Comparative proteomic analysis of cerebrospinal fluid and brain tissue using Orbitrap Astral and timsTOF Pro 2 platforms with data-independent acquisition

OBJECTIVES: Cerebrospinal fluid (CSF) contains thousands of proteins, and some of them are useful biomarkers for brain diseases, including neurodegeneration. Our objective was to compare two state-of-the-art mass spectrometers in revealing non-Alzheimer's disease (AD) to AD CSF variability. METHODS: We qualitatively compared Orbitrap Astral and timsTOF Pro 2. We analyzed CSF and brain tissue extracts from non-AD individuals and patients with AD without prior fractionation of the samples. RESULTS: The Orbitrap Astral detected twice as many proteins as the timsTOF Pro 2. The combined CSF proteome included 2,317 proteins. The reproducibility of the analysis was 74-92 % with both machines. There were no significant sex differences in the number of CSF proteins detected, but there was an age-related increase. Fifty-two proteins were exclusively present in the CSF of non-AD but absent in AD patient samples. We compared our non-pathological CSF proteome data obtained by Orbitrap Astral with three previously published datasets. The combined CSF proteome was 5,861 unique proteins. Our study identified 558 proteins that were not found in any of the other studies. CONCLUSIONS: New mass spectrometers are significantly more sensitive and can be used to extend the proteome of biological fluids and tissues in the quest for new disease biomarkers.