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Chemoproteomic profiling of targets of lipid-derived electrophiles by bioorthogonal aminooxy probe

机译：生物正交氨氧基探针对脂类亲电目标的化学计量分析

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Redox imbalance in cells induces lipid peroxidation and generates a class of highly reactive metabolites known as lipid-derived electrophiles (LDEs) that can modify proteins and affects their functions. Identifying targets of LDEs is critical to understand how such modifications are functionally implicated in oxidative-stress associated diseases. Here we report a quantitative chemoproteomic method to globally profile protein targets and sites modified by LDEs. In this strategy, we designed and synthesized an alkyne-functionalized aminooxy probe to react with LDE-modified proteins for imaging and proteomic profiling. Using this probe, we successfully quantified >4000 proteins modified by 4-hydroxy-2-nonenal (HNE) of high confidence in mammalian cell lysate and combined with a tandem-orthogonal proteolysis activity-based protein profiling (TOP-ABPP) strategy, we identified ~400 residue sites targeted by HNE including reactive cysteines in peroxiredoxins, an important family of enzymes with anti-oxidant roles. Our method expands the toolbox to quantitatively profile protein targets of endogenous electrophiles and the enlarged inventory of LDE-modified proteins and sites will contribute to functional elucidation of cellular pathways affected by oxidative stress.
机译：细胞中的氧化还原失衡会引起脂质过氧化，并产生一类称为脂质衍生亲电试剂（LDE）的高反应性代谢产物，可修饰蛋白质并影响其功能。确定LDE的靶标对于了解这种修饰在功能上如何与氧化应激相关疾病有关至关重要。在这里，我们报告了一种定量的化学计量学方法，可以对LDEs修饰的蛋白质靶标和位点进行整体分析。在此策略中，我们设计并合成了炔烃官能化的氨氧基探针，以与LDE修饰的蛋白反应进行成像和蛋白质组分析。使用该探针，我们成功地定量了> 4000种在哺乳动物细胞裂解物中具有高可信度的4-羟基-2-壬烯醛（HNE）修饰的蛋白质，并结合了基于串联正交蛋白水解活性的蛋白质谱分析（TOP-ABPP）策略，确定了HNE靶向的〜400个残基位点，包括过氧化物酶中的反应性半胱氨酸，这是具有抗氧化作用的重要酶家族。我们的方法扩展了工具箱，可定量分析内源性亲电体的蛋白质靶标，并且LDE修饰的蛋白质和位点的扩大库存将有助于阐明受氧化应激影响的细胞途径。

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