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Published on: September 25, 2011
Development and application of a method for identification of isothiocyanate-targeted molecules in colon cancer cells
Noriyuki Miyoshi1, Takumi Yonemochi, Susumu Tomono
1Department 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
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)
Insights
This study introduces a new method to find isothiocyanate (ITC)-targeted molecules by analyzing differences between benzyl ITC (BITC) and phenethyl ITC (PEITC). The technique successfully identified intracellular ITC targets in human colon cancer cells.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Proteomics
Background:
- Isothiocyanates (ITCs) are compounds with significant biological activity.
- Identifying specific molecular targets of ITCs is crucial for understanding their mechanisms of action.
- Existing methods may lack the specificity to differentiate between similar ITCs.
Purpose of the Study:
- To develop a novel method for identifying isothiocyanate (ITC)-targeted molecules.
- To leverage the subtle differences between benzyl ITC (BITC) and phenethyl ITC (PEITC) for target identification.
- To validate the method using a model protein and apply it to complex biological samples.
Main Methods:
- Incubation of dithiothreitol-reduced bovine insulin with BITC or PEITC.
- Analysis of digested peptides using ultra-performance liquid chromatography time-of-flight mass spectrometry (UPLC-TOF-MS) and liquid chromatography quadrupole TOF-MS (LC-Q-TOF-MS).
- Development of computational mathematical schemes to identify ITC-adducted peptides based on retention time and mass differences.
Main Results:
- Identification of three specific peptides (NYCN, FVNQHLCGSHLVE, ALYLVCGE) adducted with BITC or PEITC on cysteine residues.
- Observed distinct retention times (RT(BITC)
- Successful identification of intracellular ITC targets in HCT116 cells, including glutathione and macrophage migration inhibitory factor, as well as free cysteines and lysines.
Conclusions:
- The developed method effectively identifies ITC-targeted molecules by exploiting differences between similar ITCs.
- The method is capable of detecting intracellular thiocarbamoylation of cysteine, N-terminal proline, and lysine residues.
- This approach provides a valuable tool for investigating the molecular targets of ITCs in biological systems.
