Development and application of a method for identification of isothiocyanate-targeted molecules in colon cancer cells
Development and application of a method for identification of isothiocyanate-targeted molecules in colon cancer cells
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DOI：
10.1016/j.ab.2012.07.018
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发表时间：
2012-10-15
影响因子：
2.9
通讯作者：
Ohshima, Hiroshi
中科院分区：
文献类型：
--
作者：
Miyoshi, Noriyuki;Yonemochi, Takumi;Ohshima, Hiroshi
在这项研究中，我们已经开发了一种新的方法来确定异硫氰酸酯（ITC）的目标分子使用两个研究良好的ITC：苄基ITC（BITC）和苯乙基ITC（PEITC）。这种方法的原理是基于识别BITC和PEITC之间的差异模式，因为它们显示出相似的化学和生物学行为。对于方法验证，将二硫苏糖醇还原的牛胰岛素作为模型分子与BITC或PEITC孵育，并通过超高效液相色谱飞行时间质谱法（UPLC-TOF-MS）和液相色谱四极杆TOF-MS（LC-Q-TOF-MS）分析消化的肽。三个肽-NYCN，FVNQHLCGSHLVE，和BLVCGGE-被确定为在其半胱氨酸残基上与BITC或PEITC加合。用BITC或PEITC加合的每组肽通过反相柱色谱显示保留时间（RTBITC < RTPEITC），分子量差异（Δ 14.01565）。在这些发现的基础上，构建了计算数学方案来提取满足上述标准的MS离子集。应用所开发的方法提取的ITC处理的人结肠癌HCT 116细胞，谷胱甘肽的半胱氨酸残基的硫代氨基甲酰化，和N-末端脯氨酸残基的PMFIVNTNVPR从巨噬细胞迁移抑制因子被成功地确定为ITC的细胞内目标之一。此外，该方法还检测了ITC与细胞内游离半胱氨酸和赖氨酸的硫代氨基甲酰化缀合物。(C)2012 Elsevier Inc. All rights reserved.
In this study, we have developed a novel method to identify isothiocyanate (ITC)-targeted molecules using two well-studied ITCs: benzyl ITC (BITC) and phenethyl ITC (PEITC). The principle of this method is based on identifying a pattern of differences between BITC and PEITC given that they show similar chemical and biological behaviors. For method validation, dithiothreitol-reduced bovine insulin as a model molecule was incubated with either BITC or PEITC, and digested peptides were analyzed by ultra-performance liquid chromatography time-of-flight mass spectrometry (UPLC-TOF-MS) and liquid chromatography quadrupole TOF-MS (LC-Q-TOF-MS). Three peptides-NYCN, FVNQHLCGSHLVE, and ALYLVCGE-were identified as being adducted with BITC or PEITC on their cysteine residues. Each set of peptides adducted with either BITC or PEITC showed retention times (RTBITC < RTPEITC) by reverse-phase column chromatography with a difference of molecular mass (Delta 14.01565). On the basis of these findings, computational mathematical schemes were constructed to extract sets of MS ions satisfying the above criteria. Application of the developed method to an extract of ITC-treated human colon cancer HCT116 cells, thiocarbamoylation of cysteine residues of glutathione, and the N-terminal proline residues of PMFIVNTNVPR from macrophage migration inhibitory factor were successfully identified as one of the intracellular targets of ITCs. Moreover, the method also detected the thiocarbamoylated conjugates of ITCs with intracellular free cysteines and lysines. (C) 2012 Elsevier Inc. All rights reserved.