MARBL: A Live-Cell Method for Profiling Bioenergetic Heterogeneity by Noncanonical Methionine Labeling of the Cell Surface Proteome.

Heterogeneity is a hallmark of biological systems, where cell-to-cell variability supports adaptation to changing environments, but also enables maladaptive states such as drug resistance. Many sources of non-genetic variation, particularly bioenergetics and metabolism, remain difficult to measure in living cells and connect to functional outcomes. Here, we introduce MARBL (Methionine Analogues for Ratiometric Bioenergetics in Live cells), a method that encodes translationally-coupled energetic responses to metabolic stress as an internally normalized signal within the surface proteome of living cells. Applying MARBL to primary immune cells reveals that differences in baseline translational activity can underlie apparent metabolic vulnerabilities, underscoring the importance of ratiometric measurements. We demonstrate that MARBL can enrich pathogenic from non-pathogenic TH17 cells based on resilience to bioenergetic stress, which functionally distinguishes cells that produce IFNγ upon restimulation. Overall, MARBL offers a versatile platform to profile metabolic resilience in living cells and link bioenergetic state to cellular function.