Strata x

Plasma was assayed for total protein (Bio-Rad Protein Assay) and 5 µl was digested with trypsin (1:10 ratio enzyme to protein) for 18 h at 37°C following following reduction and alkylation as we previously described [37 (link)]. Peptides were desalted using solid phase extraction cartridges (Strata-x, Phenomenex) and eluted sequentially in 25% acetonitrile/0.1% formic acid and 45% acetonitrile/0.1% formic acid. Each peptide fraction was diluted 1:4000 in 0.1% formic acid and 10 µl injected on to a 2-cm c18 trap column (Dionex pepmap100, Thermo Scientific). Peptides were separated on a c18 analytical column (15 cm × 75 µm, Dionex pepmap 100) for 60 min from 0.1% formic acid to 50% acetonitrile/0.1% formic acid. Data were acquired in IDA mode on a SCIEX 5600. Wiff files were uploaded to Progenesis Qi or converted into .MGFs for MASCOT searching (v2.4) against the human proteome database (SwissProt/UniProt; 2015) before being loaded to Scaffold Q+. Both Progenesis Qi and Scaffold Q+ were utilized to determine significant protein differences to construct a consensus differential protein list to reduce analytical bias of a single software platform. Proteomics data of the present study has been loaded into the ProteomeXchange database (Submission Reference No.: 1-20190126-112361).

Plasma samples were centrifuged at 9.5 x g. Thereafter, both urine and plasma samples (1000 μl each) were spiked with a mixture of isotopically labeled standards (10 μl, 15 ng of each per sample), diluted with 1 ml sodium acetate solution (100 mM, pH 4.6 ml) and vortexed. All samples were cleaned on SPE cartridges (Strata-X, 60 mg, Phenomenex) using the following protocol: (1) conditioning with 1 ml methanol; (2) equilibration with 1 ml methanol/water (5:95); (3) loading the sample; (4) washing with 2 ml methanol/water (5:95); (5) drying was performed under full vacuum for 15 min; (6) elution with 1.3 ml acetonitrile.
Milk samples (2000 μl) were spiked with a mixture of isotopically labeled standards (20 μl, 30 ng of each per sample) diluted with 1 ml sodium acetate solution (100 mM, pH 4.6 ml) and vortexed. The samples were extracted with ethyl acetate (3 ml), vortexed and the emulsion was separated into two phases by centrifugation at 9.5 x g for 15 min. The organic phase layer was separated and ethyl acetate was evaporated under a nitrogen stream. Fatty residue was extracted with 200 μl acetonitrile. The acetonitrile phase was analyzed by LC–MS/MS.

Cell-free supernatants were collected, by means of centrifugation, for the DM fermented by LABs and for the non-fermented DM (control), (4000× g, 20 min, 4 °C) and filtered (0.22 µm filter, Millipore, Burlington, MA, USA). The supernatant was split into 2 mL acetone-compatible Eppendorf tubes, and four volumes of cold (−20 °C) acetone were added. The tubes were vortexed and incubated o/n at −20 °C. The supernatants were collected, by means of centrifugation (9400× g, 10 min at 4 °C), and dried using a vacuum concentrator (Christ RVC-2-18, Osterode am Harz, Germany). The dried pellets were resuspended in ddH20 0.1% TFA. Samples were purified, by means of SPE, on a reverse phase column (Phenomenex, Strata-X) as reported by Piovesana et al. [21 (link)]. Briefly, after conditioning the column with acetonitrile (ACN), peptides were rinsed with a 0.1% TFA aqueous solution and then eluted with ACN/ddH2O (70/30, v/v) with 0.1% TFA, and dried in the vacuum concentrator.

The reagents and solvents were of analytical or chromatographic grade. Purified microcystin-LR (MC-LR) was purchased from Cayman Chemicals (Ann Abor, USA). Erythro-2-methyl-3-methoxy-4-phenylbutyric acid (MMPB) was purchased from TCI America (Portland, USA) and 4-phenylbutryic acid (4PB) was purchased from Sigma-Aldrich (St. Louis, MO). Potassium permanganate, potassium bicarbonate, sodium metaperiodate, and sodium carbonate were purchased from MilliporeSigma (Milwaukee, USA). Sodium metabisulfite was purchased from Fisher Scientific (Chicago, USA). Cartridges used for SPE included ENV-Bond Elut (Agilent), 100 mg sorbent mass, 125 µM particle size, 3 mL loading volume; Plexa-Bond Elut (Agilent), 200 mg, 40 µM, 3 mL; C18 (Agilent), 200 mg, 40 µM, 3 mL; HLB (Oasis), 200 mg, 60 µM, 6 mL; and Strata-x (Phenomenex), 200 mg, 33 µM, 6 mL.