Activity Based Probe Developed by Sequential Dehydroalanine Strategy on Expressed Proteins Reveals a Potential α-globin Modulating Deubiquitinase

The development of ubiquitin based reagents to study and monitor deubiquitinase’s activity is highly desired goal for many laboratories in academia and industry due to the importance of these enzymes in many pathological states. However, due to the limitations of total chemical synthesis and a shortage of suitable semisynthetic tools for the preparation of large protein-ubiquitin conjugates, the field of ubiquitin-based probes is essentially restricted. We report a general and novel semisynthetic strategy for the preparation of ubiquitinated protein activity based probe applying sequential dehydroalanine formation on an expressed target. We applied this approach to construct physiologically and therapeutically relevant ubiquitinated α-globin probe, which was used for the enrichment and proteomic identification of α-globin modulating deubiquitinases. We found USP15 as a potential deubiquitinase, modulating α-globin, which excess leads to beta-thalassemia symptoms. This development opens new opportunities for activity based probe design to shed light on the important aspects underlining ubiquitination and deubiquitination in health and disease.