA) equipped with an Accela autosampler (for HPLC conditions, see paragraph above). The flow from LC was split using an analytical fixed flow splitter (split ratio = 1:10, post-column) from Analytical Scientific Instruments (El Sobrante, CA, USA). The data were processed using Xcalibur software (ThermoFinnigan). The source spray head was oriented at an angle of 90°C orthogonal to the ion-transfer tube. The mass spectrometer was operated in the negative ion mode in the range of m/z 90–900 with a scan time of 1 s.
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