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A Quantitative Chemoproteomic Platform to Monitor Selenocysteine Reactivity within a Complex Proteome
Cell Chemical Biology ( IF 8.6 ) Pub Date : 2018-07-05 , DOI: 10.1016/j.chembiol.2018.05.017
Daniel W. Bak , Jinjun Gao , Chu Wang , Eranthie Weerapana

Mammalian selenocysteine (Sec)-containing proteins, selenoproteins, are important to (patho)physiological processes, including redox homeostasis. Sec residues have been recalcitrant to mass spectrometry-based chemoproteomic methods that enrich for reactive cysteine (Cys) residues with electrophilic chemical probes, despite confirmed reactivity of Sec with these electrophiles. Highly abundant Cys peptides likely suppress low-abundant Sec peptides. By exploiting the decreased pKaof Sec relative to Cys, we have developed a chemoproteomic platform that relies on low pH (pH 5.75) electrophile labeling, reducing Cys reactivity and enhancing identification of Sec-containing peptides across mouse tissues and cell lines. The utility of this Sec-profiling platform is underscored by evaluation of the selectivity of auranofin, an inhibitor of the selenoprotein, thioredoxin reductase, against both reactive Cys- and Sec-containing proteins. Platform limitations pertain to the non-physiological low-pH conditions that could perturb protein structure and function. Future work necessitates the discovery of Sec-selective electrophiles that function at physiological pH.

中文翻译:

监测复杂蛋白质组中硒代半胱氨酸反应性的定量化学计量学平台

含哺乳动物硒代半胱氨酸(Sec)的蛋白质硒蛋白对(病理)生理过程(包括氧化还原稳态)很重要。尽管已证实Sec与这些亲电试剂具有反应性,但Sec残留物仍难以用于基于质谱的化学计量学方法,该方法可通过亲电化学探针富集反应性半胱氨酸(Cys)残基。高含量的Cys肽可能会抑制低含量的Sec肽。通过利用相对于Cys降低的pKaof Sec,我们开发了一种化学旋转平台,该平台依赖于低pH(pH 5.75)亲电试剂标记,降低Cys反应性,并增强了小鼠组织和细胞系中含有Sec的肽的鉴定。通过评估金诺芬的选择性,强调了该Sec分析平台的实用性,硒蛋白的一种抑制剂,硫氧还蛋白还原酶,针对反应性含Cys和Sec的蛋白质。平台限制涉及可能扰乱蛋白质结构和功能的非生理性低pH条件。未来的工作需要发现在生理pH下起作用的Sec选择性亲电试剂。
更新日期:2018-09-20
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