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Abstract
The study of metabolically labeled or probe-modified proteins is an important area in chemical proteomics. Isolation and purification of the protein targets is a necessary step before MS identification. The biotin-streptavidin system is widely used in this process, but the harsh denaturing conditions also release natively biotinylated proteins and non-selectively bound proteins. A cleavable linker strategy is a promising approach for solving this problem. Though several cleavable linkers have been developed and tested, an efficient, easily synthesized, and inexpensive cleavable linker is a desirable addition to the proteomics toolbox. Here, we describe the chemical proteomics application of a vicinal diol cleavable linker. Through easy-to-handle chemistry we incorporate this linker into an activity-based probe and a biotin alkyne tag amenable for bioorthogonal ligation. With these reagents, background protein identifications are significantly reduced relative to standard on-bead digestion.
Highlights
The covalent modification of proteins by small molecules within a complex proteome is a major theme in chemical biology and proteomics
ABPP makes use of small molecules (activity-based probes (ABPs)) that react with the active form of a specific enzyme or enzyme class by means of a “warhead,” which is often derived from a mechanism-based enzyme inhibitor (Fig. 1A)
For tandem labeling of azide probes, we designed the alkyne containing cleavable biotin reagent 3, which was synthesized in solution in four steps (Fig. 2C)
Summary
The covalent modification of proteins by small molecules within a complex proteome is a major theme in chemical biology and proteomics. Bulky fluorophore or biotin tags on chemical probes might interfere with efficient protein binding They can negatively influence the cell permeability of probes, which limits their applicability in in vitro experiments. Bioorthogonal chemistries, such as the Bertozzi-Staudinger ligation [7] and the 1,3-bipolar cycloaddition of an azide and an alkyne (click chemistry) [8], allow tandem labeling strategies in which a biotin or a fluorophore is attached to an enzyme probe complex in a separate step. An essential step in ABPP, as well as in other chemical proteomics approaches, is the elucidation of the tagged proteins This usually involves a biotin-mediated enrichment step followed by mass-spectrometry-based identification. We show that the synthesis of the linker and its probe derivatives is straightforward, that the linker is compatible with tandem click labeling, that enrichment and release of probe targets is efficient, and that the identification of targets takes place with significantly lower background than in on-bead digestion protocols
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