Xu Guozhong, Chance Mark R
Center for Synchrotron Biosciences, Albert Einstein College of Medicine of Yeshiva University, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
Anal Chem. 2005 Apr 15;77(8):2437-49. doi: 10.1021/ac0484629.
Protein footprinting based on hydroxyl radical-mediated modification and quantitative mass spectroscopic analysis is a proven technique for examining protein structure, protein-ligand interactions, and structural allostery upon protein complex formation. The reactive and solvent-accessible amino acid side chains function as structural probes; however, correct structural analysis depends on the identification and quantification of all the relevant oxidative modifications within the protein sequence. Sulfur-containing amino acids are oxidized readily and the mechanisms of oxidation are particularly complex, although they have been extensively investigated by EPR and other spectroscopic methods. Here we have undertaken a detailed mass spectrometry study (using electrospray ionization mass spectrometry and tandem mass spectrometry) of model peptides containing cysteine (Cys-SH), cystine (disulfide bonded Cys), and methionine after oxidation using gamma-rays or synchrotron X-rays and have compared these results to those expected from oxidation mechanisms proposed in the literature. Radiolysis of cysteine leads to cysteine sulfonic acid (+48 Da mass shift) and cystine as the major products; other minor products including cysteine sulfinic acid (+32 Da mass shift) and serine (-16 Da mass shift) are observed. Radiolysis of cystine results in the oxidative opening of the disulfide bond and generation of cysteine sulfonic acid and sulfinic acid; however, the rate of oxidation is significantly less than that for cysteine. Radiolysis of methionine gives rise primarily to methionine sulfoxide (+16 Da mass shift); this can be further oxidized to methionine sulfone (+32 Da mass shift) or another product with a -32 Da mass shift likely due to aldehyde formation at the gamma-carbon. Due to the high reactivity of sulfur-containing amino acids, the extent of oxidation is easily influenced by secondary oxidation events or the presence of redox reagents used in standard proteolytic digestions; when these are accounted for, a reactivity order of cysteine > methionine approximately tryptophan > cystine is observed.
基于羟基自由基介导的修饰和定量质谱分析的蛋白质足迹法是一种经证实的技术，可用于研究蛋白质结构、蛋白质-配体相互作用以及蛋白质复合物形成时的结构变构。具有反应活性且可溶剂接触的氨基酸侧链充当结构探针；然而，正确的结构分析取决于对蛋白质序列中所有相关氧化修饰的鉴定和定量。含硫氨基酸很容易被氧化，尽管它们已通过电子顺磁共振（EPR）和其他光谱方法进行了广泛研究，但其氧化机制特别复杂。在此，我们使用伽马射线或同步加速器X射线对含有半胱氨酸（Cys-SH）、胱氨酸（二硫键连接的半胱氨酸）和蛋氨酸的模型肽进行氧化后，开展了详细的质谱研究（使用电喷雾电离质谱和串联质谱），并将这些结果与文献中提出的氧化机制所预期的结果进行了比较。半胱氨酸的辐射分解导致半胱氨酸磺酸（质量位移+48 Da）和胱氨酸成为主要产物；还观察到其他次要产物，包括半胱氨酸亚磺酸（质量位移+32 Da）和丝氨酸（质量位移-16 Da）。胱氨酸的辐射分解导致二硫键的氧化断裂以及半胱氨酸磺酸和亚磺酸的生成；然而，氧化速率明显低于半胱氨酸。蛋氨酸的辐射分解主要产生甲硫氨酸亚砜（质量位移+16 Da）；它可以进一步氧化为甲硫氨酸砜（质量位移+32 Da）或另一种质量位移为-32 Da的产物，这可能是由于γ-碳处形成了醛。由于含硫氨基酸的高反应活性，氧化程度很容易受到二次氧化事件或标准蛋白酶解消化中使用的氧化还原试剂的影响；当考虑到这些因素时，观察到的反应活性顺序为半胱氨酸>蛋氨酸≈色氨酸>胱氨酸。
