Department of Food and Nutritional Sciences, Graduate School of Nutritional and Environmental Sciences, and Global Center of Excellence Program, University of Shizuoka, Shizuoka, Japan.
Anal Biochem. 2012 Oct 15;429(2):124-31. doi: 10.1016/j.ab.2012.07.018. Epub 2012 Jul 24.
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 (RT(BITC)<RT(PEITC)) by reverse-phase column chromatography with a difference of molecular mass (Δ14.01565). 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.
在这项研究中，我们开发了一种新的方法，使用两种研究充分的异硫氰酸盐（ITC）：苄基异硫氰酸盐（BITC）和苯乙基异硫氰酸盐（PEITC）来鉴定 ITC 靶向分子。该方法的原理基于识别 BITC 和 PEITC 之间的差异模式，因为它们表现出相似的化学和生物学行为。为了方法验证，用模型分子二硫苏糖醇还原的牛胰岛素与 BITC 或 PEITC 孵育，并通过超高效液相色谱飞行时间质谱（UPLC-TOF-MS）和液相色谱四极杆飞行时间质谱（LC-Q-TOF-MS）分析消化肽。鉴定出三个肽-NYCN、FVNQHLCGSHLVE 和 AYLVCGE-在其半胱氨酸残基上与 BITC 或 PEITC 缀合。用反相柱色谱法，每个与 BITC 或 PEITC 缀合的肽组都显示出保留时间（RT(BITC)<RT(PEITC)），差异为分子量（Δ14.01565）。基于这些发现，构建了计算数学方案来提取满足上述标准的 MS 离子集。将所开发的方法应用于 ITC 处理的人结肠癌细胞 HCT116 的提取物、谷胱甘肽半胱氨酸巯基的硫代氨基甲酰化以及巨噬细胞迁移抑制因子 PMFIVNTNVPR 的 N-末端脯氨酸残基中，成功鉴定为 ITC 之一的细胞内靶标。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。
