Sera were fractionated using a ProteinChip Q strong anion-exchange resin filtration plate (Bio-Rad Laboratories, Hercules, CA). The filtration plate was re-hydrated and equilibrated with rehydration buffer (50 mM Tris-HCl, pH = 9) and the resin washed with rehydration buffer and U1 solution (1 M urea, 0.2% CHAPS, 50 mM Tris-HCl, pH = 9) [Additional file 3: Figure S1B ]. Serum samples were then mixed with U1 solution and added to the equilibrated filtration plate. Successive elutions with different buffers with decreasing pH and a final organic solvent (= different fractions) were collected by centrifugation. The buffers used included pH = 9 (50 mM Tris-HCl, 0.1% n-octyl β-D-glucopyranoside (OGP)), pH = 7 (50 mM 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), 0.1% OGP), pH = 5 (100 mM Na acetate, 0.1% OGP), pH = 4 (100 mM Na acetate, 0.1% OGP), pH = 3 (100 mM Na citrate, 0.1% OGP), and organic solvent (33.3% isopropanol, 16.7% acetonitrile, 0.1% trifluoroacetic acid) [Additional file 3: Figure S1C ].