Abstract
Activity-based probes (ABPs) that specifically target subsets of related enzymatic proteins are finding increasing use in proteomics research. One of the main applications for these reagents is affinity isolation of probe-labeled targets. However, the use of cheap and efficient biotin affinity tags on ABPs can be problematic due to difficulty in release of captured proteins. Here we describe the evaluation of activity-based probes carrying a chemically cleavable linker that allows selective release of probe-labeled proteins under mild elution conditions that are compatible with mass spectrometric analysis. Specifically, we compare results from standard on-bead digestion of probe-labeled targets after affinity purification with the results obtained using chemoselective cleavage. Results are presented for multiple APBs that target both serine and cysteine proteases. These results highlight significant improvements in the quality of data obtained by using the cleavable linker system.
Highlights
Activity-based probes (ABPs) that target subsets of related enzymatic proteins are finding increasing use in proteomics research
Activity-based probes have been mainly used for profiling the serine and cysteine families because both utilize catalytic nucleophiles in their active site that can be effectively targeted by a number of classes of synthetically accessible electrophiles (6 –12)
We present here the analysis of this cleavable linker system applied to ABPs that target both cysteine and
Summary
Activity-based probes (ABPs) that target subsets of related enzymatic proteins are finding increasing use in proteomics research. When compared with the on-bead digestion of labeled targets, specific chemical elution produces more reliable proteomics data that are free of background protein contamination. Labeled cathepsin active site peptides were eluted by incubation of the beads in cleavage buffer (100 mM ammonium hydrogen carbonate, 25 mM sodium hydrosulfite) for 15 min.
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