Pierce c18 columns

In-solution protein digestion was processed according to the protocol described by Morelle and Michalski (2007) [85 (link)] with some modifications. Protein precipitates were dissolved in 100 µL of 50 mM NH₄HCO₃ (pH 8.0) with 0.1% SDS. Freshly prepared DTT solution was added so that the final concentration of DTT was 20 mM, and the sample was incubated at 60 °C for 1 h. The freshly prepared iodoacetamide solution was then added so that the final concentration was 20 mM, and the sample was incubated for 1 h in the dark. The sample was diluted twice, and 5 µg of trypsin were added. The sample was then left overnight with gentle shaking at 37 °C for complete digestion. After trypsinolysis, 1% formic acid was added to adjust the pH to 3–4, and the sample was desalted on Pierce™ C18 columns (Thermo Fisher Scientific, Waltham, MA, USA), then vacuum dried and resuspended in acetonitrile for further LC-MS analysis.

Tears and saliva were used for shotgun proteomics analysis. Protein samples were lysed with 1% SDS, 0.1 M EDTA in 200 nM HEPES (pH 8), protease cOmplete™ inhibitor tablets (Sigma-Aldrich, ON, Canada). Proteins were denatured with the addition of a final concentration of 10 mM dithiothreitol (DTT). Samples were alkylated by incubation with a final concentration of 15 mM iodoacetamide (IAA) in the dark for 25 min at room temperature. Samples were next digested with Trypsin (Promega, Madison, WI, United States ). With HCl the pH adjusted to 6.5. Next, to label peptide α- and ε-amines, samples were incubated for 18 h at 37°C with isotopically heavy (40 mM ¹³CD₂O+20 mM NaBH₃CN (sodium cyanoborohydride)) or light labels (40 mM light formaldehyde (CH₂O) + 20 mM NaBH₃CN), all final concentrations. Samples were subjected to C18 chromatography using Pierce™ C18 columns (Thermo Scientific™, ON, Canada) before being subjected to liquid chromatography and tandem mass spectrometry (LC-MS/MS).