Activity based probe with a diazo cleavable linker-a novel tool in proteomic analysis of cysteine proteases

摘要

Activity based proteomics had become a prominent tool for profiling of enzyme activities in complex protein samples. This approach utilizes small organic molecules (activity based probes) which covalently bind to the active sites of specific enzyme classes. Activity based probe is generally composed of three parts: a reactive group which binds to the active site, a linker region which increases specificity of the probe and prevents steric hindrance between reactive group and the tag and the tag molecule which enables detection and affinity enrichment of labeled proteins. Biotin is most commonly used as a tag, but denaturing elution conditions that are required to elute labeled proteins from immobilized avidin, can cause contamination of the sample with nonspecifically bound and natively biotinylated proteins. In this work we present a novel activity based probe SV1 which enables specific elution free of background protein contamination (Figure 2). We have used cleavable linker probe to profile cysteine proteases in various mouse tissue homogenizates by LC-MS/MS. We eluted labeled proteins by reduction cleavage and prepared samples for MS analysis by 'in solution' digestion. We compared quality of protein identification of our chemically eluted proteins with 'on bead' digestion which is commonly used for preparation protein samples enriched with immobilized avidin. Lysates were labeled with fluorescent version of SV1 probe and analysed by SDS-PAGE, for easier visualization. Labeling profile showed presence of four targeted cathepsins and their labeling could be competed away by pretreatment with cysteine protease inhibitor E-64 (Figure 3).