Mass Spectrometry-Based Quantification of Orexin Species in Human Cerebrospinal Fluid Reveals Differential Dynamics Associated with Sleep

Orexin neuropeptides are central to sleep-wake regulation. However, current cerebrospinal fluid (CSF) assays primarily detect inactive orexin-A fragments using radioimmunoassay (RIA), which limits mechanistic understanding. We developed a high-sensitivity mass spectrometry (MS) assay that can comprehensively quantify human CSF orexin species, including the biologically active long orexin-A and orexin-B, as well as their metabolites and prepro-orexin that are biologically inactive. In participants with narcolepsy type 1 (NT1), all orexin species were significantly reduced compared to those with narcolepsy type 2 (NT2) or idiopathic hypersomnia (IH). MS detected all orexin peptides in NT1 samples below the RIA detection threshold. Notably, the ratio of short to long orexin-B peptides differentiated NT2 from IH, suggesting altered orexin peptide metabolism. Sleep deprivation increased all orexin species with distinct temporal dynamics across peptide forms. The stable isotope labeling kinetics method using MS revealed increased turnover of orexin-A and orexin-B, but not prepro-orexin, during sleep deprivation, indicating activity-dependent regulation. These findings demonstrate that MS enables comprehensive profiling of orexin dynamics and reveals biologically relevant differences in peptide processing and turnover under different sleep conditions. This approach is a powerful tool for advancing our understanding of orexin physiology and its role in sleep-wake pathophysiology.