Université Paris-Saclay, CEA, INRAE, Medicaments et Technologies pour la Sante (MTS), SIMoS, 91191, Gif-sur-Yvette, France.
CNRS, Laboratoire des Biomolécules, LBM, Sorbonne Université, Ecole Normale Supérieure, PSL University, 75005, Paris, France.
Angew Chem Int Ed Engl. 2021 Aug 9;60(33):18272-18279. doi: 10.1002/anie.202106117. Epub 2021 Jul 9.
Activity-based probes enable discrimination between the active enzyme and its inactive or inactivated counterparts. Since metalloproteases catalysis is non-covalent, activity-based probes targeting them have been systematically developed by decorating reversible inhibitors with photo-crosslinkers. By exploiting two types of ligand-guided chemistry, we identified novel activity-based probes capable of covalently modifying the active site of matrix metalloproteases (MMPs) without any external trigger. The ability of these probes to label recombinant MMPs was validated in vitro and the identity of the main labelling sites within their S ' region unambiguously assigned. We also demonstrated that our affinity probes can react with rhMMP12 at nanogram scale (that is, at 0.07 % (w/w)) in complex proteomes. Finally, this ligand-directed chemistry was successfully applied to label active MMP-12 secreted by eukaryote cells. We believe that this approach could be transferred more widely to many other metalloproteases, thus contributing to tackle their unresolved proteomic profiling in vivo.
基于活性的探针能够区分活性酶与其非活性或失活的对应物。由于金属蛋白酶的催化是非共价的，因此已经通过用光交联剂修饰可逆抑制剂来系统地开发针对它们的基于活性的探针。通过利用两种类型的配体引导化学，我们鉴定出了新型的能够在没有任何外部触发的情况下共价修饰基质金属蛋白酶（MMPs）活性位点的基于活性的探针。这些探针在体外标记重组 MMPs 的能力得到了验证，并且明确确定了它们 S'区域内主要标记位点的身份。我们还证明，我们的亲和探针可以在纳克级（即 0.07%（w/w））在复杂的蛋白质组中与 rhMMP12 反应。最后，这种配体导向化学成功地应用于标记真核细胞分泌的活性 MMP-12。我们相信，这种方法可以更广泛地应用于许多其他金属蛋白酶，从而有助于解决它们在体内未解决的蛋白质组学分析问题。
