Noninvasive Wound Swab Proteomics Enables Prognostic Stratification of Diabetic Foot Ulcer Healing: The HEAL-DFU Proof-of-Concept Study

Objective: Diabetic foot ulcers (DFUs) affect up to 34% of people with diabetes and precede 80% of lower extremity amputations, yet no ulcer-level molecular biomarkers distinguish healing trajectories. We aimed to identify wound swab proteins associated with DFU healing time and characterize their prognostic potential. Research Design and Methods: LC-MS/MS proteomics was performed on wound swabs from 112 people with diabetes presenting with new DFUs, followed for up to 6 months. Participants were divided into fast-healers (<40), slow-healers (41-180 days) and chronic wounds (>180 days). Proteins associated with ''time to healing'' were identified using Firth-penalized Cox regression (FDR < 0.2; HR<1 indicated slow healing) and used to construct a cross-validated Protein Healing Score (PHS). Sensitivity and causal mediation analyses examined clinical variable effects on protein abundance, with independent dataset validation. Results: Participants had a mean ({+/-}SD) age of 67.5 ({+/-}12.3) years, and diabetes duration of 27.5 ({+/-}14.9) years. Twenty-seven proteins formed two clusters: a slow-healing cluster enriched in neutrophil granule and innate immune proteins (HR<1), and a fast-healing cluster enriched in proteostatic stress proteins (HR>1). Triglyceride levels differed between healing groups (p=0.001); seven slow-healing proteins showed triglyceride-mediated effects (21-28%), suggesting a dyslipidaemia-innate immunity axis. The PHS significantly stratified prognosis (adjusted HR=1.47, 95%CI 1.14- 1.89; p=0.003), outperforming clinical variables alone (AUC-OVR 0.71 vs. 0.62). Transcriptomic validation confirmed directional concordance. Conclusions: Wound swab proteomics at DFU onset identified a 27-protein healing signature linked to neutrophil-innate immunity and triglyceride associated dyslipidaemia, offering a potential non-invasive prognostic tool.