A Mild Chemically Cleavable Linker System for Functional Proteomic Applications

Abstract

One of the primary goals in the field of proteomics is finding ways to enrich specific protein targets from complex mixtures. Generally, this is accomplished with activity-based probes (ABPs) that allow specific modification by the formation of a stable covalent bond with reactive groups on a target protein. Such proteomic probes often carry biotin tags and can either be generally reactive towards free nucleophiles, such as thiols (i.e, isotope-coded affinity tagging (ICAT) reagents), or react through a specific enzymatic process with a key catalytic residue. Although enrichment by immobilized streptavidin allows efficient isolation of even highly dilute targets, one of the primary limitations is the need for harsh, denaturing conditions to disrupt the biotin–streptavidin interaction. This elution generally results in contamination of the desired probe-labeled proteins with avidin monomers, proteins that were nonselectively bound to the streptavidin, and endogenously biotinylated proteins. Hence, additional purification techniques such as gel electrophoresis are required prior to identification with MS. The incorporation of a cleavable linker between the biotin tag and the site of attachment to the target protease provides a significant advance as it allows specific elution of probelabeled proteins or peptides. Recently, a number of cleavable linkers have been reported with a focus on applications in MS and ICAT. However, these reagents require a strong acid (trifluoroacetic acid), making cleavage of labeled proteins directly from a strepavidin resin problematic as nonselective release can also occur. Disulfide cleavable linker systems, though useful for a variety of applications, are subject to disulfide exchange and show premature cleavage in cellular systems and reducing buffer solutions. Herein, we report a novel diazobenzene derivative and its application as a chemoselective cleavable linker system (Scheme 1). This linker can be easily incorporated into small-molecule probes that allow the isolation of specific protein targets by streptavidin affinity purification