Fluorogenic derivatization reagents suitable for isolation and identification of cysteine-containing proteins utilizing high-performance liquid chromatography-tandem mass spectrometry
Fluorogenic derivatization reagents suitable for isolation and identification of cysteine-containing proteins utilizing high-performance liquid chromatography-tandem mass spectrometry
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DOI：
10.1021/ac034840i
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发表时间：
2004-02-01
影响因子：
7.4
通讯作者：
Imai, K
中科院分区：
文献类型：
--
作者：
Masuda, M;Toriumi, C;Imai, K
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。用这些试剂对蛋白质混合物进行衍生化后，利用高效液相色谱-荧光检测和串联质谱法以及基于概率的蛋白质鉴定算法进行一系列蛋白质分离、消化和鉴定的标准过程。 DAABD 和 TAABD 衍生物均在飞摩尔水平上进行荧光检测，并且通过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，灵敏度提高了 100 倍以上。对存储有 DAABD 连接的 Cys 残基片段信息的 MASCOT 数据库搜索系统进行修改，可以识别衍生牛血清白蛋白 (BSA) 的蛋白水解肽片段，估计 BSA 的序列覆盖率为 38%。利用 DAABD-Cl 作为衍生化试剂，还可以鉴定秀丽隐杆线虫可溶性提取物（10 杯蛋白质）中的多种蛋白质。因此，对于复杂蛋白质基质中蛋白质的鉴定，DAABD-Cl 可能是比先前报道的 7-氟-2,1,3-苯并恶二唑-4-磺酸铵更合适的试剂。
The fluorogenic derivatization reagents with a positive charge, 4-(dimethylaminoethylaminosulfonyl)-7-chloro-2,1,3-benzoxadiazole (DAABD-Cl) and 7-chloro-2,1,3-benzoxadiazole-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 mug 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.