Analysis of Biomarkers in Diabetic Foot Ulcer Patients With Dampness-Heat Syndrome Based on 4D-DIA Proteomics Technology.

A total of 201 differentially expressed proteins (DEPs) were identified between DFU-DHS patients and healthy controls using 4D-DIA proteomics.

• The study included 16 DFU-DHS patients and six healthy controls (HCs) for the proteomics discovery phase.
• DEPs were screened using criteria of |fold change (FC)| > 1.2 and p < 0.05.
• Serum samples were analyzed using 4D-data-independent acquisition (DIA) proteomics technology.

Bioinformatics analyses revealed that DEPs were enriched in lipid metabolism pathways and complement-coagulation cascades.

• Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and protein-protein interaction (PPI) analyses were conducted on the 201 DEPs.
• Specific lipid metabolism pathways enriched included high-density lipoprotein (HDL) remodeling and cholesterol metabolism.
• Complement-coagulation cascades were also identified as a significant enriched pathway among the DEPs.

PPI network analysis revealed a core functional module centered on four proteins: APOA1, LCAT, PLTP, and CETP.

• The four proteins identified — APOA1, LCAT, PLTP, and CETP — are classified as apolipoproteins involved in cholesterol metabolism.
• These four proteins were selected as candidate key biomarkers for further validation.
• The core module was identified through protein-protein interaction (PPI) network analysis of the 201 DEPs.

ELISA validation confirmed significant dysregulation of APOA1, LCAT, PLTP, and CETP in an independent DFU-DHS cohort.

• Validation was performed in 28 independent DFU-DHS cases using enzyme-linked immunosorbent assay (ELISA).
• All four biomarkers showed statistically significant dysregulation compared to HCs (all p < 0.05 vs. HCs).
• This validation cohort was independent from the 16 patients used in the proteomics discovery phase.

The combination of APOA1, LCAT, PLTP, and CETP exhibited high diagnostic efficacy for DFU-DHS with an AUC of 0.9672.

• Diagnostic performance was assessed using receiver operating characteristic (ROC) analysis.
• The combined panel of four biomarkers yielded an area under the curve (AUC) of 0.9672.
• This is described as the first study to employ 4D-DIA proteomics specifically on DFU-DHS patients.