Targeted mass spectrometry to monitor nuclear accumulation of endogenous Nrf2 and its application to SH-SY5Y cells stimulated with food components.

The Nrf2 signaling pathway is highly significant for redox homeostasis. Hence, nutrients and drugs activating Nrf2 can prevent oxidative stress-mediated medical conditions. After activation, Nrf2 accumulates in the cell nucleus; therefore, stimulation of Nrf2 by food components and drugs is usually monitored by measuring nuclear Nrf2 levels. The present study developed a targeted mass spectrometry method for the highly reliable quantification of nuclear Nrf2 levels. Three Nrf2-specific peptides were detected after enzymatic digestion of the nuclear fraction by the developed protocol for micro-liquid chromatography-tandem mass spectrometry in scheduled multiple reaction monitoring mode (microLC-MS/MS-sMRM). The method also identified nuclear Nrf2 unequivocally and specifically in the SDS-PAGE fraction of 100-150 kDa. Moreover, highly precise and linear relative quantification was achieved (mean relative standard deviation 8.3%; coefficient of determination 0.998). Incubation experiments in SH-SY5Y neuroblastoma cells revealed significantly up to 6-fold elevated nuclear Nrf2 levels after stimulation with 10 µM carnosol (rosemary), 10 µM sulforaphane (broccoli), or 20 µM cinnamaldehyde (cinnamon). Our results were in very good accordance with conventional Nrf2 western blotting and were highly correlated with the food components' effect on the expression levels of NAD(P)H dehydrogenase [quinone] 1 and thioredoxin reductase 1, two major Nrf2-regulated cytoprotective enzymes. The newly developed microLC-MS/MS-sMRM method shows broad applicability and can serve as a highly selective and reliable method to analyze Nrf2 activation. Graphical abstract ᅟ.

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. SIGNIFICANCE: 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.

Profiling of antioxidative enzyme expression induced by various food components using targeted proteome analysis.

SCOPE: A method was developed for targeted proteome analysis of the expression profile of a set of antioxidative enzymes in rat macrophages and applied to screen the antioxidative potential of several food components/foods. METHODS AND RESULTS: Expression profiles of heme oxygenase 1, peroxiredoxin 1, thioredoxin reductase 1, glutathione reductase, glutathione-S transferase P1, and superoxide dismutase 1 were analyzed by nanoLC-MS/MS in selected scheduled reaction monitoring (sSRM) mode monitoring two to three peptides per protein and three transitions per peptide. Relative quantification was performed by metabolic labeling. The validated method was used to profile the activity of capsaicin, carnosol, diallyl trisulfide, maslinic acid, quercetin, sulforaphane, cinnamaldehyde and coffee extract to modulate the expression levels of antioxidative enzymes. Carnosol and sulforaphane most effectively induced protein expression, leading to upregulation of at least five out of the six antioxidative enzymes by a maximum factor of 22.80 ± 6.71 (heme oxygenase 1 by carnosol). Heme oxygenase 1 was most susceptible to nutritive modulation, whereas glutathione reductase expression rates were hardly affected. CONCLUSION: Targeted mass proteome analysis allows comprehensive evaluation of antioxidative effects by food ingredients. Simultaneous expression analysis of a set of proteins provided valuable insights how various enzymes were differently affected by food components.