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首页> 外文期刊>Analytical chemistry >Fluorogenic Derivatization Reagents Suitable for Isolation and Identification of Cysteine-Containing Proteins Utilizing High-Performance Liquid Chromatography-Tandem Mass Spectrometry
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Fluorogenic Derivatization Reagents Suitable for Isolation and Identification of Cysteine-Containing Proteins Utilizing High-Performance Liquid Chromatography-Tandem Mass Spectrometry

机译：适用于高效液相色谱-串联质谱法分离和鉴定含半胱氨酸蛋白的荧光衍生试剂

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摘要

The fluorogenic derivatization reagents with a positive charge, 4-(dimethylaminoethylaminosulfonyl)-7-chloro-2,1,3-benzoxadiazole (DAABD-Cl) and 7-chloro-2,1,3-benz-oxadiazole-4-sulfonylaminoethyltrimethylammonium chloride (TAABD-Cl), are proposed for use in proteomics studies. Following derivatization of protein mixtures with these reagents, a series of standard processes of isolation, digestion, and identification of the proteins were performed utilizing high-performance liquid chromatography-fluorescence detection and tandem mass spectrometry with the probability-based protein identification algorithm. Both DAABD and TAABD derivatives were detected fluorometrically at the femtomole level and showed more than 100-fold improvement in sensitivity compared to the underivatized original compounds with an electrospray ionization ion trap mass spectrometer analysis. The modification of the MASCOT database search system memorized with the fragment information of a DAABD-attached Cys residue allowed the identification of the proteolytic peptide fragments of the derivatized bovine serum albumin (BSA) with an estimated 38% sequence coverage of BSA. Utilizing DAABD-Cl as a derivatization reagent, identification of several proteins was also possible in a soluble extract of Caenorhabditis elegans (10 μg of protein). Consequently, for identification of proteins in the complex matrixes of proteins, DAABD-Cl could be a more appropriate reagent than ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate as reported previously.
机译：带正电荷的荧光衍生试剂4-（二甲基氨基乙基氨基磺酰基）-7-氯-2,1,3-苯并恶二唑（DAABD-Cl）和7-氯-2,1,3-苯并恶二唑-4-磺酰基氨基乙基三甲基氯化铵（TAABD-Cl），建议用于蛋白质组学研究。用这些试剂衍生蛋白质混合物后，使用高效液相色谱-荧光检测和串联质谱以及基于概率的蛋白质鉴定算法，进行了一系列标准的蛋白质分离，消化和鉴定过程。用电喷雾电离离子阱质谱仪分析，与未衍生化的原始化合物相比，DAABD和TAABD衍生物均在氟虫灵水平上进行了荧光检测，灵敏度提高了100倍以上。 MASCOT数据库搜索系统的修改存储了与DAABD相连的Cys残基的片段信息，从而可以鉴定具有38％的BSA序列覆盖率的衍生牛血清白蛋白（BSA）的蛋白水解肽片段。利用DAABD-Cl作为衍生试剂，还可以鉴定秀丽隐杆线虫的可溶性提取物（10μg蛋白）中的几种蛋白。因此，与先前报道的7-氟-2,1,3-苯并恶二唑-4-磺酸铵相比，DAABD-Cl可能是更合适的试剂，可用于鉴定蛋白质复杂基质中的蛋白质。

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