B-351 Analytical Assessment of Fibronectin and Collagen I Fragment Biomarkers in Hypermobile Ehlers–Danlos Syndrome Using Immunoenrichment and LC–MS/MS

Abstract

Hypermobile Ehlers–Danlos syndrome (hEDS) and hypermobility spectrum disorders (HSD) lack objective laboratory biomarkers, contributing to prolonged diagnostic delays and reliance on subjective clinical criteria. Ritelli et al. recently reported disease-associated ∼45–52 kDa fibronectin (FN) and type I collagen (COL1) plasma fragments detected by immunoprecipitation (IP) and Western blotting, suggesting a potential protein-based diagnostic marker. However, Western blotting is a laborious, low throughput technique not ideally suited for routine clinical analysis. Alternatively, liquid chromatography-mass spectrometry has the potential to enable translation of this observation into a scalable, quantitative routine clinical assay. Serum samples from individuals with hEDS/HSD and controls were analyzed using IP with the same commercial anti-FN and anti-COL1 antibodies reported by Ritelli et al., covalently coupled to magnetic beads. Two complementary LC–MS/MS workflows were evaluated: (1) direct in-solution trypsin digestion of immunoprecipitated proteins followed by discovery proteomics, and (2) SDS-PAGE separation of IP-enriched material, excision of the ∼45–55 kDa gel region, in-gel trypsin digestion, and LC–MS/MS analysis. Peptide identification was performed using high-resolution LC–MS/MS with database searching at 1% false discovery rate. Candidate disease-associated peptides identified by discovery software were subjected to stringent manual validation, including extracted ion chromatogram inspection, signal-to-noise assessment, isotopic pattern evaluation, and reproducibility across biological replicates. Across both analytical workflows, fibronectin- and collagen I–derived peptides were reproducibly detected following immunoenrichment, with many peptides exhibiting well-defined chromatographic peaks, appropriate m/z, isotopic distributions, and consistent retention times. Database searching resulted in identification of multiple candidate peptides with robust signal intensity and apparent statistically significant differences between hEDS/HSD and control samples, suggesting potential disease association at the initial screening level. However, subsequent manual review of extracted ion chromatograms in TraceFinder with re-integration based on peak area demonstrated that the majority of these discovery-stage candidates exhibited comparable signal intensity in both cases and controls, indicating detection of shared protein regions rather than disease-specific fragments. These apparent group differences were largely attributable to limitations in database searching or low-level noise influencing discovery statistics rather than true biological separation. Remaining candidates failed analytical validation due to low signal strength, poorly defined chromatographic peaks, inconsistent detection across samples, or mismatch with expected m/z and isotopic profiles. Analysis of peptides derived from the excised ∼45–55 kDa gel region yielded similar findings, with either uniformly detected peptides across groups or signals insufficient for reliable quantification. No peptides demonstrated case-restricted presence, reproducible enrichment, or fragment-specific mapping consistent with a disease-associated fibronectin or collagen I fragmentation signature. Consequently, no candidates met predefined analytical and biological criteria required for advancement to targeted assay development. Using antibody reagents and enrichment strategies aligned with prior Western blot–based reports, LC–MS/MS analysis did not identify reproducible fibronectin or collagen I peptide signatures specific to hEDS/HSD. These findings highlight challenges in translating gel-based fragment observations into peptide-level mass spectrometry assays and underscore the importance of rigorous analytical validation. Further work is needed to clarify whether the reported fragments represent low-abundance, heterogeneous proteoforms, pre-analytical artifacts, or disease features not readily captured by bottom-up proteomics.