Identification and quantification of Hypocretin-1/Orexin-A 1-14 and 1-16 fragments in immunopurified cerebrospinal...

Hypocretin-1/Orexin-A is associated with stabilization of the sleep/wake cycle. Reductions of cerebrospinal fluid (CSF) concentrations of orexin-A, as determined by radioimmunoassay (RIA), are considered consistent with the diagnosis of narcolepsy type 1 (NT1). However, measuring full-length orexin-A by other methods such as liquid chromatography-tandem mass spectrometry (LC-MS/MS) has proven technically challenging. In this investigation, abundant fragments of orexin-A in CSF were identified and quantified using LC-MS, which allowed assessment of the concordance with clinical RIA measurements.

Orexin-A-related peptides were immunopurified using the antibody employed in the RIA from 80 residual CSF samples submitted to our reference laboratory for RIA testing (n = 31, ≤110 pg/mL (low); n = 15, 111-200 pg/mL (intermediate); n = 34, >200 pg/mL (normal)). Enriched peptides then underwent LC-MS/MS analysis, resulting in identification of abundant orexin-A fragments. Following the identification of fragments, quantitative targeted LC-MS measurements were compared to a clinically relevant RIA assay.

Full-length orexin-A was not detected in CSF, but two relatively abundant N-terminal orexin-A fragments, comprising amino acids 1-14 and 1-16, were identified by LC-MS/MS. Comparison with RIA yielded Spearman's correlation coefficients (ρ) of 0.91 and 0.94 for the 1-14 and 1-16 fragments, respectively. Additionally, the LC-MS and RIA method were 88% concordant for orexin-A deficient categorizations characteristic of NT1.

Quantitative measurement of the identified orexin-A peptide fragments via LC-MS may represent a significant advance in diagnostic capabilities for evaluating central hypersomnolence disorders. These findings also lay the foundation for future studies on the diagnostic utility and clinical relevance of these orexin-A fragments.