Abstract
<jats:title>Abstract</jats:title> <jats:p>Nasal fluid collected at the brain-nose interface (BNI) may provide a minimally invasive window into central nervous system (CNS) biology, but its suitability for deep, reproducible proteomics remains unclear. Here, we establish and benchmark a standardized liquid chromatography-tandem mass spectrometry workflow for BNI-proximal nasal fluid and compare its proteome to matched cerebrospinal fluid (CSF) from 40 individuals with cognitive impairment. Using pooled nasal fluid, we identified single-pot, solid-phase sample preparation combined with data-independent acquisition (DIA) as the optimal strategy, with low coefficients of variation. In cognitively impaired cohort, DIA-based proteomics on a timsTOF pro quantified on average 5,210 proteins in nasal fluid and 1,800 in CSF, with 4,901 nasal fluid proteins detected in at least 75% of samples. Nasal fluid contained nearly 50% of the CSF proteome (876 shared proteins), including numerous CNS- and neurodegeneration-associated species. The result of this exploratory study position BNI-derived nasal fluid as a robust, proteome-rich biofluid for proteomics-driven CNS biomarker research.</jats:p>