DIA-based serum proteomics of the complement, coagulation cascade identifies candidate biomarkers for myasthenia gravis

ObjectiveThis study aims to analyze serum protein changes in patients with myasthenia gravis (MG) via proteomics to identify and validate potential biomarkers.MethodsSerum samples were collected from 10 MG patients and 10 healthy controls. The data-independent acquisition (DIA) quantitative proteomics technique was used to measure serum protein levels and identify differentially expressed proteins. Functional enrichment analyses, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and gene set enrichment analysis (GSEA), were performed to determine the enrichment trends of the differentially expressed proteins. On the basis of the p values from the enrichment analysis, hierarchical clustering was used to identify significantly enriched pathways in each group. The STRING database was used to construct protein, protein interaction networks of the differentially expressed proteins, identifying the top 50 most closely interacting protein subnetworks and their associated pathways. The target proteins were then validated via mass spectrometry-based targeted proteomics (PRM). Receiver operating characteristic (ROC) curves were plotted for visual analysis. Additionally, serum samples from 20 MG patients and 20 healthy controls (HCs) were collected to validate the proteomic results by enzyme-linked immunosorbent assay (ELISA).ResultsA total of 220 proteins met the exploratory nominal differential-expression criteria between the MG and healthy control groups, including 49 upregulated and 171 downregulated proteins. After Benjamini, Hochberg FDR correction, 138 proteins remained statistically significant, including 23 upregulated and 115 downregulated proteins. Pathway analysis suggested enrichment of complement/coagulation cascades, tight junctions, regulation of the actin cytoskeleton, and Rap1 signaling. Five prioritized proteins, PROS1, PROC, C4BPA, PFN1, and TLN1, were further evaluated by PRM and ELISA. PRM supported their differential abundance and pathway-level association with complement/coagulation regulation and cytoskeleton-related processes, but did not demonstrate a causal damage cascade. ROC analysis showed AUC values greater than 0.8 in this exploratory cohort; however, these estimates should be interpreted cautiously because of the limited sample size. ELISA validation further supported increased PROS1, PROC, and C4BPA levels and decreased PFN1 and TLN1 levels in MG patients compared with controls.ConclusionPROS1, PROC, C4BPA, PFN1, and TLN1 represent exploratory candidate serum biomarkers for AChR-positive MG and require validation in larger, multicenter cohorts before clinical application.