Abstract by Jun Cai

Wound healing is a fundamental survival mechanism and failure may cause significant disease, such as seen in acute wound infections and non-healing ulcers. Besides a reduced life quality for patients, failed skin repair has resulted in a huge economic burden for society. Therefore, the development of improved methods to measure wound status, predict healing outcomes,
and monitor healing progression, is essential.

Proper wound repair is characterized by balanced proteolytic events, whereas in wound infection, aberrant protease activity may lead to deteriorated wound healing. The downstream products of the proteolytic processing of proteins are peptides, which are of great importance, since they act as signals of the levels of proteolytic actions by endogenous and bacterial proteases. Therefore, they could be potential biomarkers for early diagnosis and intervention, to optimize treatment of wounds.

The main goal of this thesis is to investigate the wound peptidome for the presence of potential peptide-based diagnostic biomarkers. Furthermore, the studies also help to extend our knowledge of normal wound healing processes and those during wound infection.

In the first study, we developed a robust mass spectrometry based peptidomics workflow for the characterization of the wound peptidome. Using this method, we found that sterile postsurgical wound fluids have a high degree of peptides compared to human plasma.
Furthermore, comparisons of the peptidome of uninfected healing wounds and Staphylococcus aureus infected wounds revealed unique peptide patterns, demonstrating the usefulness of the method for further qualitative studies of the wound peptidome.

As the observed differences may be explained by the levels and activities of individual proteases, which could degrade proteins into peptides with enzyme specific sequences, in our second study, we selected two endogenous protease (neutrophil elastase, cathepsin G) and two bacterial proteases (Pseudomonas aeruginosa elastase (LasB), and Staphylococcus aureus V8) and compared their individual effects on thecleave of proteinsin plasma and sterile wound fluids. As expected, we found a significant increase in the number of identified peptides and proteins after enzymaticdigestion, and show enzyme-specific effects on the peptidome, which can be explained by the observed substrate specificities. Further investigations of the peptide patterns revealed enzyme-specific unique peptides.

Taken together, the work in this thesis defines a workflow for the analysis of qualitative differences in peptidomes of plasma and wound fluids, due to the levels and activities of endogenous and bacterial proteases and provides proof for its usefulness in discovering potential diagnostic and prognostic biomarkers for wound healing and infection.