Wound exudate degradomics in normal and impaired healing: Exploring the proteomic and proteolytic landscape of human tissue repair in acute and chronic wounds through the lens of wound exudates

Wound healing in higher organisms comprises a plethora of biological processes, protein interaction networks and signaling pathways, which exhibit tightly coordinated activities to achieve tissue repair in a timely and orderly manner. Immediately upon injury and tissue damage, the hemostasis phase generates a fibrin clot to prevent hemorrhage and restore the physical barrier between the environment and the organism. In the inflammatory stage, immune cells migrate to the wound, fighting against pathogens and clearing damaged cells. In the proliferative stage, fibroblasts and other cell types migrate to the wound site, proliferating and producing extracellular matrix proteins. Finally, in the remodeling stage, the extracellular matrix proteins are organized, and tissue scarring is formed. Although much is known about the process of acute wound healing, the extremely convoluted landscape of involved proteases and signaling mechanisms has not been fully elucidated. Therefore, it is important to investigate such complex systems with state-of-the-art methods and instrumentation, enabling the concomitant measurement of biomolecules at a system-wide scale, and the analysis of such readouts from a systems biology perspective.

Chronic or impaired wounds are wounds that are unable to heal in the expected timeline and manner. Impaired wound healing constitutes a huge societal and economic burden to healthcare systems worldwide. Patients with chronic wounds not only need continuous treatment, but also have mortality and morbidity rates often comparable to cancer. Notably, individuals suffering from impaired wounds are typically elderly, diabetic, or patient groups with other co-morbidities, populations that are expected to increase in size in the coming decades. It is therefore of great interest to discover biomarkers for chronic wound evaluation and healing trajectory, as well as mechanisms that could be therapeutically targeted in impaired wound healing.

In this PhD project, the wound healing proteome and degradome (the protein products of proteolysis, which either stem from protein degradation or activation of latent proteoforms through proteolytic processing) were interrogated with MS based proteomics workflows, using wound exudates of human impaired or acute healing patients. The samples used were exudates extracted from wound dressings applied to wound healing patients over the course of their treatment regimen. They originated from a double-blinded clinical study with two groups, chronic wound patients with venous leg ulcers, and normal healing patients undergoing split-thickness skin grafting. Overall, the main objectives of the project were three-fold. The first objective was to develop an efficient and robust protein extraction and sample preparation workflow, as well as analytical methods for the investigation of dressing-derived wound fluids with MS-based proteomics. The second objective was to employ such methods in the quantification of known, relevant biomarkers for the wound healing process from the samples of the clinical study. The final objective was to dissect the wound healing protease network and its substrates, as well as signaling networks and other processes involved in wound healing using system-wide unbiased proteomics methodologies.

During the first work package of the project, a protocol for the efficient and robust extraction of the proteome fraction contained in the wound exudates was developed. The novel methodology was optimized and streamlined for use in large sample studies. In addition, a new analytical workflow was established for the highly sensitive and accurate quantification of protein biomarkers by targeted proteomics, combining elements from recent advances in mass spectrometry instrumentation and sample preparation. The second objective was achieved with the methods developed in the initial work package, detecting and quantifying proteins in wound exudates, spanning six orders of magnitude in concentration. Fibronectin, collagen I, S100A8/S100A9, MMP-2, MMP-9, neutrophil elastase, as wells as TNFα and IL-1β were quantified in 365 wound exudate samples from acute and impaired healers, with the last two proteins to our knowledge quantified for the first time by targeted proteomics in complex body fluids and without prior enrichment. The third goal was attained by employing a TMT-TAILS methodology for the investigation of the proteome and N-terminome of samples at a global scale, optimizing the sample preparation workflow for plasma-like protein dynamic range and off-line fractionation for deeper coverage. The results of this study provided a comprehensive longitudinal dataset for acute wound healing, obtaining a map of the wound healing protease network with temporal resolution at protein as well as enzyme activation levels, and discovering several putative biomarkers for wound healing progression.