Evaluating Protein Enrichment Methods to Improve Biomarker Discovery in Equine Cerebrospinal Fluid

Cerebrospinal fluid (CSF) is a valuable source of biomarkers for neurological diseases, but detection of low-abundance proteins is often masked by highly abundant proteins. Enrichment strategies can enhance proteomic coverage and improve biomarker discovery, yet comparative evaluations of such methods in equine CSF are limited.

This study compared the ProteoMiner Small-Capacity Enrichment Kit and the PreOmics Enrich-iST Kit for their ability to deplete high-abundance proteins and enhance detection of low-abundance proteins relevant to neuropathology.

Equine CSF samples were processed with either a native in-solution trypsin digestion without further enrichment, ProteoMiner Small-Capacity Enrichment Kit, or PreOmics enrichment. Samples were analysed by label-free liquid chromatography-tandem mass spectrometry. Proteins were identified and quantified using emPAI scores, and gene ontology pathway analyses were performed to evaluate enrichment efficiency and biological relevance.

The PreOmics Enrich-iST Kit identified the highest number of proteins overall, including neurobiology-relevant low-abundance proteins not detected by other methods, and achieved superior depletion of high-abundance proteins. Gene ontology pathway analysis revealed broader enrichment of neuropathology-relevant pathways.

The PreOmics Enrich-iST Kit outperformed the ProteoMiner Small-Capacity Enrichment Kit and native digestion in equine CSF proteomics, providing greater depletion of high-abundance proteins and enhanced detection of neurobiology-relevant low-abundance proteins. This method offers a robust tool for comprehensive proteomic profiling and may facilitate the discovery of novel biomarkers for equine neurological disorders.