Cerebrospinal fluid (CSF) has been proven a useful matrix for neurological diseases research, not only to identify biomarkers for diagnostic purposes, treatment prediction, or treatment response, but also to understand the molecular mechanisms involved in disease progression. However, CSF proteome profiling by mass spectrometry faces two main analytical challenges. Firstly, the broad dynamic range of all liquid biopsies, which hinders the sensitivity of protein measurements. Secondly, the need to analyze hundreds of human samples in a reproducible and timely manner. Indeed, there has been a trade-off among sensitivity, number of analytes, number of samples, sample amount, and data quality. Recently developed mass spectrometry instrumentation allows for the rapid acquisition of samples, aligning with the need of large patient cohorts essential for translational clinical projects. On the other hand, several commercial solutions have appeared to address the limitations associated with processing of liquid biopsies improving in-depth protein identification. One such solution is the ENRICH-iST kit by PreOmics, originally designed to diminish the dynamic range in plasma samples by enriching low-abundance proteins onto paramagnetic beads.

In this study, we adapted the ENRICH-iST kit protocol to process cerebrospinal fluid. We assessed several amounts of CSF starting material, and evaluated the reproducibility and the sensitivity of this enrichment strategy by means of the coefficient of variation and the number of identified proteins and peptides detected. A pool of healthy human CSF sample was processed in triplicates and different amounts with either the iST-BCT or ENRICH-iST PreOmics kits. Digested samples were analyzed by liquid chromatography coupled to mass spectrometry using data-independent acquisition and short gradients across three instruments: an Orbitrap Eclipse Tribrid, an Orbitrap Astral, and a timsTOF HF. Chromatography settings and data-acquisition schema were tailored for each platform.

Our findings highlight the suitability of the ENRICH-iST sample preparation kit for processing cerebrospinal fluid. The kit significantly increases both proteome coverage and depth in cerebrospinal fluid, while maintaining technical reproducibility and low coefficients of variation. Therefore, our work shows how using such sample preparation kits, like ENRICH-iST, ensures accurate and high-quality profiling of CSF samples. In summary, we show how new developments in sample preparation together with last generation mass spectrometry instrumentation allow for fast and sensitive analysis of CSF samples required for large-scope neurological disease studies.