Online monitoring of immunoaffinity-based depletion of high-abundance blood proteins by UV spectrophotometry using enhanced green fluorescence protein and FITC-labeled human serum albumin

In this study, we developed a method of measuring recovery yields of a multiple affinity removal system column easily and rapidly using enhanced green fluorescence protein as an indicator of flow-through proteins. Also, we monitored the capture efficiency through depletion of a high-abundance protein, albumin labeled with fluorescein isothiocyanate.This simple method can be applied easily to common high-abundance protein depletion methods, effectively reducing experimental variations in biomarker discovery studies.Extracellular body fluids that contain plasma and serum are the most valuable sources for biomarker discovery and application, but useful protein biomarkers exist at low concentrations (ng/ml or lower) in blood; approximately 30 high-abundance proteins constitute ~99% of the total protein mass in human plasma, rendering the detection of low-abundance proteins difficult. Several methods have been developed to deplete high-abundance proteins from blood, most of which are based on the immunoaffinity of antibodies toward high-abundance proteins, for example, multiple affinity removal system (MARS, Agilent, CA, USA) columns which can remove several specific high-abundance proteins [1-5], but some reports have demonstrated that these systems incur problems, such as incomplete removal of high-abundance proteins [3,6,7]. Recently, Bellei at al. showed that untargeted proteins are removed concomitantly [8] and Chengjian et al. also reported that untargeted human plasma proteins, such as alpha-1-acid glycoprotein 1, alpha-1-acid glycoprotein 2, and alpha-1-antichymotrypsin, bind to MARS columns [9].In fact, the reproducibility of the removal and recovery of flow-through proteins is a critical aspect of high-abundance protein depletion and although Veronica et al. reported that in a triplicate 2-DE experiment, the coefficient of variation (C.V.) of flow-through and bound fractions by MARS depletion was 5.31% and 4.12%, respectively, 3 depletions were not sufficient t