Figure 6
Depletion efficiency using FITC-labeled albumin as a high-abundance protein indicator. (A) Structural model of FITC-HSA, drawn using Pymol software [24]. Using a protein databank file, HSA (PDB ID: 1E7I) was processed to be single molecule, and the backbone of HSA was drawn in cartoon mode in magenta. Yellow circles indicate FITC on lysine residues. FITC-HSA (10, 20, and 30 μg) was subjected to SDS-PAGE (12%, 7 cm) and visualized under UV488 nm illumination without any staining. There were non-HSA protein bands in the lane, which were considered to be HSA-binding proteins. (B) Three chromatograms at UV280 nm were overlain in the three repeated depletion runs using FITC-HSA as the high-abundance protein indicator. (C) Three chromatograms at UV488 nm were overlain in the three repeated depletion runs using FITC-HSA as the high-abundance protein indicator. (D) Three chromatograms at UV488 nm were overlain in the three repeated depletion using FITC-HSA-spiked depleted plasma. The 6 most abundant proteins (HSA, immunoglobulin G, alpha-1-antitrypsin, immunoglobulin A, transferrin, and haptoglobin) were depleted in prior and 600 μg of FITC-HSA was used to spike depleted plasma, which was intended to simulate real plasma. In (B), (C), and (D), there were small unbound peaks at 17 min, which were considered to be the unbinding of FITC-HSA caused by epitopes blocking.