Enrichment and separation of acidic and basic proteins using the centrifugal ultrafiltration followed by nanoparticle‐filled capillary electrophoresis

Abstract

This report describes a method for enrichment and separation of acidic and basic proteins using the centrifugal ultrafiltration followed by nanoparticle-filled capillary electrophoresis. To improve stacking and separation efficiencies of proteins, the separation buffer containing 1.6% poly(diallyldimethylammonium chloride) was added with gold nanoparticles (AuNP), poly(ethylene oxide), cetyltrimethylammonium bromide, and poly(vinyl alcohol). As a result, the use of AuNP as additives exhibited better efficiency in separation, stacking, and analysis time. Even for large-volume samples (110 nL), the separation efficiencies of acidic and basic proteins remained greater than 10(4) and 10(5) plates/m, respectively. To further enhance detection sensitivity, protein samples were enriched using the centrifugal ultrafiltration, followed by our proposed stacking method. The detection sensitivity was improved up to 314-fold compared to normal hydrodynamic injection. Additionally, the limits of detection at a signal-to-noise of 3 for most proteins were down to nanomolar range. We have validated the application of our method by means of analyses of 50 nM lysozyme in saliva samples. The proposed method was also successfully applied to the analyses of egg-white proteins, which have large differences in molecular weight and pI.