Analytical Biochemistry 2012-10-15

Development and application of a method for identification of isothiocyanate-targeted molecules in colon cancer cells.

Noriyuki Miyoshi, Takumi Yonemochi, Susumu Tomono, Ryuuta Fukutomi, Yoshimasa Nakamura, Hiroshi Ohshima

Index: Anal. Biochem. 429(2) , 124-31, (2012)

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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)