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Veterinary medicine and science2026; 12(3); e70933; doi: 10.1002/vms3.70933

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

Abstract: 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. Objective: 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. Methods: 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. Results: 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. Conclusions: 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.
© 2026 The Author(s). Veterinary Medicine and Science published by John Wiley & Sons Ltd.
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Publication Date: 2026-04-10 PubMed ID: 41961066PubMed Central: PMC13067976DOI: 10.1002/vms3.70933Google Scholar: Lookup
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Summary

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Overview

  • This study compares two protein enrichment methods to improve the detection of low-abundance proteins in equine cerebrospinal fluid (CSF), aiming to enhance biomarker discovery for neurological diseases in horses.
  • The researchers evaluated the ProteoMiner Small-Capacity Enrichment Kit and the PreOmics Enrich-iST Kit for their effectiveness in depleting high-abundance proteins and enriching low-abundance proteins relevant to neuropathology.

Background and Objective

  • Cerebrospinal fluid (CSF) is a critical biological fluid used in studying neurological diseases because it reflects changes in the central nervous system.
  • One major challenge in CSF proteomics is that highly abundant proteins, such as albumin, often mask the detection of low-abundance proteins that could be vital biomarkers.
  • Protein enrichment strategies help overcome this by selectively depleting high-abundance proteins to improve the identification of rarer proteins.
  • Despite the importance, there is a lack of comparative studies assessing these enrichment methods specifically in equine CSF.
  • The objective of the study was to compare the ProteoMiner Small-Capacity Enrichment Kit and PreOmics Enrich-iST Kit in terms of:
    • Ability to deplete high-abundance proteins
    • Capacity to enhance detection of low-abundance, neuropathology-relevant proteins
    • Overall proteomic coverage and biological pathway enrichment

Methods

  • Equine CSF samples were divided into three processing groups:
    • Native in-solution trypsin digestion without enrichment (control)
    • ProteoMiner Small-Capacity Enrichment Kit treatment
    • PreOmics Enrich-iST Kit treatment
  • Label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed to analyze peptide and protein profiles from each group.
  • Protein identification and relative quantification were performed using the emPAI (exponentially modified Protein Abundance Index) scores, which estimate protein abundance based on peptide detection.
  • Gene ontology (GO) pathway analyses were conducted to interpret the biological relevance of the identified proteins and assess enrichment efficiency.

Results

  • The PreOmics Enrich-iST Kit outperformed both the ProteoMiner Kit and the native digestion method by identifying the highest total number of proteins.
  • This kit was particularly effective in detecting low-abundance proteins relevant to neurobiology and neuropathology, which were not found using the other methods.
  • It achieved superior depletion of high-abundance proteins, reducing their masking effect and allowing better access to the low-abundance proteome.
  • Gene ontology analysis revealed that the PreOmics method enriched a broader set of neuropathology-related biological pathways compared to the other methods.
  • The ProteoMiner Kit showed some improvements over native digestion but was less effective than the PreOmics Kit in both protein identification and depletion capacity.

Conclusions and Implications

  • The study concluded that the PreOmics Enrich-iST Kit is a robust and superior tool for proteomic profiling of equine CSF.
  • Its enhanced performance in depleting high-abundance proteins and detecting low-abundance, neurobiology-relevant proteins makes it particularly valuable for biomarker discovery in equine neurological disorders.
  • Using such enrichment methods could facilitate the identification of novel diagnostic or therapeutic biomarkers, potentially advancing veterinary neurology research and clinical practice.
  • Overall, the findings support adopting advanced enrichment methodologies like PreOmics Enrich-iST in future equine CSF proteomics studies to gain comprehensive insights into neurological disease mechanisms.

Cite This Article

APA
Federico F, Amie W, Alzbeta C, Anders J, David LP, Mandy PJ. (2026). Evaluating Protein Enrichment Methods to Improve Biomarker Discovery in Equine Cerebrospinal Fluid. Vet Med Sci, 12(3), e70933. https://doi.org/10.1002/vms3.70933

Publication

Veterinary medicine and science
ISSN: 2053-1095
NlmUniqueID: 101678837
Country: England
Language: English
Volume: 12
Issue: 3
Pages: e70933
PII: e70933

Researcher Affiliations

Federico, Foti
  • Institute of Infection, Veterinary & Ecological Sciences, University of Liverpool, Neston, UK.
Amie, Wilson
  • Institute of Infection, Veterinary & Ecological Sciences, University of Liverpool, Neston, UK.
Alzbeta, Chabronova
  • Institute of Life Course and Medical Sciences, Faculty of Health & Life Sciences, University of Liverpool, Liverpool, UK.
Anders, Jensen
  • Institute of Life Course and Medical Sciences, Faculty of Health & Life Sciences, University of Liverpool, Liverpool, UK.
David, Lunn Paul
  • Institute of Infection, Veterinary & Ecological Sciences, University of Liverpool, Neston, UK.
Mandy, Peffers Jayne
  • Institute of Life Course and Medical Sciences, Faculty of Health & Life Sciences, University of Liverpool, Liverpool, UK.

MeSH Terms

  • Animals
  • Horses / cerebrospinal fluid
  • Biomarkers / cerebrospinal fluid
  • Proteomics / methods
  • Horse Diseases / cerebrospinal fluid
  • Chromatography, Liquid / veterinary
  • Tandem Mass Spectrometry / veterinary
  • Cerebrospinal Fluid Proteins
  • Nervous System Diseases / veterinary
  • Nervous System Diseases / cerebrospinal fluid
  • Nervous System Diseases / diagnosis

Grant Funding

  • vet/prj/811 / Horserace Betting Levy Board (HBLB)
  • Prj812 / Anders Jensen Horserace Betting Levy Board

Conflict of Interest Statement

The authors declare no conflicts of interest.

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