5 mL glass vials, dried under a nitrogen stream at 30 °C and stored at −20 °C. For analysis, the samples were reconstituted in ACN/MeOH/CHCl3 (49:49:2, v/v/v). 3.3. High Performance Liquid Chromatography/Mass Spectrometry For HPLC/ESI-LIT-FTICRMS analysis buffer was added to the reconstituted sample to

a final concentration of 5 mM NH4Ac/50 mM HAc (pH 3.75). Chromatographic inhibitor Tofacitinib separations were performed using a Surveyor MS pump and Surveyor autosampler (Thermo Fisher Scientific, San Jose, CA, USA). A 5 µL full loop injection was used. Separation Inhibitors,research,lifescience,medical was carried out on a microbore C4-Nucleodur Gravity column (150 mm × 1 mm i.d., particle size 5 µm) from Macherey-Nagel (Düren, Germany). The following binary gradient Inhibitors,research,lifescience,medical was used at a flow rate of 115 µL/min: 0 min 50% B, 2 min 50% B, 35 min 75% B, 45 min 100% B, 65 min 100% B, 66 min 50% B, 80 min 50% B. Mobile phase A consisted of 2.5% ACN, 2.5% MeOH and 95% H2O (v/v/v) with 5 mM NH4Ac/50 mM HAc (pH 3.75). Mobile phase B contained 5% H2O, 25% n-propanol, 35% ACN and 35% MeOH (v/v/v/v) with 5 mM NH4Ac/50 mM HAc (pH 3.75). The ESI-LIT-FTICRMS experiments were carried out using a LTQ FT Fourier transform ion cyclotron resonance hybrid-mass spectrometer

(Thermo Fisher Scientific, Bremen, Germany), www.selleckchem.com/products/17-AAG(Geldanamycin).html equipped with a 7.0 T actively shielded superconducting magnet and electrospray Inhibitors,research,lifescience,medical ionization source. All analyses were carried out in negative ionization mode. The instrument was operated in the data-dependent Inhibitors,research,lifescience,medical mode. Survey centroid MS spectra in the mass range m/z 185–1,850 were acquired in the FTICR with a resolution R = 25,000 at m/z 400 (target accumulation value 5,000,000, maximal ion accumulation time 750 ms). The two most intensive ions were sequentially isolated for accurate mass measurements by a FTICR “SIM scan” in a narrow mass window Inhibitors,research,lifescience,medical (±5 Da, R = 50,000, target accumulation value 100,000, maximal ion accumulation time 750 ms) in the profile mode. Subsequent fragmentations (MS2, MS3) were carried out in the linear ion trap by collision-induced dissociation (CID; target

accumulation value 10,000, maximal ion accumulation time 150 ms). Former target ions selected for MS/MS were dynamically excluded for 60 s. The total cycle time was approximately 4.6 s. The general mass spectrometric Brefeldin_A conditions were: Spray voltage: −3.5 kV, sheath gas flow: 30 arbitrary units, auxiliary gas flow: 5 arbitrary units and sweep gas flow: 2 arbitrary units. Ion transfer capillary temperature was set to 225 °C and the following parameters were used for CID MS2 and MS3 experiments: Normalized collision energy: 30%, activation: q = 0.25 and activation time: 30 ms. Ion selection thresholds were 500 counts for SIM scans, 500 counts for MS2 and 100 counts for MS3 experiments. 3.4.