Dionex ultimate 3000 uhplc

The analysis of biogenic amine was performed through the implementation of the HPLC method, including dansyl chloride derivatisation. The samples were prepared according to the methods proposed by Kulawik, Dordevic, Gambuś, Szczurowska and Zając (2018). Sample separation was performed using the Dionex Ultimate 3000 UHPLC (Thermo Scientific, Waltham, MA, USA), FLD 3400RS four-channel fluorescent detector (Thermo Scientific) on a Kromasil 100-5-C18 4.6 × 250 mm column (Akzo Nobel, Amsterdam, The Netherlands). In an earlier study by Jamróz, Kulawik, Guzik, and Duda (2019), specific chromatographic conditions were presenting. The Supelco biogenic amine standards were used for referencing (Sigma-Aldrich, St. Louis, MO, USA).

Extraction, digestion and purification according to previously published protocols^{105 (link)}, and are detailed in Supplementary SI-3_Proteomics. Protein analysis was conducted by nanoLC-Orbitrap MS/MS using a THERMO SCIENTIFIC DIONEX Ultimate 3000 UHPLC (equipped with THERMO BioBasic C18 precolumn (30 mm × 75 µm i.d.) and in-house packed analytical column (210 mm × 75 µm i.d.) made of the same stationary phase). The UHPLC was directly coupled to a THERMO SCIENTIFIC LTQ Orbitrap Velos mass spectrometer which analyzed the peptides in positive mode. For bioinformatics analysis, two fractions of each sample were combined into one search to create one output file. PEAKS 8.5 (BIOINFORMATICS SOLUTIONS INC.) was used to search the RAW data for matches against publicly available sequences of mammals, birds and fish species in imported UniProt (www.uniprot.org) and NCBI (https://www.ncbi.nlm.nih.gov/protein) databases.
Full details of the Methods are reported in Supplementary SI-1.B.

High-performance liquid chromatography (HPLC) measurement of Kyn was performed in cell culture supernatants collected from each treatment type. One mL of supernatant sample was first treated with 168.6 μL of 72% trichloroacetic acid to precipitate out dissolved proteins; subsequently, samples were centrifuged at maximum speed for 10 min, and the resulting protein-free supernatants were transferred into glass HPLC vials for further HPLC analysis. Chromatographic separation of Kyn was achieved on a Dionex Ultimate^® 3000 UHPLC (Thermo Scientific) on a reversed-phase Accucore^TM aQ column (Thermo Scientific^TM) with 2.6-μm particle size. The mobile phase gradient consisted of 0.1% trifluoroacetic acid (TFA) in water (A) and 0.1% TFA in acetonitrile (B). Standards for all analytes were utilized to determine the retention time and UV emission spectrum at 365 nm for Kyn. Analyte concentration in samples was analyzed by comparison with the respective standards using the Chromeleon^TM 7.2 Chromatography Data System (Thermo Scientific^TM Dionex^TM).

Media of four- and eight-day cultures were entirely retrieved and their AFB₁ content determined after extraction with 25 and 40 mL chloroform respectively. Extracts were held for 2 h on a horizontal shaking table at 200 rpm and were then filtered through a Whatman 1PS phase separator filter (GE Healthcare Life Sciences, Vélizy-Villacoublay, France, 150 mm diameter). Filtrates were evaporated to dryness and dissolved in 1 mL of a water-acetonitrile-methanol mixture (65:17.5:17.5; v/v/v). Extracts were filtered using 0.45 µm porosity disks (Thermo Scientific Fisher, Villebon-Sur-Yvette, France) before analysis. HPLC analysis was performed using a Dionex Ultimate 3000 UHPLC (Thermo Scientific, France) using a 125 × 2 mm, 5 µm, 100 Å, Luna^® C18(2) LC column (Phenomenex, Torrance, CA, USA). Aflatoxins were separated using the program described by Fu, Huang, & Min, 2008, with minor modifications [62 (link)]. A mixture of water (acidified with 0.2% acetic acid)-acetonitrile (79:21, v/v) is eluent A and methanol is eluent B. Separation program consists of a 30 min A:B (82.5:17.5) isocratic flow at 0.2 mL/min. Aflatoxins were detected using a fluorescent detector at wavelengths of 365/430 nm (excitation/emission). UV Spectra were confirmed by an additional diode array detector (DAD) coupled to the apparatus. Sample concentrations were calculated based on a standard calibration curve.

Total cell lysates were reduced with DTT and cysteines alkylated with iodacetamide followed by precipitation and digestion with Trypsin (Promega) as described previously [78 (link)]. Resulting peptides were desalted (SOLA RP) before analysis. Mass spectrometric analysis of digested cell lysates was conducted on a Q-Exactive (Thermo) mass spectrometer with a resolution of 70,000 (at 200 M/z) coupled to a Dionex Ultimate 3000 UHPLC (Thermo) system. Peptide separation was archived on an Easy column (2μm Pepmap, C18, 75μm x 500mm) using a linear gradient from 2-40% of buffer B (composition as above) in 57 minutes. Precursors with an M/z between 380 and 1800 were selected for MS/MS with an isolation width of 1.6 Da and 28% normalized collision energy using the 15 most abundant precursor ions. Selected precursors (Threshold 10,000 counts) were excluded for 27s. MS/MS spectra were searched using the Mascot search engine (Matrixscience), and quantitation was performed using LC Progenesis software (Non-Linear Dynamics) as described [78 (link)]. Raw intensities are reported in table S6.