Reversed phase sep pak c18 cartridges

AFB₁-lysine adduct standard was synthesized and purified as previously described by Sabbioni et al. [39 (link)]. Albumin determination reagent (bromocreosol purple), and normal human serum were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO). Pronase (25 kU, Nuclease-free) was purchased from Calbiochem (La Jolla, CA). Protein assay dye reagent concentrate and protein standards were purchased from Bio-Rad Laboratories Inc. (Hercules, CA). Boric acid, o-phthaldialdehyde (OPA), 2-mercaptoethanol, FB₁ from F. verticilioides (~ 98% purity, TLC), 10× phosphate buffered saline (PBS), ammonium hydroxide, ammonium acetate, sodium chloride, sodium phosphate monobasic, hydrochloric acid, and formic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). OPA reagents were prepared by dissolving 10 mg of OPA and 30 μl of 2-mercaptoethanol in 250 μl of methanol and mixing with 4.75 ml of 3% Boric acid buffer (pH 10.5) and stored at 4 °C avoiding light before use. Mixed mode solid phase extraction (SPE) cartridges, as well as Sep-Pak reversed phase C18 cartridges were purchased from the Waters Corp. (Milford, MA). All other chemicals and solvents were of highest grade and purity available.

The saliva proteome samples were prepared as described in (Jersie-Christensen, Sultan & Olsen, 2016 (link)) with a few modifications. Briefly, 1 ml of saliva was mixed with 1.5 ml lysis buffer (9 M Guanidine hydrochloride, 10 mM Chloroacetamide, 5 mM tris(2-carboxyethyl)phosphine in 100 mM Tris pH 8.5) and heated for 10 min (99 °C) followed by 4 min of sonication.
Protein concentration was measured with Bradford protein assay and ranged from 1–2.5 mg/ml. All samples were digested with the same amount of Lysyl Endoproteinase (Wako, Osaka, Japan) in a ratio of 1:100 w/w calculated from the highest concentration for 2 h. Samples were diluted to a final volume of 10 ml with 25 mM Tris pH8 and digested overnight with Trypsin (modified sequencing grade; Sigma) in a 1:100 w/w ratio.
Digestion was quenched by adding 1 ml of 10% trifluoroacetic acid and centrifuged at 2,000 g for 5 min. The resulting soluble peptides in the supernatant were desalted and concentrated on Waters Sep-Pak reversed-phase C₁₈ cartridges (one per sample) and the tryptic peptide mixtures were eluted with 40% acetonitrile (ACN) followed by 60% ACN. Peptide concentrations were determined by NanoDrop (Thermo, Wilmington, DE, USA) measurement.

Cells were stimulated with H₂O₂ (Sigma Aldrich) for 10 min in PBS at 37 °C, collected by washing with ice-cold PBS and lysed in modified RIPA buffer (50 mM Tris pH 7.5, 400 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.1% Na-deoxycholate), protease inhibitor mixture (Roche) supplemented with 2 mM Na-orthovanadate, 5 mM NaF, 5 mM Glycero-2-phosphate, 1 μM ADP-HPD (Millipore) and 40 μM PJ-34 (Enzo Life Sciences) and cleared by high-speed centrifugation. Proteins were precipitated by adding fourfold excess volumes of ice-cold acetone and stored at −20 °C overnight. Subsequently, proteins were solubilized in a urea solution (6 M urea/2 M thiourea/10 mM HEPES pH 8.0). Protein concentrations in lysates were measured using Bradford assay (Bio-Rad). Next, proteins were reduced by adding dithiothreitol to a final concentration of 1 mM, and alkylated with chloroacetamide at 5.5 mM. Proteins were digested using endoproteinase Lys-C (1:100 w/w) and modified sequencing grade trypsin (1:100 w/w) after a fourfold dilution in 50 mM ammonium bicarbonate solution. Protease digestion was terminated by slow addition of trifluoroacetic acid to pH 2. Precipitates were removed by centrifugation for 10 min at 3,000g. Peptides were purified using reversed-phase Sep-Pak C18 cartridges (Waters). Peptides were eluted off the Sep-Pak with 50 and 80% acetonitrile.