title: Development and application of a method for identification of isothiocyanate-targeted molecules in colon cancer cells.
abstract: 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.
authors: Department of Food and Nutritional Sciences, Graduate School of Nutritional and Environmental Sciences, and Global Center of Excellence Program, University of Shizuoka, Shizuoka, Japan. miyoshin@u-shizuoka-ken.ac.jp
authors: Noriyuki
authors: N
authors: Miyoshi
authors: Takumi
authors: T
authors: Yonemochi
authors: Susumu
authors: S
authors: Tomono
authors: Ryuuta
authors: R
authors: Fukutomi
authors: Yoshimasa
authors: Y
authors: Nakamura
authors: Hiroshi
authors: H
authors: Ohshima
mesh_terms: D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D003110:Colonic Neoplasms / Q000378:metabolism / Q000473:pathology*; D045325:HCT116 Cells; D006801:Humans; D007328:Insulin / Q000737:chemistry / Q000378:metabolism; D017879:Isothiocyanates / Q000737:chemistry*; D010455:Peptides / Q000032:analysis*; D025341:Principal Component Analysis; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D007328:Insulin; D017879:Isothiocyanates; D010455:Peptides; C058305:phenethyl isothiocyanate; C031403:benzyl isothiocyanate