Trypsin lysc mixture

Bradykinin (SC090), culture and reagents were all purchased from Sigma-Aldrich (MO, St Louis, USA). DC Protein^TM assay, electrophoresis reagents and Clarity^TM Western ECL were purchased from Bio-Rad (Hercules, California, USA). PD 98,059 and Ibuprofen were ordered from Cayman Co (MI, Ann Arbor, USA). Antibodies used as follow: Anti-β-Actin (Abcam, Cambridge, UK, ab8227), Phospho-p44/42 MAPK (ERK1/2) rabbit mAb (Cell Signaling Technology, Danvers, MA, USA, 4370 s), p42/44 MAPK rabbit mAb (ERK1/2) (Cell Signaling Technology, 4695 s), Peroxidase-conjugated affinipure donkey anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA) 711-035-152), or affinipure donkey anti-mouse IgG (Jackson Immune, 715–035-150), mouse monoclonal antibody anti-COX-2 (clone COX214). HPLC water was purchased from Avantor (Valley Center, PA, USA), acetonitrile (ACN) from Thermo Fisher Scientific (Waltham, MA, USA), trypsin/Lys-C mixture was produced by Promega (Madison, WI, United States) and formic acid (FA) was obtained from (TCI America, Portland, OR, USA).

Acetonitrile (ACN), methanol (MeOH), acetic acid (HAc), formic acid (FA), and ammonium bicarbonate (NH₄HCO₃) were obtained from Merck. Protease inhibitor cocktail and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich. For tryptic digestion, iodoacetamide (IAA), urea, and dithiothreitol (DTT) were obtained from Sigma-Aldrich and trypsin/Lys-C mixture (mass spectrometry grade; Promega) were used. Ultrapure water was prepared by Milli-Q water purification system (Millipore).

For the proteomic analysis, cells were seeded in 6-well plates, allowed to settle for 48 h and incubated with DON (0.1, 1, 10 µM) or solvent control (DMSO 1:1000) for 24 h. At the end of the incubation, samples were processed using a classical bottom up proteome analysis strategy; to this aim high-resolution mass spectrometry was employed as described in detail previously^{87 (link)}. In short, cells were lysed, protein content in the cytoplasmic fraction was determined using a Bradford assay (Bio-Rad Laboratories, Vienna, Austria; Fig. S1b), and sample aliquots were digested with Trypsin/Lys-C mixture (Promega Corporation, Madison, WI, USA). Peptides were separated by nano-flow UHPLC (Ultimate 3000RSLC Thermo Fisher Scientific, Austria) and analyzed with a Q Exactive orbitrap mass spectrometer (Thermo). Data analysis was accomplished using the MaxQuant 1.6.0.1 software^{88 (link)} including the Andromeda search engine and the UniProt database for human proteins (version 102014 with 20,195 entries). For protein identification, a FDR < 0.01 was applied both on peptide and protein level with at least two peptides identified per protein. For label free quantification, a FDR < 0.05 was applied to a two-sided T-test and a minimum of a twofold abundance difference.

Protein digestion was performed using 2 μl of each plasma sample as previously described, with some modifications (14 , 15 ). Briefly, 23 μl of protein digestion buffer, including reduction and alkylation reagents, was added to 2 μl plasma samples in 96-well plates. The mixture was boiled for 25 min at 60 °C to denature and alkylate the proteins. After cooling samples to room temperature, protein digestion was performed at 37 °C overnight using a trypsin/LysC mixture (Promega) at a 100:1 protein-to-protease ratio. The second digestion was performed at 37 °C for 2 h using trypsin (enzyme-to-substrate ratio [w/w], 1:1000). All resulting peptides were acidified with 10% trifluoroacetic acid (TFA). The acidified peptides were loaded onto custom-made styrene divinylbenzene reversed-phase sulfonate-StageTips according to previously described procedures (15 , 16 ). The StageTip was washed three times with 100 μl 0.2% TFA. Three fractionations were performed using elution buffers with a step gradient of increasing acetonitrile (40%, 60%, and 80%) in 1% ammonium hydroxide. All the eluted peptides were dried using a SpeedVac centrifuge (Thermo Fisher Scientific).

Cell pellets were lysed with lysis buffer (4% SDS and 2 mM TCEP in 0.1 M Tris pH 8.5). Protein concentration was measured by a BCA-reducing compatible kit (Thermo Fisher Scientific, Waltham, MA, USA). Protein digestion was performed using a filter-aided sample preparation (FASP) procedure as described previously [80 (link),81 (link)]. After 200 μg of proteins was precipitated overnight at −20 °C using ice-cold acetone, protein digestion was performed via the two-step FASP procedure as described with some modifications [80 (link),81 (link)]. Protein pellets were dissolved in SDT buffer (4% SDS, 10 mM TCEP, and 50 mM CAA in 0.1 M Tris pH 8.0) and loaded onto a 30 K Amicon filter (Millipore, Jaffrey, NH, USA). The buffer exchanges were performed with UA solution (8 M urea in 0.1 M Tris pH 8.5) via centrifugation at 14,000× g for 15 min. Following the exchange of buffer with 50 mM TEAB, protein digestion was performed at 37 °C overnight using a trypsin/Lys-C mixture (Promega, Madison, WI, USA) at a 100:1 protein-to-protease ratio. The digested peptides were collected by centrifugation. After the filter units were washed with 40 mM ABC, the second digestion was performed at 37 °C for 2 h using trypsin (enzyme-to-substrate ratio (w/w) of 1:1000). The peptide concentration was measured by tryptophan assay [82 (link)].