Detection of reversible protein thiol modifications in tissues
Detection of reversible protein thiol modifications in tissues
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DOI：
10.1016/j.ab.2006.08.020
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发表时间：
2006-11-15
影响因子：
2.9
通讯作者：
Smith, Charles V.
中科院分区：
文献类型：
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作者：
Rogers, Lynette K.;Leinweber, Barbara L.;Smith, Charles V.
蛋白质巯基（PSH）的氧化/还原反应参与了许多生理和病理过程。虽然许多新的技术已被开发用于分离和鉴定蛋白质中的修饰半胱氨酰残基，试剂和样品处理的关键评估往往被忽视。我们仔细比较了N-乙基马来酰亚胺（NEM）、碘乙酰胺（IAM）和碘乙酸（IAA）在烷基化蛋白质硫醇中的有效性，发现与IAM和IAA相比，NEM需要更少的试剂（125对1000 mol：mol过量），需要更少的时间（4 min对4 h），并且在较低pH下更有效（4.3对8.0）。评估了二硫苏糖醇（DTT）和三（2-羧乙基）膦（TCEP）还原悬浮在NaPO 4缓冲液或MeOH中的蛋白质二亚硫酸酯的相对功效，并且在测试的浓度（10100 mM）下未检测到总归一化荧光的差异;然而，在用DTT在MeOH中还原的样品中，单个条带分辨率似乎更好。此外，我们发现，与在含有NEM的MeOH中均质化的样品相比，在含有过量摩尔量NEM的水性缓冲液中均质化的组织样品中离体氧化最小化。使用NEM用于硫醇烷基化，DTT用于二硫键还原，mBBr用于标记还原的二硫键和荧光检测，我们能够生成凝胶内标准曲线并定量生物样品中的总二硫键含量，以及通过扫描光密度法鉴定特定蛋白条带的变化。我们证明，我们已经确定的复杂样品中的二硫化物检测试剂和技术也适用于二维电泳分离。(c)2006年爱思唯尔公司All rights reserved.
Oxidation/reduction reactions of protein thiol groups (PSH) have been implicated in many physiological and pathological processes. Although many new techniques for separation and identification of modified cysteinyl residues in proteins have been developed, critical assessment of reagents and sample processing often are overlooked. We carefully compared the effectiveness of N-etliylmaleimide (NEM), iodoacetamide (IAM), and iodoacetic acid (IAA) in alkylating protein thiols and found that NEM required less reagent (125 vs. 1000 mol:mol excess), required less time (4 min vs. 4 h), and was more effective at lower pHs (4.3 vs. 8.0) in comparison with IAM and IAA. The relative efficacy of dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP) for reducing protein disulficles suspended in NaP04 buffer or MeOH was assessed, and no differences in total normalized fluorescence were detected at the concentrations tested (10100 mM); however, individual band resolution appeared better in samples reduced with DTT in MeOH. In addition, we found that oxidation ex Vivo was minimized in tissue samples that were homogenized in aqueous buffers containing excess molar quantities of NEM compared with samples homogenized in MeOH containing NEM. Using NEM for thiol alkylation, DTT for disulfide reduction, and mBBr for labeling the reduced disulfide and fluorimetric detection, we were able to generate an in-gel standard curve and quantitate total disulfide contents within biological samples as well as to identify changes in specific protein bands by scanning densitometry. We demonstrated that reagents and techniques we have identified for disulfide detection in complex samples are also applicable to two-dimensional electrophoresis separations. (c) 2006 Elsevier Inc. All rights reserved.