Rebrin Igor, Bregere Catherine, Gallaher Timothy K, Sohal Rajindar S
Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA, USA.
Methods Enzymol. 2008;441:283-94. doi: 10.1016/S0076-6879(08)01215-9.
Nitration and oxidation of tyrosine, tryptophan, and methionine residues in proteins are potential markers of their interaction with peroxynitrite. This chapter describes the procedure for the detection of these nitro-oxidative modifications by tandem mass spectrometry. The peptide YGDLANWMIPGK, shown to contain a nitrohydroxytryptophan in the mitochondrial enzyme succinyl-CoA:3-ketoacid coenzyme A transferase (SCOT) in vivo, was synthesized and exposed to peroxynitrite in order to test whether an identical tryptophan derivative could be generated in vitro. Data show that the occurrence of specific fragmented ions corresponding to the oxidation of methionine, nitration of tyrosine, and nitration/oxidation of tryptophan residues can be used to identify the sites of the nitration and oxidation of proteins in vitro and in vivo. It is also demonstrated that a nitrohydroxy addition to the tryptophan, similar to that present in SCOT in vivo, can be produced in vitro.
蛋白质中酪氨酸、色氨酸和蛋氨酸残基的硝化和氧化是其与过氧亚硝酸盐相互作用的潜在标志物。本章介绍了通过串联质谱检测这些硝基氧化修饰的方法。肽YGDLANWMIPGK在体内线粒体酶琥珀酰辅酶A：3-酮酸辅酶A转移酶（SCOT）中含有硝基羟基色氨酸，合成该肽并使其暴露于过氧亚硝酸盐中，以测试体外是否能产生相同的色氨酸衍生物。数据表明，对应于蛋氨酸氧化、酪氨酸硝化和色氨酸残基硝化/氧化的特定碎片离子的出现，可用于鉴定蛋白质在体外和体内的硝化和氧化位点。还证明了在体外可以产生类似于体内SCOT中存在的色氨酸硝基羟基加成物。
