Cyclic acetals as cleavable linkers for affinity capture

Abstract

Labeling proteins with biotin is a widely used method to identify target proteins due to biotin’s strong binding affinity for streptavidin. Combined with alkyne-azide cycloaddition, which enables the coupling of probes to targeted proteins, biotin tags linked to an alkyne or azide have become a powerful tool for purification and analysis of proteins in proteomics. However, biotin requires harsh elution conditions to release the captured protein from the bead matrix. Use of these conditions reduces signal to noise and complicates the analysis. To improve affinity capture, cleavable linkers have been introduced. Here, we demonstrate the use of a cyclic acetal biotin probe that is prepared easily from commercially available starting materials, is stable to cell lysates, yet is cleaved under mildly acidic conditions, and which provides an aldehyde for further elaboration of the protein, if desired.

Traceless Cleavage of Protein–Biotin Conjugates under Biologically Compatible Conditions

“…Alternatively the use of cleavable biotinylation reagents allows selective release of the protein through proteolytic, photolytic or chemical cleavage . Chemically cleavable linkers include diazobenzenes (cleavage: sodium dithionite), vicinal diols (sodium periodate), bisarylhydrazones (catalytic transamination), acylhydrazones, silyl ethers, maleic anhydrides and acetals (acid), levulinoyl esters (hydrazine), and dithanes, bromomaleimides and conjugate acceptors (thiol). However, upon cleavage, residual atoms from the linker remain attached to the protein, potentially complicating downstream processes such as activity assays or proteomic analysis.…”

Invention of stimulus‐responsive peptide‐bond‐cleaving residue (Spr) and its application to chemical biology tools

“…With these issues in mind, some groups , including ours , designed a traceable linker that enables not only target elution by linker cleavage as similar to the cleavable linker, but also selective labeling of the target for discrimination of the target from contaminated non‐targets. Design of our Spr‐based traceable linker 18 is depicted in Scheme .…”

Development of a Turn-On Fluorescent Traceable Linker Employing <i>N</i>-Sulfanylethylcoumarinyl Amide for the Enrichment and Visualization of Target Proteins

“…[17][18][19] Because the contamination of non-target proteins hampers the subsequent identification of target proteins, several research groups including ours were prompted to engage in the development of an advanced cleavable linker. [20][21][22][23][24][25][26] The new cleavable linkers were designed to selectively label a target protein for facile discrimination of the target from non-targets. These linkers enable label-based tracing of the target; therefore, we named this type of cleavable linker a "traceable linker."…”

“…We previously developed an N-sulfanylethylanilide (SEAlide)-based traceable linker (1) as shown in Chart 1A. 20,27) Traceable linker 1 is first connected with an alkynylated target protein by CuAAC, and the generated conjugate is then adsorbed on SAv beads, similar to the case of the cleavable linkers. Because phosphate activates the SEAlide unit from an amide to a thioester form, 28,29) the addition of a phosphate buffer containing reductant for the removal of a t-BuS group of the SEAlide moiety was necessary, and a labeling reagent with a cysteine moiety induced bioothogonal Native Chemical Ligation (NCL) 30) to elute a labeled target protein.…”

“…After adsorption on SAv beads, treatment with phosphate buffer containing a non-fluorescent nucleophile and a reductant would induce the elution of the target via removal of the t-BuS group, activation of the SECmide moiety to the corresponding thioester form, and subsequent nucleophilic cleavage of the thioester via NCL or a thiolysis reaction. The eluted target has an aminocoumarin A) Previously reported SEAlide-based traceable linker20) ; B) SECmide-based traceable linker developed in this study.Chart 1. Traceable Linker-Based Enrichment and Selective Labeling of a Target Protein Structures of Turn-On Fluorescent Traceable Linker 3 and Its Negative Control SDS-PAGE Analyses for the Optimization of Reaction/Elution Conditions (CBB: Coomassie Brilliant Blue Staining; WB: Western Blotting Using SAv-HRP; FL: Fluorescence Imaging, λ ex = 302 nm, λ em = 530 ± 14 nm) A) Introduction of 3 or 4 onto alkynylated BSA.…”