All materials mentioned in this section were sourced from Sigma-Aldrich, except where noted below. Plasma samples (HUPO PPP No. BDAA01-Cit) were thawed and either water or protease inhibitor cocktail (P 8340) added at a 1% level (1:100 dilution). The protease inhibitor stock solution consists of 104 mm 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), aprotinin (80 mm), leupeptin (2 mm), bestatin (4mm), pepstatinA (1.5 mm), and E-64 (1.4 mm). The final concentrations in the plasma samples were 1/100th of these values. Plasma was also treated with each of the individual protease inhibitors, as detailed above. These inhibitors were dissolved in DMSO (D 8418) at the concentrations indicated above for the cocktail. The treated plasma samples (50 mL each) were depleted of albumin and IgG using an antibody based depletion resin, (Proteo-Prep Immunoaffinity Albumin and IgG Depletion Kit (PROT-IA)), then equilibrated with 1% protease inhibitors or water in equilibration buffer. Depleted plasma was precipitated in TCA/deoxycholate (PROT-PR) and the pellets dissolved in 7 m urea, 2 m thiourea, 1% w/v C7BzO, and 40 mm Trizma base (C 0356). Protein quantitation of each depleted sample was determined using a Bradford protein assay (B 6916), with a 1 mg/mL BSA (P 0914) standard. Each depleted plasma sample was reduced and alkylated with a final concentration of 5 mm tributylphosphine and 15 mm iodoacetamide (PROT-RA) respectively for 30 min each, at room temperature. For first dimension IEF, bromophenol blue (B 0126) was added as a tracking dye to each sample at a final concentration of 0.001% w/v. The IPG strips, 11cm, pH 4-7, (GE Healthcare) were passively rehydrated for 4 h at room temperature, until the entire sample was absorbed into the strips. The strips were then focused at 6000 V for a total of 85 000 Vh at 207C, using a PROTEAN IEF cell (Bio-Rad). For second-dimension SDS-PAGE, the IPG strips were equilibrated with an SDS equilibration buffer (I 7281) for 15 min, then loaded onto 4-20% SDS-PAGE gels (Bio-Rad) and electrophoresed at 170 V for 80 min in Tris-glycine-SDS running buffer (T 7777). SigmaMarker™ Wide Range molecular weight markers (M 4038) were added to the lane on the extreme right of the gels. The gels were then Coomassie stained with EZBlue Gel Staining Reagent (G 1041), and subsequently destained in water. Stained gels were imaged and then evaluated using the Phoretix Expression 2-DE imaging software (Nonlinear Dynamics). Spots of interest were manually excised from the gel and the proteins tryptically digested overnight at 377C using the Trypsin Profile IGD Kit (PP0100). The extracted peptide digests were dried at 307C using a vacuum centrifuge.
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