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

摘要

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 100-150kDa. 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 10M carnosol (rosemary), 10M sulforaphane (broccoli), or 20M 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.