Methods for the determination and quantification of the reactive thiol proteome.
Methods for the determination and quantification of the reactive thiol proteome.
复制标题
DOI：
10.1016/j.freeradbiomed.2009.06.012
复制
发表时间：
2009-09-15
影响因子：
7.4
通讯作者：
Landar, Aimee
中科院分区：
文献类型：
--
作者：
Hill, Bradford G.;Reily, Colin;Oh, Joo-Yeun;Johnson, Michelle S.;Landar, Aimee
蛋白质硫醇修饰发生在生理和病理条件下，并已被证明有助于蛋白质结构、功能和氧化还原信号的变化。大多数蛋白质的硫醇修饰发生在具有低pKA的半胱氨酸残基上；这些亲核蛋白质组成了“反应性硫醇蛋白质组”。这个蛋白质组中最活跃的成员通常是低丰度蛋白。因此，需要灵敏和定量的方法来检测和测量生物样品中的硫醇修饰。为了实现这一点，我们标准化了生物素化和荧光团标记的烷基化试剂的使用，如生物素化的碘乙酰胺(BIAM)、N-乙基马来酰亚胺(BT-NEM)和BODIPY标记的IAM和NEM，用于一维和二维蛋白质组策略。将纯化的细胞色素c和甘油醛-3-磷酸脱氢酶组分与已知数量的生物素或BODIPY荧光团偶联，创建可在标准SDS-PAGE凝胶上运行的外部标准，允许通过Western blotting或荧光成像来定量生物样品中的蛋白质硫醇。提供了使用硫醇反应探针和制定外部标准的详细方案，用于可视化和测量生物样品中的蛋白质硫醇修饰。
Protein thiol modifications occur under both physiological and pathological conditions and have been shown to contribute to changes in protein structure, function, and redox signaling. The majority of protein thiol modifications occur on cysteine residues that have a low pKa; these nucleophilic proteins comprise the “reactive thiol proteome.” The most reactive members of this proteome are typically low abundance proteins. Therefore, sensitive and quantitative methods are needed to detect and measure thiol modifications in biological samples. To accomplish this, we have standardized the usage of biotinylated and fluorophore-labeled alkylating agents, such as biotinylated iodoacetamide (BIAM), N-ethylmaleimide (Bt-NEM), and BODIPY-labeled IAM and NEM, for use in one- and two-dimensional proteomic strategies. Purified fractions of cytochrome c and glyceraldehyde-3-phosphate dehydrogenase were conjugated to a known amount of biotin or BODIPY fluorophore to create an external standard that can be run on standard SDS-PAGE gels, which allows for the quantification of protein thiols from biological samples by Western blotting or fluorescence imaging. A detailed protocol is provided for using thiol-reactive probes and making external standards for visualizing and measuring protein thiol modifications in biological samples.
登录
查看更多内容
影响因子：
4.8
作者：
Forrester, Michael T.;Foster, Matthew W.;Stamler, Jonathan S.
通讯作者：
Stamler, Jonathan S.
影响因子：
2.9
作者：
Rogers, Lynette K.;Leinweber, Barbara L.;Smith, Charles V.
通讯作者：
Smith, Charles V.
影响因子：
15.9
作者：
RUTSTEIN, DD;INGENITO, EF;BURKE, JM
通讯作者：
BURKE, JM
影响因子：
2.9
作者：
Kim, JR;Yoon, HW;Rhee, SG
通讯作者：
Rhee, SG
影响因子：
4.1
作者：
Wong, Hansen L.;Liebler, Daniel C.
通讯作者：
Liebler, Daniel C.