Identification of photooxidation sites in bovine alpha-crystallin
Identification of photooxidation sites in bovine alpha-crystallin
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DOI：
10.1111/j.1751-1097.1997.tb03200.x
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发表时间：
1997-11-01
影响因子：
3.3
通讯作者：
Schey, KL
中科院分区：
文献类型：
--
作者：
Finley, EL;Busman, M;Schey, KL
由于蛋白质的UV照射可产生活性氧物质，并且暴露于UV光与白内障发生有关，因此使用串联质谱（MS/MS）鉴定牛α-晶状体蛋白（具有分子伴侣活性的主要透镜蛋白）的光氧化位点。用UV光（>293 nm）照射牛α-晶状体蛋白1、4和8 h，用胰蛋白酶消化并通过基质辅助激光解吸电离、飞行时间质谱（MALDI）分析以鉴定氧化序列，通过反相HPLC纯化胰蛋白酶肽并通过MS/MS测序氧化肽以确定氧化位点，色氨酸荧光随着UV暴露时间的增加而呈指数下降，并且含有α A-晶状体蛋白残基1-11和1-11，12-22和57-69的α B-晶状体蛋白被确定为通过比未修饰的肽的质量高16 D或16 Da的倍数的位移而被氧化。MALDI分析揭示了所有四个序列的单一氧化，其随着UV暴露时间的增加和α B 12-22的可能的双重氧化而增加，光氧化的特定位点表明α A-和α B-的N-末端区域晶状体蛋白暴露于水性环境中，并且位于邻近亚基的色氨酸残基附近。
Because UV irradiation of proteins can produce reactive oxygen species and exposure to UV light has been implicated in cataractogenesis, the sites of photooxidation of bovine alpha-crystallin, a major lens protein with molecular chaperone activity, were identified using tandem mass spectrometry (MS/MS), Bovine alpha-crystallin was irradiated with UV light (>293 nm) for 1, 4 and 8 h, digested with trypsin and analyzed by matrix-assisted laser desorption ionization, time-of-flight mass spectrometry (MALDI) to identify the oxidized sequences, Tryptic peptides were purified by reverse-phase HPLC and oxidized peptides were sequenced by MS/MS to determine the sites of oxidation, Tryptophan fluorescence decreased exponentially with increasing time of UV exposure and peptides containing residues 1-11 of alpha A-crystallin and 1-11, 12-22 and 57-69 of alpha B-crystallin were determined to be oxidized by shifts of 16 D or multiples of 16 Da above the mass of the unmodified peptide, The MALDI analysis revealed single oxidation of all four sequences, which increased with increasing time of UV exposure and possible double oxidation of alpha B 12-22, The specific sites of photooxidation indicate that the N-terminal regions of alpha A- and alpha B-crystallin are exposed to an aqueous environment and are in the vicinity of tryptophan residues from neighboring subunits.