A bioorthogonal enrichment tag for purification of biomolecules from complex mixtures

Abstract

Enrichment methods to purify target biomolecules from complex mixtures such as cell lysates are quite important to make high-quality results. To date, the biotin enrichment tag is widely used, but it has several problems such as too harsh enrichment conditions, low yield, and interference from endogenous biotin. Alternatively, phosphate tags have recently been employed for IMAC-based enrichment of the target molecules, but it also has interference issues from endogenous phosphoproteins, and its negative charge hinders mass-spectrometric analysis. To address these issues, herein we report a simple IMAC-based enrichment tag with orthogonal binding characteristics with phosphates. Our enrichment tag with an alkyne handle can be applied to the labeling of azido-containing biomolecule through CuAAC (Copper(I)-catalyzed Azide-Alkyne Cycloaddition) in aqueous buffers and the subsequent enrichment worked well with commercial IMAC resins.