Department of Chemistry, Boston College, Chestnut Hill, MA, United States.
Methods Enzymol. 2022;662:187-225. doi: 10.1016/bs.mie.2021.10.003. Epub 2021 Nov 15.
Selenoproteins comprise a small group of selenocysteine (Sec) containing proteins, often involved in redox homeostasis. While Sec is functionally similar to cysteine (Cys), with both acting as protein-centered nucleophiles, chemoproteomic strategies employing electrophilic probes have often failed to rigorously identify Sec residues, due to their relatively low abundance with respect to Cys across a proteome. To improve the enrichment and detection of selenoproteins, herein we describe a chemoproteomic strategy that relies on the unique properties of Sec as compared to Cys, such as reduced pK and the unique isotopic distribution of selenium. Low pH electrophilic probe labeling of mouse proteomes reduces Cys reactivity, resulting in increased identification of most soluble selenoproteins. This quantitative chemoproteomic platform provides a method to reliably measure changes in selenoprotein abundance across growth conditions as well as quantify inhibition by selenoprotein specific inhibitors, such as Auranofin.
硒蛋白包含一小群含有硒代半胱氨酸 (Sec) 的蛋白质，通常参与氧化还原稳态。虽然 Sec 在功能上与半胱氨酸 (Cys) 相似，两者都作为蛋白质中心亲核试剂，但由于相对于整个蛋白质组中的 Cys 而言，Sec 的丰度相对较低，因此采用亲电探针的化学蛋白质组学策略通常无法严格鉴定 Sec 残基。为了提高硒蛋白的富集和检测，本文描述了一种化学蛋白质组学策略，该策略依赖于 Sec 与 Cys 相比的独特性质，例如降低的 pK 和硒的独特同位素分布。低 pH 亲电探针标记小鼠蛋白质组可降低 Cys 的反应性，从而增加大多数可溶性硒蛋白的鉴定。这种定量化学蛋白质组学平台提供了一种方法，可以可靠地测量生长条件下硒蛋白丰度的变化，以及定量测定硒蛋白特异性抑制剂（如金诺芬）的抑制作用。
