Broccoli & Signalling Pathways Figure 4. LC-MS of insulin incubated with and without SF in human plasma. Extracted ion LC-MS chromatograms (m/z 1183.6–1184.1) of insulin-SF MH55+ in (A) unmodified insulin (20 mg/ml) in human plasma control and (B) human plasma incubated with insulin (20 mg/ml) and 50 mM SF for 4 h at 37uC, showing the appearance of two different insulin-SF conjugates at retention times of 6.46 and 7.08 min. The enhanced product ion (EPI)-MS spectra of these two insulin-SF conjugates are shown in Figure 5. doi:10.1371/journal.pone.0002568.g004 analysis of mRNA and protein turnover, and post translational protein modifications such as phosphorylation, associated with components of the signal transduction pathway and downstream targets. Furthermore, the effect of pathway perturbation may depend upon the precise cell type, with potentially differential effects occurring in epithelial and stromal cells. Despite these reservations, it is of considerable interest that broccoli intervention is associated with perturbation of TGFb1, EGF and insulin signalling, each of which has been associated with prostate carcinogenesis [31–35], in addition to carcinogenesis at other sites [28,36,37], and inflammation associated with myocardial infarc- tion [38]. It is noteworthy that broccoli consumption was also associated with alterations in mRNA processing, and this is being further explored. It is likely that the major bioactive products derived from broccoli are the isothiocyanates, sulforaphane and iberin. These have been shown to have a multitude of biological activities in cell models consistent with anticarcinogenic activity [15]. However, these studies largely involve exposing cells to concentrations of SF and IB far in excess of those which occur transiently in the plasma after broccoli consumption, and are mediated by the intracellular activity of the ITCs by, for example, perturbing intracellular redox status, depletion of glutathione and perturbation of the Keap1- Nfr2 complex. We question whether these processes would occur in vivo, as any of the ITCs entering cells would immediately be inactivated through conjugation with glutathione that would be present in relatively high concentration. Thus, we explored whether the biological activity of ITCs may be mediated through their chemical interaction with signalling peptides within the extracellular environment of the plasma, which has a low glutathione concentration. We demonstrated that ITCs readily form thioureas with signalling proteins in the plasma through PLoS ONE | www.plosone.org 9 July 2008 | Volume 3 | Issue 7 | e2568