6340 ion trap

To determine serum fluoxetine and norfluoxetine concentrations in dams, liquid chromatography coupled with mass spectrometry (LC-MS/ MS) was used as described [41, 50, 51, 54] . The chromatographic separation was achieved using an Ultra High Capacity chip including a 500 nL trapping column and a 150 mm × 75 μm analytical column, both packed with a Zorbax 80SB 5 μm C18 phase (Agilent Technologies) on a 1200 series LC-chip system (Agilent Technologies, Germany). The mobile phase was composed of H2O/FA (100:.1, v/v) (A) and ACN/ H2O/FA (90:10:.1, v/v/v) (B) which were used in gradient elution mode. Mass spectrometric detection was carried out using a 6340 Ion Trap consisting of a nanoelectrospray ionization source operating in positive mode (Agilent Technologies, Waldbronn). An Oasis μElution MCX 96-well plate (Waters, UK) was used to prepare the samples for the analysis. All conditions were performed in duplicate and back-calculated using a calibration curve. Twenty-five μL of serum was needed per animal. Mean ( ± SEM) medication levels for the dams at sacrifice were 19.3 ± 6.7 ng/ml for fluoxetine and 110.1 ± 26.1 ng/ml for norfluoxetine (n = 13 treated dams).

A. niger CBS 513.88 mycelial lysates were obtained following 48 h growth in PDB media (45 h culture followed by supplementation with GT (2.5 μg/ml) (Induced) or methanol (Uninduced) for 3 h. Mycelia were snap-frozen and ground in liquid N 2 , and lysed by sonication and bead-beating in lysis buffer A. Protein lysates were clarified by centrifugation prior to use in assays. Reaction mixtures were carried out essentially as described by [11] (link) with modifications: Tris (2-carboxyethyl) phosphine (TCEP)-reduced GT (1 mg/ml; 2 μl), SAM (25 mg/ml in PBS, 3 μl), A. niger lysate (20 μl; 3 mg/ml protein) and PBS (75 μl). Negative controls used 20 μl lysis buffer instead of lysate. Reactions were incubated at 37 °C for 3 h and terminated by adjustment to 15% (v/v) TCA, vortexed briefly and incubated on ice for 30 min. Samples were centrifuged at 10,000 × g for 10 min, 4 °C and supernatants were diluted 1/10 in 0.1% (v/v) formic acid and spin filtered prior to LC-MS/MS analysis (Agilent Ion Trap 6340) to detect thiomethylated GT derivatives [11] (link).

A. fumigatus wild-type, DgtmA and gtmA c strains (Dolan et al., 2014) (link) were grown (10 8 conidia/ml) in quadruplicate (Czapek-Dox broth, 200 rpm, 3 d; then static, 25 d 37 C). Culture supernatants and ethyl acetate organic extracts (100 ml) were analysed by RP-HPLC with UV detection (Agilent 1200 system), using a C18 RP-HPLC column (Agilent Zorbax Eclipse XDB-C18 Semi-Preparative; 5 mm particle size; 4.6 Â 250 mm) at a flow rate of 2 ml/min (Fig 1 ).
A mobile phase of water and acetonitrile with 0.1 % (v/v) trifluoroacetic acid, was used under various gradient conditions. For LCeMS analysis, organic extracts were diluted 1/10 in 0.1 % (v/v) formic acid and spin filtered prior to LCeMS analysis (Agilent Ion Trap 6340). Gliotoxin (purity: 98 %) and BmGT (purity: 99 %) standards were obtained from Sigma-Aldrich and Enzo Life Sciences, respectively. Fumagillin, pseurotin A, tryprostatin B and fumitremorgin C were identified based on published m/z ratios, retention times and fragmentation patterns as described previously (O'Keeffe et al., 2014) (link). All data were analysed using built-in GraphPad prism version 5.01 functions, as specified. The level of significance was set at p < 0.05 (*), p < 0.001 (**), and p < 0.0001 (***), unless otherwise stated.