Removal of the six most abundant proteins in human plasma was achieved with a single 4.6 x 50 mm multiple affinity removal system (MARS) HPLC column (Agilent Technologies, Wilmington, DE, USA). The MARS column contains polyclonal antibodies to human albumin, transferrin, haptoglobin, a-1 antitrypsin, IgG, and IgA. Typically, plasma was diluted five-fold with the manufacturer's equilibration buffer and filtered through a 0.22 mm microcentrifuge filter tube, and aliquots containing ~1 mg total protein were injected onto the antibody column. A total of 193 mL (14.5 mg) of plasma was depleted. The flow-through fractions from sequential injections were collected, pooled, and concentrated to 200 mL (2.4 mg) using a 5 K MWCO spin concentrator (Millipore). Affinity-bound major proteins were eluted with the manufacturer's elution buffer, neutralized with 1 m NaOH, concentrated as above, and stored at — 70°C. The concentrated unbound fraction (depleted plasma) was reduced with 10 mM DTT for 1 h at 23°C in 1 mL of buffer (final volume) containing 8 m urea, 10 mM glycine, pH 8.5. The reaction volume was subsequently reduced to 200 mL using a 5 K MWCO spin concentrator, and alkylated with 25 mM iodoacetamide in 1 mL of buffer (final volume) containing 8 Murea, 10 mM glycine, pH 8.5, for 2 h at 23°C. Reaction was quenched by adding DTT to 1% final concentration. Prior to MicroSol-IEF, salts and reagents were removed by buffer exchange using a 5 K MWCO spin concentrator.
For the analysis of human serum, the major proteins from 415 mL (35.3 mg) of serum were depleted using two MARS columns, where the 50 mm column was connected in tandem to another 4.6 x 100 mm column. All buffers used in the depletion were supplemented with protease inhibitors (1 mM DFP, 1 mg/mL leu-peptin, 1 mg/mL pepstatin, 5 mM EDTA). Each injection contained ~200 mL of fivefold diluted serum. The unbound fractions were pooled and concentrated to 240 mL (4.3 mg). Proteins were reduced and alkylated in the presence of 100 mM Tris-Cl,
8 m urea, pH 8.3 with 20 mM DTT, and 60 mM iodoacetamide for 1 h at 37°C each. Reaction was terminated with 60 mM DTT for 15 min at 37°C. Salts and reagents were removed by precipitation with 9 vol of acetone.
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