Trypsin singles proteomic grade

Enriched fractions were obtained by preparative RP-HPLC (Dionex Ultimate 3000 instrument, ThermoFisher Scientific, Sunnyvale CA) of pools of WD saliva. The chromatographic column was a reversed phase Vydac (Hesperia, CA) C8 column with 5 µm particle diameter (250 x 10 mm). The solutions used for preparative RP-HPLC were the same utilized for analytical HPLC-ESI-MS experiments. The gradient was linear from 0 to 60% B in 40 minutes and from 60 to 100% B in 5 minutes, with a flow rate of 2.8 ml/min. Four fractions corresponding to peaks eluting between 39 and 44 minutes were collected separately and lyophilized. Each fraction was solubilized in 100 µl of ultrapure H 2 O, and 1/3 of the solution was acidified with 0.2% TFA (1:1 v/v ratio) to be checked by low-resolution HPLC-ESI-MS. The remaining sample was submitted to digestion by the kit "Trypsin Singles Proteomic Grade" (Sigma-Aldrich) according to the manufacturer's instructions. Digestion was stopped after 12 h by acidification with 0.1% TFA (final concentration), and the solution stored at -80° C until the analysis by high-resolution HPLC-ESI-MS/MS.

For proteolytic cleavage, 0.4 μg of proteins were mixed with 0.1 μg of trypsin or endoproteinase Glu-C. The tryptic digestion was performed by using the kit "Trypsin Singles Proteomic Grade" (Sigma-Aldrich) according to the manufacturer's instructions. The Glu-C digestion was performed in 100 mM ammonium bicarbonate (pH 8.0) at 37 °C overnight. Digestions was stopped by acidification with 0.1% formic acid (FA) final concentration, and the solutions were stored at -80 °C until the analysis by HPLC-ESI-high-resolution MS/MS.