C18 sep pack cartridges

DC samples stored in sterile PBS at −80°C were dried in a vacuum concentrator, and proteins were denatured in 8 M urea for 30 minutes at room temperature. Extracted proteins were reduced by incubation with 5 mM dithiothreitol at 56°C for 25 minutes and alkylated by incubation with 14 mM iodoacetamide at room temperature for 30 minutes in the dark, and further incubation in 5 mM dithiothreitol for 15 minutes at room temperature. Samples were then diluted into 50 mM ammonium bicarbonate to a final concentration of 1.6 M urea and 0.1 M calcium chloride was added to the mixture of proteins. The content was mixed by vortex and centrifuged briefly, and the supernatants were transferred to a new 2.0 mL tube. Samples were digested by incubation with 2 μg of sequencing-grade modified trypsin (Promega, Madison, WI, USA) overnight at 37°C, and digested peptide mixtures were equilibrated to pH 2.0 by addition of 0.4% trifluoroacetic acid. The tryptic peptide samples were desalted using C18 Sep-pack cartridges (Waters Corporation, Milford, MA, USA), dried in a vacuum concentrator, and then reconstituted in 0.1% formic acid, centrifuged at 10,000 rpm for 10 minutes, and stored at −20°C for analysis.