I1149

Synthetic peptides designed on the tryptic Rif1 peptide spanning residues 463–479 were obtained from ThermoFisher Scientific, and contained palmitoylated C466/CAM-labeled C473 (IYQC[PALMITOYL]IMLSPVC[CAM]ETIPEK), CAM-labeled C466/palmitoylated C473 (IYQC[CAM]IMLSPVC[PALMITOYL]ETIPEK) or palmitoylated C466/ palmitoylated C473 (IYQC[PALMITOYL]IMLSPVC[PALMITOYL]ETIPEK). Peptides were diluted in 20% CH₃CN, aliquoted, and stored at −20 °C. To monitor acyl-CAM and acyl-NEM exchange following treatment with reducing agents, peptide aliquots were diluted in a solution containing 50 mM Tris-HCl pH 8, 40% CH₃CN, and either 40 mM DTT or 40 mM TCEP, and reduction was carried out for 2 h at 56 °C. Alkylation with either 90 mM iodoacetamide (IAA, Sigma-Aldrich, I1149) or 90 mM NEM was performed for 1 h at room temperature. 1% trifluoroacetic acid (TFA, Pierce Perbio, 28904) was added, and samples were analyzed by mass spectrometry.

Samples of the three individual cow’s milk allergens; BLG, ALA and β-casein as well as samples of digoxigenin-coupled BLG and ALA were concentrated by evaporation in SpeedVac. A solution of 6 M guanidine-HCl (G4505, Sigma), 0.5 M Tris-HCl (A1087.1000, AppliChem, Darmstadt, Germany) and 0.01 M. The EDTA concentration is 0.01 M (1.08418, Merck, Darmstadt, Germany) pH 8.6 was added to the allergens to obtain an allergen concentration of 5 mg/mL. Subsequently dithiothreitol (DTT, D0632, Sigma) was added to a final concentration of 0.1 M and the solutions were saturated with argon, capped and incubated for 2 h at 50°C. Iodoacetamide (I1149, Sigma) diluted in 0.5 M Tris-HCl, pH 8.6 was added to the solution to give a final concentration of 0.24 M. After incubation for 30 min at room temperature (RT) 2-mercaptoethanol (M7522, Sigma) was added to give a concentration of 2.4 M. Lastly, the solutions were placed in dialysis-tubes with a pore size of 6-8 kDa (Spectra/Por®Dialysis Membrane MWCO: 6-8000, Spectrum Laboratories, Inc., Rancho Domingues, CA, USA), and dialysed against PBS (137 mM NaCl, 3 mM KCl, 8 mM Na₂HPO₄, 1 mM KH₂PO₄, pH 7.2) for three days at 4°C and afterwards stored at -20°C until further use.

Fifteen thousand A549 cells were seeded in each well of a 6-well plate and the cells were grown in 80 ppm DDW medium or treated with, 0.5 μm CAMP, 2 nm PCTL or 0.5 μm MTX in four replicates. After 48 h treatment, the cells were collected and lysed using 50 mm Tris (741883, Sigma) buffer and 8 m urea (U5378, Sigma), 1% SDS, and protease inhibitor (5892791001, Sigma) at pH 8.5. For the time course experiment, the cells were grown in either NW or 80 ppm DDW and treated with either MTX or PCTL for 4, 15, 26, 38, and 48 h. After protein reduction using 8 mm DTT (10708984001, Sigma) and alkylation using 25 mm IAA (I1149, Sigma), the proteins were precipitated using cold acetone at −20 °C overnight followed by centrifugation and resuspension. Proteins were then digested by Lys C (125–05061, Wako Chemicals GmbH, Neuss, Germany) (1:75 enzyme to protein ratio) at 30 °C for 6 h and trypsin (V5111, Promega) (1:50 enzyme to protein ratio) at 37 °C overnight. After labeling using the TMT-10 reagent (90110, Thermo Fisher Scientific), desalting on C18 Sep-pak columns (WAT054960, Waters, Milford, MA) and fractionation using high pH reversed-phase peptide fractionation kit (84868, Thermo Fisher Scientific) according to manufacturer's instructions, the obtained 10 fractions of peptides in each sample were analyzed by shotgun proteomics using nanoLC-MS/MS.