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

 
28 January 2019

Bio-orthogonally tagged activity-based probes for proteomics 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 2018, 9, 1162. https://www.nature.com/articles/s41467-018-03511-6
(2) El Oualid et al. 2010, 49, 10149. https://onlinelibrary.wiley.com/doi/full/10.1002/anie.201005995
(3) Kwasna et al. Molecular Cell 2018, 70, 150. https://www.cell.com/molecular-cell/fulltext/S1097-2765(18)30142-4
(4) Haahr et al. Molecular Cell 2018, 70, 165. https://www.cell.com/molecular-cell/fulltext/S1097-2765(18)30143-6
(5) Hermanns et al. Nature Communications 2018, 9, 799. https://www.nature.com/articles/s41467-018-03148-5
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