Integrated proteomics and glycoproteomics reveal metabolic reprogramming in Nannochloropsis oceanica under CO₂ limitation

Nannochloropsis oceanica , a model oleaginous microalga with emerging applications in food and bioenergy, relies on carbon-concentrating mechanisms (CCMs) to adapt to low CO₂ conditions, yet the role of glycosylation in this process remains unclear. Here, we applied 4D-DIA proteomics and glycoproteomics to investigate protein expression and N-glycosylation dynamics in N. oceanica under high- (5% CO₂) and low-carbon (0.04% CO₂) conditions. A total of 5,136 protein groups were identified, including 454 differentially expressed proteins, most of which were downregulated under carbon limitation. Functional analyses revealed that photosynthesis, ribosome, and fatty acid biosynthesis pathways were suppressed, whereas oxidative phosphorylation and catabolic processes were enhanced, indicating a shift toward a survival-oriented metabolic state. Glycoproteomic analysis identified 459 N-glycosylation sites, with 131 showing significant changes. Differentially expressed glycosylated proteins were enriched in energy metabolism, translation, membrane transport, and signaling pathways. Network analysis further highlighted central metabolic enzymes and regulatory proteins as key glycosylation targets. Notably, lipid biosynthesis and photosynthetic components were downregulated, whereas central carbon metabolism underwent extensive remodeling under low CO₂ conditions. Overall, our results demonstrate that protein glycosylation plays a critical role in enabling N. oceanica to respond to carbon limitation by reprogramming metabolic networks. These findings provide new insights and potential targets for improving carbon fixation efficiency and stress tolerance in microalgae.