Crude serum was diluted five times with phosphate buffer (pH 7.4) and subjected to mild sonication in a Vibra cell sonicator using the following settings: 6 cycles of 5 sec pulse; 30 sec gap in between; at 20% amplitude. After sonication, the top two highabundance serum proteins (albumin and IgG) were removed using Albumin & IgG Depletion SpinTrap (GE Healthcare) following the manufacturer's instructions. Extraction of protein from depleted serum samples was performed employing TCA/acetone precipitation method as described by Chen et al., with slight modifications [37] . In brief, depleted serum samples were diluted (1:4 ratio) with ice-cold acetone containing 10% (w/v) TCA. Uniform mixing was performed using mild vortexing for 15 sec and the mixture was allowed to incubate at 220uC for 2 hrs for protein precipitation. After completion of the incubation period, tubes were centrifuged at 1000 g for 15 min at 4uC. Supernatants were separated and kept in fresh microcentrifuge tubes, and the pellets were dissolved in rehydration buffer [8 M urea, 2 M thiourea, 4% (w/v) CHAPS, 2% (v/v) IPG buffer (pH 4-7; Linear), 40 mM DTT and traces of bromophenol blue]. In order to precipitate the remaining amount of proteins present in the collected 10% TCA/acetone-containing supernatants, 1 mL icecold acetone was added to each tube and the samples were subjected one additional round of precipitation and extraction process. In all cases, prior to re-suspension in rehydration buffer, the pellet was briefly air-dried. Prior to proteomic analyses, protein concentration in the samples was quantified using the 2D-Quant kit (GE Healthcare) following the manufacturer's instructions. A total of 600 mg of depleted serum protein extract dissolved in 350 mL of rehydration buffer was loaded on 4-7 pH range IPG strips (18 cm) and underwent passive rehydration for 14-16 hrs. Isoelectric focusing (IEF) was performed on an Ettan IPGphore 3 isoelectric focusing unit (GE Healthcare) for overall approximately 78 kVh using the following voltage settings: 200 V for 4 h (step and hold), 500 V for 1 h (step and hold), 1000 V for 1 h (step and hold), 8000 V for 3 h (gradient), and 8000 V for 7:30 h (step and hold). After completion of IEF, the focused IPG strips were stored at 220uC until the second dimensional analysis was performed. Preceding to the second dimensional separation, each strip was equilibrated to reduce and alkylate the proteins (for 15 min each) using equilibration buffer containing 6 M Urea, 75 mM Tris-HCl pH 8.8, 29.3% (v/v) glycerol, 2% (w/v) SDS, and 0.002% (w/v) bromophenol blue. Just prior to use, 1% (w/v) DTT or 2.5% (w/v) IAA was added in the first (reducing) and second (alkylating) equilibration buffer, respectively. The second dimension was performed on 12.5% SDS polyacrylamide gels using an Ettan DALTsix electrophoresis unit (GE Healthcare). After electrophoresis GelCode Blue Safe Protein Stain (Thermo Scientific, USA) was utilized for visualization of the protein spots. Proteins extracted from each of the subjects were run in duplicate to verify the reproducibility and curtail technical artifacts.