Measurement of Melanin Metabolism in Live Cells by [U-13C]-L-Tyrosine Fate Tracing Using Liquid Chromatography-Mass Spectrometry

Abstract

Melanin synthesis occurs within a specialized organelle called the melanosome. Traditional methods for measuring melanin levels rely on the detection of chemical degradation products of melanin by high-performance liquid chromatography (HPLC). Although these methods are robust, they are unable to distinguish between melanin synthesis and degradation, and are best suited to measure melanin changes over long periods of time. We developed a method that actively measures both eumelanin and pheomelanin synthesis by fate tracing [U-13C] tyrosine using liquid chromatography-mass spectrometry (LC-MS). Using this method, we confirmed previous reports of melanin synthesis differences between melanocytes derived from individuals with different skin color or MC1R genotype, and uncovered new information regarding the differential de novo synthesis of eumelanin and pheomelanin, also called mixed melanogenesis. We also revealed that distinct mechanisms that alter melanosomal pH differentially induce new eumelanin and pheomelanin synthesis. Finally, we revealed that the synthesis of L-DOPA, an important metabolite of tyrosine, is differentially controlled by multiple factors. Because tyrosine fate tracing is compatible with untargeted LC-MS based metabolomics, this approach enables the broad measurement of cellular metabolism in combination with melanin metabolism, and we anticipate that this approach will shed new light on multiple mechanisms of melanogenesis.