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Objective: To catalog protein markers and develop quantitative expression signatures for functional and metabolic glucose responsiveness of insulin-producing beta cells. Methods: Data independent, alternate scanning LC-MS was used to quantitatively profile and calculate within-sample protein amounts of the proteomes of FACS-purified rat pancreatic beta and alpha cells, compared to liver and brain, with focus on beta cell-specific expressions. The proteomes of beta cell subpopulations with different metabolic and functional responsiveness were compared following their FACS-separation on basis of absence or presence of a rapid rise in cellular NAD(P)H-fluorescence at 7.5 mM glucose. Results: For quantitative tissue comparison, 6 stably-expressed reference proteins (PPIA, HSPA8, TUBB5, PFN1, YWHAE, RAB1B) were selected for geometric normalization of abundances. All tissues combined, 943 proteins were reliably quantified. 93 out of 467 proteins detected in beta cells were unique to this cell type. Several other proteins showed beta cell selectivity in terms of relatively high molar abundance after normalization. The proteome of beta cell subpopulations with higher glucose responsiveness reflected their functional properties: (i) higher glucose-induced insulin synthesis correlated with 50% higher expression of the protein biosynthetic machinery: a concerted up-regulation of 40s and 60s ribosomal constituents, NADPH-dependent protein folding and translation elongation; (ii) higher rates of glucose utilization were associated with 50% higher levels of glycolytic enzymes, and of the cytosolic arm of the malate/aspartate-shuttle, which transports glycolysis-derived NADH into mitochondria. Their higher glucose oxidation rates are, however, not associated with a higher expression of mitochondrial catabolic enzymes (Krebs cycle, β-oxidation, respiratory chain) which thus appear not rate-limiting for oxidative flux. Conclusion: Label-free LC-MS analysis using data-independent, alternate scanning discloses molecular differences between functionally different beta cell subpopulations, providing quantitative and stoichiometric data within a single experiment. Insulin-producing beta cells with higher glucose responsiveness exhibited a higher expression of glycolytic enzymes but not of mitochondrial enzymes.