Won Sang Joon, Eschweiler Joseph D, Majmudar Jaimeen D, Chong Fei San, Hwang Sin Ye, Ruotolo Brandon T, Martin Brent R
Program in Chemical Biology and Department of Chemistry, University of Michigan , 930 North University Avenue, Ann Arbor, Michigan 48109, United States.
ACS Med Chem Lett. 2016 Dec 9;8(2):215-220. doi: 10.1021/acsmedchemlett.6b00441. eCollection 2017 Feb 9.
Activity-based protein profiling (ABPP) has revolutionized the discovery and optimization of active-site ligands across distinct enzyme families, providing a robust platform for in-class selectivity profiling. Nonetheless, this approach is less straightforward for profiling reversible inhibitors and does not access proteins outside the ABPP probe's target profile. While the active-site competitive acyl protein thioesterase 2 inhibitor ML349 ( = 120 nM) is highly selective within the serine hydrolase enzyme family, it could still interact with other cellular targets. Here we present a chemoproteomic workflow to enrich and profile candidate ML349-binding proteins. In human cell lysates, biotinylated-ML349 enriches a recurring set of proteins, including metabolite kinases and flavin-dependent oxidoreductases that are potentially enhanced by avidity-driven multimeric interactions. Confirmatory assays by native mass spectrometry and fluorescence polarization quickly rank-ordered these weak off-targets, providing justification to explore ligand interactions and stoichiometry beyond ABPP.
基于活性的蛋白质谱分析（ABPP）彻底改变了跨不同酶家族的活性位点配体的发现和优化，为同类选择性分析提供了一个强大的平台。尽管如此，这种方法在分析可逆抑制剂时不太直接，并且无法检测ABPP探针目标谱之外的蛋白质。虽然活性位点竞争性酰基蛋白硫酯酶2抑制剂ML349（Kd = 120 nM）在丝氨酸水解酶家族中具有高度选择性，但它仍可能与其他细胞靶点相互作用。在这里，我们提出了一种化学蛋白质组学工作流程，以富集和分析候选ML349结合蛋白。在人细胞裂解物中，生物素化的ML349富集了一组反复出现的蛋白质，包括代谢物激酶和黄素依赖性氧化还原酶，这些蛋白质可能通过亲和力驱动的多聚体相互作用而增强。通过原生质谱和荧光偏振进行的验证性分析迅速对这些弱脱靶进行了排序，为探索ABPP之外的配体相互作用和化学计量提供了依据。
