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.

Highlights

• Why did we undertake this study?

  No prior study has examined wound swab protein associations with DFU healing time as a continuous outcome, offering finer prognostic resolution than discrete healing categories.

• What is the specific question(s) we wanted to answer?

  Can wound swab proteins at DFU presentation predict healing trajectories, and do clinical factors mediate these associations?

• What did we find?

  Twenty-seven proteins formed two prognostic clusters: a slow-healing cluster enriched in neutrophil/innate immune proteins and a fast-healing cluster linked to proteostatic stress. A Protein Healing Score outperformed clinical variables in stratifying healing outcomes (AUC-OVR 0.71 vs. 0.62). Triglyceride-mediated and neuropathy-driven effects on wound proteins revealed mechanistic links to impaired healing.

• What are the implications of our findings?

  Wound swab proteomics offers a non-invasive prognostic tool at DFU onset, potentially enabling earlier targeted intervention.