Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063239/checksum.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063239/MP_190924_preomic6neat.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063239/MP_190924_native3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063239/MP_190924_proteominer_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063239/MP_190924_preomic5neat.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063239/MP_190924_proteominer_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063239/MP_190924_native4.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063239/MP_190924_proteominer_2.mzidftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063239/MP_190924_preomic5neat.mzidftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063239/MP_190924_proteominer_1.mzidftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063239/MP_190924_preomic6neat.mzidftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063239/MP_190924_native3.mzidftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063239/MP_190924_native4.mzidftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063239/MP_190924_preomic5neat.mgfftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063239/MP_190924_native4.mgfftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063239/MP_190924_proteominer_1.mgfftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063239/MP_190924_preomic6neat.mgfftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063239/MP_190924_native3.mgfftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD063239/MP_190924_proteominer_2.mgfprimaryOK200peffs@liverpool.ac.ukFederico FotiData-dependent acquisitionMass SpectrometryBottom-up proteomicsCerebrospinal fluid (csf)proteomicspreomics enrich-ist™Cerebrospinal fluid (csf); proteomics; native digestion; proteominer™ small-capacity enrichment kit; preomics enrich-ist™; liquid chromatography with tandem mass spectrometry (lc-ms/ms)proteominer™ small-capacity enrichment kitliquid chromatography with tandem mass spectrometry (lc-ms/ms)native digestionhttps://www.ebi.ac.uk/pride/archive/projects/PXD063239Cerebrospinal FluidCerebrospinal fluid (CSF) was collected post-mortem from a 13-year-old mare euthanised for non-neurological reasons. Collection was performed via atlanto-occipital centesis, followed by centrifugation to remove cells and debris. The CSF was aliquoted and stored at -80°C until further use. Prior to processing, samples were thawed and centrifuged at 13,000 × g for 10 minutes to remove particulates. All analyses were conducted in technical duplicate. Protein Quantification and Sample Preparation Total protein concentration was determined using the ThermoScientific Pierce™ BCA Protein Assay Kit (Cat. No. 23225). Three sample preparation strategies were employed: 1. Native Digestion: 100 µg of protein was subjected to in-solution tryptic digestion following established protocols. This approach served as the control, with no enrichment or depletion steps prior to digestion. 2. ProteoMiner™ Small-Capacity Enrichment Kit (Bio-Rad®, Cat. No. 163-3006): 1 mg of protein was enriched using the manufacturer's protocol, adapted for equine CSF. The kit uses a combinatorial hexapeptide ligand library immobilised on chromatographic beads to compress the protein dynamic range. Abundant proteins saturate their ligands, allowing excess to be washed away and low-abundance proteins to be proportionally enriched. On-bead digestion was performed using the same method as native digestion. 3. PreOmics® ENRICH-iST Kit (Cat. No. P.O.00163): 20 µg of protein was processed following the manufacturer’s instructions. This kit uses paramagnetic beads functionalised with charge-based and hydrophobic chemistries to bind peptides post-digestion, thereby depleting high-abundance proteins and contaminants while enriching low-abundance peptides for downstream LC-MS/MS analysis. Mass Spectrometry LC-MS/MS was performed using a ThermoScientific Q-Exactive HF-X Hybrid Quadrupole-Orbitrap mass spectrometer. Data-dependent acquisition (DDA) involved a full MS scan at 60,000 resolution (AGC: 3e6 ions; max fill time: 100 ms). Peptides were separated over a 1-hour gradient with 30-minute blanks between runs to avoid carryover. Sample runs were performed as previously described (DOI: 10.1074/jbc.M114.566554) . Data Processing and Analysis MS/MS spectra were searched against the UniHorse protein database using a local MASCOT server (Matrix Science, London, UK). Search parameters included: • Peptide mass tolerance: 10.0 ppm • Fragment mass tolerance: 0.01 Da • Enzyme: trypsin • One missed cleavage allowed • Fixed modification: carbamidomethylation (C) • Variable modification: oxidation (M) Protein abundance was estimated using the exponentially modified Protein Abundance Index (emPAI). emPAI values were averaged across duplicates, and enrichment or depletion was assessed based on fold-change relative to native digestion. Functional Classification and Pathway Analysis Identified proteins were analysed using the PANTHER Classification System (version 17.0), applying protein accession numbers to retrieve Gene Ontology (GO) terms and pathway annotations. Pathway enrichment was assessed using the PANTHER Overrepresentation Test with FDR correction. Graphical outputs were generated in R Studio (v4.1.2) using ggplot2 (v3.3.5).PrideRaw LC-MS/MS data were processed using an in-house Mascot server (Matrix Science, London, UK). Spectral searches were conducted against the UniHorse database using the following parameters: peptide mass tolerance of 10.0 ppm, fragment mass tolerance of 0.01 Da, enzyme specificity set to trypsin with one missed cleavage allowed, fixed modification of carbamidomethylation (C), and variable modification of oxidation (M). Quantitative analysis was performed using the exponentially modified Protein Abundance Index (emPAI), which provided semi-quantitative estimates of protein abundance. emPAI scores were averaged across technical duplicates for each condition and used to assess the degree of enrichment or depletion by calculating percentage changes relative to native digestion. Proteins were annotated and functionally classified using the PANTHER Classification System (v17.0), and pathway enrichment analysis was performed using the PANTHER Overrepresentation Test (release 2024-01-01), with statistical significance based on the false discovery rate (FDR) threshold of <0.05. The GO Biological Process ontology was selected to evaluate functional enrichment. All statistical analyses and data visualisations were carried out in R Studio (v4.1.2) using the ggplot2 package (v3.3.5).ProteomicsMandy PeffersCOMPLETEHead of Department Musculoskeletal Ageing Science |Institute of Life Course and Medical Sciences | Faculty of Health & Life Sciences |University of Liverpool | United KingdomEquus Caballus (horse)41961066 Federico F, Amie W, Alzbeta C, Anders J, David LP, Mandy PJ. Evaluating Protein Enrichment Methods to Improve Biomarker Discovery in Equine Cerebrospinal Fluid. Vet Med Sci. 2026 12(3):e70933 10.1002/vms3.70933federico.foti@liverpool.ac.ukUniversity of LiverpoolUnited Kingdom10.6019/PXD063239<h4>Background</h4>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.<h4>Objectives</h4>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.<h4>Methods</h4>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.<h4>Results</h4>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.<h4>Conclusions</h4>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.Evaluating Protein Enrichment Methods to Improve Biomarker Discovery in Equine Cerebrospinal Fluid.Federico Foti F, Amie Wilson W, Alzbeta Chabronova C, Anders Jensen J, David Lunn Paul LP, Mandy Peffers Jayne PJfalseEvaluating Protein Enrichment Methods to Improve Biomarker Discovery in Equine Neurological DisordersThis study evaluated and compared three sample preparation strategies for proteomic analysis of equine cerebrospinal fluid (CSF): native in-solution digestion, the ProteoMiner™ Small-Capacity Enrichment Kit (Bio-Rad®), and the PreOmics Enrich-iST™ Kit (PreOmics®). CSF is in direct contact with the central nervous system (CNS), and its protein content reflects both physiological and pathological states, making it an ideal fluid for biomarker discovery in neurological diseases. However, its analysis is hindered by the wide dynamic range of protein concentrations and the predominance of highly abundant proteins (HAPs), such as albumin, which can obscure the detection of diagnostically relevant low-abundance proteins (LAPs). To address these limitations, enrichment kits such as ProteoMiner and PreOmics have been employed to reduce HAPs and enhance LAP detection prior to mass spectrometry. While ProteoMiner uses a combinatorial hexapeptide ligand library to compress the dynamic range by saturating binding sites of abundant proteins, PreOmics relies on paramagnetic beads with hydrophobic and charge-based retention to selectively bind peptides and improve sample purity. This is the first study to apply the PreOmics Enrich-iST kit to equine CSF. Label-free LC-MS/MS was used to analyse the samples. A total of 849 unique proteins were identified across all methods, with the PreOmics kit identifying the highest number of proteins and achieving the most consistent depletion of HAPs. emPAI scores were used to quantify protein abundance, revealing that albumin levels decreased by over 60% with PreOmics but slightly increased with ProteoMiner. Pathway enrichment analysis using PANTHER showed that the PreOmics kit enabled detection of a broader and more statistically significant array of biological pathways, while ProteoMiner selectively enriched for HAPs with known roles in neurodegenerative disorders, such as Dickkopf WNT signalling pathway inhibitor 3 (DKK3) and clusterin (CLU). These findings highlight that although both kits are capable of enriching LAPs, they differ in their enrichment patterns and efficiency. PreOmics provides better overall proteome coverage and greater statistical power in pathway analysis, whereas ProteoMiner may be more effective for targeted studies of specific neuropathology-related proteins. This study provides valuable insights into the applicability and complementarity of these enrichment methods for biomarker discovery in equine neurological disease.2026-04-132025-04-23PXD063239NEWT:3555NEWT:71647NEWT:330879NEWT:35554NCBITaxon:12939NEWT:32046NEWT:544496NEWT:59529NEWT:2042546NEWT:32049NEWT:1392696NEWT:259447NEWT:295546NCBITaxon:2719036NEWT:45351NEWT:445974NEWT:43179NEWT:180454NEWT:5722NCBITaxon:1280NEWT:112503NEWT:1129NEWT:309807NEWT:10245NEWT:55153NEWT:309800NEWT:281395NEWT:1211601NEWT:876138NEWT:44271NEWT:193516NEWT:351581NEWT:43186NEWT:428146NEWT:498257NEWT:10036NEWT:1590NEWT:498019NEWT:1351NEWT:1438992NEWT:1352NEWT:2649997NEWT:1147161NEWT:661410NEWT:638632NEWT:263737NEWT:224326NEWT:1333499NEWT:376619NCBITaxon:79857NEWT:1096976NEWT:5702NEWT:95648NEWT:1589NEWT:135622NEWT:1349NEWT:35786NEWT:1580NEWT:399784NEWT:96731NEWT:383379NEWT:1401257NEWT:418106NEWT:10029NEWT:913645NEWT:44491NEWT:641809NEWT:27933NEWT:556484NEWT:317447NEWT:4688NEWT:7955NEWT:7719NEWT:7959NEWT:2261NEWT:868565NEWT:31156NEWT:3112NEWT:79329NEWT:4442NEWT:192875NEWT:1214915NEWT:12637NEWT:59729NEWT:2164133NEWT:284218NEWT:295105NEWT:108061NEWT:60711NEWT:1316931NEWT:224308NEWT:3347NEWT:511145NEWT:160621NEWT:212790NEWT:931281NEWT:4432NEWT:5762NEWT:5763NEWT:658457NEWT:1310161NEWT:77133NEWT:295358NEWT:145481NCBITaxon:79824NEWT:2246NEWT:2652724NEWT:5757NEWT:68415NCBITaxon:4563NEWT:5755NEWT:44689NEWT:44447NEWT:752555NEWT:498211NEWT:5759NEWT:347256NEWT:1736231NEWT:5518NEWT:398007NEWT:1527468NEWT:1392NEWT:498217NEWT:498216NEWT:2242NEWT:11320NEWT:286NEWT:391619NEWT:246196NEWT:287NEWT:246197NEWT:145458NEWT:633149NEWT:1149786NEWT:10239NEWT:68895NEWT:44685NEWT:161934NEWT:4897NEWT:1148NEWT:5744NEWT:72901NEWT:213863NEWT:5508NEWT:3329NEWT:5507NEWT:410661NEWT:55571NEWT:35500NEWT:1235816NEWT:1140NCBITaxon:2157NEWT:1143NEWT:4896NEWT:1287689NEWT:1390NEWT:11557NEWT:1094343NEWT:1462472NEWT:1336795NEWT:644042NEWT:294NEWT:1773NEWT:1182590NEWT:3712NEWT:82380NEWT:3711NEWT:935293NEWT:375146NEWT:270643NEWT:2065263NEWT:177437NEWT:1772NEWT:10418NEWT:118698NEWT:1616117NEWT:263NEWT:118696NEWT:34865NEWT:52283NEWT:11988NEWT:284812NEWT:8175NEWT:400667NEWT:43330NEWT:1603293NEWT:408169NEWT:44664NEWT:47946NEWT:459758NEWT:3702NEWT:1245466NEWT:243277NEWT:1246791NEWT:7067NEWT:118694NEWT:586722NEWT:2850NEWT:118691NEWT:34871NEWT:33548NEWT:274NEWT:7070NCBITaxon:287NEWT:408172NEWT:408170NEWT:96794NEWT:3708NEWT:332648NEWT:44670NEWT:536231NEWT:376219NEWT:219813NEWT:1510NEWT:1436733NEWT:460519NEWT:1247411NEWT:1515NEWT:572307NEWT:1432138NEWT:1424507NEWT:1194599NEWT:272844NEWT:9483NEWT:333760NEWT:56636NEWT:1513458NEWT:1233681NCBITaxon:40559NEWT:506599NEWT:2853422NEWT:81077NEWT:84588NEWT:1679718NEWT:480NEWT:65349NEWT:67767NEWT:46835NEWT:109757NEWT:582580NEWT:300852NEWT:1502NEWT:128017NEWT:376686NEWT:95486NEWT:508771NEWT:1883446NEWT:253NCBITaxon:2759NEWT:1233435NEWT:93061NEWT:93062NEWT:34613NCBITaxon:4932NEWT:109760NEWT:943274NEWT:644223NEWT:235443NEWT:108458NEWT:5936NEWT:272623NEWT:272624NEWT:320637NEWT:886559NEWT:3750NEWT:55521NEWT:983964NEWT:118499NEWT:11706NEWT:32644NEWT:527796NEWT:1130798NEWT:55529NEWT:499175NEWT:109779NEWT:3745NEWT:1715989NCBITaxon:4751NEWT:476272NEWT:3747NEWT:195051NEWT:3988NEWT:1116234NEWT:561007NEWT:1255228NEWT:649908NEWT:410289NEWT:373153NEWT:3983NEWT:203696NEWT:352472NEWT:1071661NEWT:1202532NEWT:360094NEWT:470NEWT:41364NEWT:5911NEWT:1313NEWT:1218097NEWT:1080348NCBITaxon:50557NEWT:39251NEWT:39491NEWT:212142NCBITaxon:5811NEWT:29491NEWT:101841NEWT:153481NEWT:2014887NEWT:33952NEWT:153009NEWT:261756NEWT:571256NEWT:40483NEWT:63366NEWT:63367NEWT:688333NEWT:215402NCBITaxon:548681NEWT:318586NCBITaxon:5820NEWT:1547NEWT:272634NEWT:198107NEWT:9031NEWT:27292NEWT:1872122NEWT:1308NEWT:7091NEWT:1034015NEWT:760568NEWT:141262NEWT:108931NEWT:1293497NEWT:1055524NEWT:4087NEWT:9778NEWT:306NEWT:150475NEWT:303NEWT:267872NEWT:97477NEWT:7111NEWT:347515NEWT:172269NEWT:9534NEWT:6269NEWT:5180NEWT:256737NEWT:4090NEWT:4092NEWT:9541NEWT:8694NEWT:4093NEWT:33936NEWT:4096NEWT:8692NEWT:2903NEWT:185579NEWT:941442NEWT:536088NEWT:13076NEWT:1226408NEWT:7108NEWT:40479NEWT:43989NEWT:4076NEWT:142615NEWT:317NEWT:1006581NEWT:33940NEWT:885318NEWT:39488NEWT:550NEWT:4081NEWT:273068NEWT:554NEWT:98334NEWT:451516NEWT:552NEWT:4084NEWT:1325291NEWT:63577NEWT:36185NEWT:1225786NEWT:7574NEWT:1715256NEWT:30640NEWT:575412NEWT:929793NEWT:1507179NEWT:29204NEWT:28112NEWT:114155NEWT:6494NEWT:6491NEWT:507601NEWT:520NEWT:186441NEWT:634955NEWT:643680NEWT:214092NCBITaxon:6157NEWT:6239NEWT:13095NEWT:162425NEWT:216257NEWT:102169NEWT:208893NEWT:9986NEWT:4054NEWT:8654NEWT:8658NEWT:1268063NEWT:8657NEWT:8655NEWT:5147NEWT:98351NEWT:1263854NEWT:118503NEWT:2059687NEWT:160488NEWT:28104NEWT:207559NEWT:407821NCBITaxon:2NEWT:85057NEWT:985076NEWT:568708NEWT:1215323NEWT:986NEWT:52641NEWT:7159NEWT:267671NEWT:28532NEWT:353152NEWT:8496NEWT:7165NEWT:1257118NEWT:27448NEWT:40674NEWT:1194669NEWT:51329NEWT:243230NEWT:443906NEWT:1080772NEWT:519NEWT:8479NEWT:8485NEWT:6063NEWT:49240NEWT:1328388NEWT:104395NEWT:667127NEWT:9557NEWT:9315NEWT:6289NEWT:377586NEWT:6287NEWT:38569NEWT:300641NEWT:727NEWT:9796NEWT:9554NEWT:118562NEWT:725NEWT:7137NEWT:469008NEWT:256318NEWT:9321NEWT:206411NCBITaxon:6191NEWT:59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