New protein footprinting: fast photochemical iodination combined with top-down and bottom-up mass spectrometry.
New protein footprinting: fast photochemical iodination combined with top-down and bottom-up mass spectrometry.
复制标题
DOI：
10.1007/s13361-012-0403-1
复制
发表时间：
2012-08
影响因子：
3.2
通讯作者：
Gross, Michael L.
中科院分区：
文献类型：
--
作者：
Chen, Jiawei;Cui, Weidong;Giblin, Daryl;Gross, Michael L.
关键词：
我们报告了一种新的方法为FPOP（快速光化学氧化的蛋白质），其中碘物种被用作修饰试剂。我们通过碘苯甲酸在248 nm处的光解产生自由基;推定的碘自由基然后迅速修饰靶蛋白。这种碘自由基标记是敏感的，可调的，和位点特异性的，仅修饰组氨酸和酪氨酸残基，与修饰14个氨基酸侧链的OH自由基相反。我们碘化的肌红蛋白（Mb）和脱辅基肌红蛋白（aMb）在其自然状态和分析的结果，自上而下和自下而上的蛋白质组学策略。自上而下测序选择一定水平的修饰（添加一个I、两个I）并确定修饰反应中产生的主要组分，而自下而上测序揭示每个修饰位点的细节。Tyr 146被发现是修饰的aMb，但Mb较少。与NMR研究一致，His 82、His 93和His 97对于aMb的修饰比对于Mb的修饰多至少10倍。对于碳酸酐酶及其载脂蛋白形式，修饰程度没有显著差异，表明它们在构象上相似，为该方法提供了对照。对于赖脯胰岛素、胰岛素-EDTA和锌复合胰岛素，碘化产率对胰岛素低聚化状态的差异敏感。碘自由基标记是蛋白质足迹法的一个有前途的补充，提供比·OH和SO 4-·更高的特异性和更低的反应性，这两种自由基已经在FPOP中使用。
We report a new approach for the FPOP (fast photochemical oxidation of proteins) whereby iodine species are used as the modifying reagent. We generate the radicals by photolysis of iodobenzoic acid at 248 nm; the putative iodine radical then rapidly modifies the target protein. This iodine-radical labeling is sensitive, tunable, and site-specific, modifying only histidine and tyrosine residues in contrast to OH radicals that modify 14 amino-acid side chains. We iodinated myoglobin (Mb) and apomyoglobin (aMb) in their native states and analyzed the outcome by both top-down and bottom-up proteomic strategies. Top-down sequencing selects a certain level (addition of one I, two I's) of modification and determines the major components produced in the modification reaction, whereas bottom-up reveals details for each modification site. Tyr146 is found to be modified for aMb but less so for Mb. His82, His93 and His97 are at least 10 times more modified for aMb than for Mb, in agreement with NMR studies. For carbonic anhydrase and its apo form, there are no significant differences of the modification extents, indicating their similarity in conformation and providing a control for this approach. For lispro insulin, insulin-EDTA, and insulin complexed with zinc, iodination yields are sensitive to differences in insulin oligomerization state. The iodine radical labeling is a promising addition to protein footprinting methods, offering higher specificity and lower reactivity than ·OH and SO4-·, two other radicals already employed in FPOP.
登录
查看更多内容
影响因子：
14.8
作者：
Guan, Lan;Kaback, H. Ronald
通讯作者：
Kaback, H. Ronald
影响因子：
3.3
作者：
Furlan, A
通讯作者：
Furlan, A
影响因子：
4.4
作者：
KAWASAKI, M;LEE, SJ;BERSOHN, R
通讯作者：
BERSOHN, R
影响因子：
7.4
作者：
Gau, Brian C.;Chen, Hao;Zhang, Yun;Gross, Michael L.
通讯作者：
Gross, Michael L.
影响因子：
2.9
作者：
JEFFREY, PD;COATES, JH
通讯作者：
COATES, JH