A collaborative effort between scientists at UbiQ, Genentech, Boston Biochem (Bio-Techne) and Stanford University.


28 January 2019

Bio-orthogonally tagged activity-based probes forproteomics of deubiquitinating enzymes  

A recent paper in Nature Communications describes a collaborative effort between scientists at UbiQ, Genentech, Boston Biochem (Bio-Techne) and Stanford University (1). Here a reactive-site-centric chemoproteomics protocol is presented which allows evaluating activity and probe reactivity of deubiquitinating enzymes (DUBs, also termed deubiquitylases). The chemoproteomics protocol makes use of a new type of bio-orthogonally tagged activity-based probe (ABP) and sequential on-bead digestions to enhance the identification of probe-labeling sites on DUBs (Figure 1). Ultimately, ZUFSP (ZUP1) was identified as a previously unannotated DUB with high selectivity toward cleaving K63-linked ubiquitin chains (3-5).
Figure 1.

The bio-orthogonally tagged ABPs are based on a ubiquitin (UbiQ-193) or SUMO2 protein (UbiQ-237), which is functionalized on the C-terminus with a vinyl pentynyl sulfone (VPS) electrophile (Figure 1). By using click chemistry, the VPS building block allows post-labeling functionalization of probe bound DUBs with azide modified molecules (such as azide-biotin). The ABPs are made by UbiQ using its total chemical synthesis technology of small proteins (2), with the SUMO2 ABP representing the first reported SUMO protein made by a total linear chemical synthesis (1).

UbiQ now offers the following VPS activity-based probes:

  • HA-Ahx-Ahx-Ub-VPS (UbiQ-193)
  • HA-Ahx-Ahx-SUMO2-VPS (UbiQ-237)
(1) Hewings et al. Nature Communications 20189, 1162.
(2) El Oualid et al. 2010, 49, 10149.
(3) Kwasna et al. Molecular Cell 2018, 70, 150.
(4) Haahr et al. Molecular Cell 2018, 70, 165.
(5) Hermanns et al. Nature Communications 20189, 799.
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