Targeted proteome analysis with isotope-coded protein labels for monitoring the influence of dietary phytochemicals on the expression of cytoprotective proteins in primary human colon cells.

Cytoprotective effects by upregulating cellular expression levels of antioxidant proteins are attributed to a significant number of food ingredients. Evaluation of those cytoprotective effects and identification of the most active components requires reliable and comprehensive proteomic strategies. Thus, promising potential bioactive ingredients could be determined for the prevention of various diseases, including colon cancer formation. We established a multiplexed nanoLC-MS/MS targeted proteomic method, operated in scheduled selected reaction monitoring mode (sSRM), to record modulation of the expression levels of six major antioxidant proteins induced by dietary phytochemicals. Relative quantification was achieved by isotope-coded protein labels (ICPLs) and based on two to three proteotypic peptides per target protein. The assay provided accurate (mean relative error 6.4%) and precise (mean RSD 7.4%) quantification. Incubation experiments were carried out in primary human colon epithelial cells (HCoEpiCs) and revealed significant upregulation of NAD(P)H dehydrogenase [quinone] 1 (up to threefold) and thioredoxin reductase 1 (up to twofold) by 10μM sulforaphane (from broccoli), 5μM carnosol (rosemary), and 20μM cinnamaldehyde (cinnamon). The latter two substances additionally upregulated heme oxygenase-1 and were identified as the most active components in the test set. The results provide additional evidence for nutritive cytoprotection in human colon cells. Targeted proteome analysis using LC coupled to scheduled selected reaction monitoring (sSRM)-MS is a highly flexible and reliable method to monitor protein expression profiles. The present study screened modulators occurring in food, which may be protective against colon cancer by inducing cytoprotective enzymes. Primary human colonic epithelial cells were used because they model the conditions in healthy gut tissue better than immortalized cells. Thus, an LC-MS/MS-sSRM protocol was established and validated including relative quantification of cytoprotective protein expression by isotope-coded protein labels, because metabolic labelling cannot be applied for primary cells. The present study demonstrated that the major components of cinnamon and rosemary, respectively, i.e. cinnamaldehyde and carnosol, are potent alimentary candidates to increase the expression of cytoprotective enzymes in the human colon. Among the investigated enzymes, NAD(P)H dehydrogenase [quinone] 1 (NQO1) was most susceptible towards modulation by phytochemicals. NQO1 exerts its cytoprotective activity by detoxifying electrophilic and oxidative xenobiotics with quinone structure.