Abstract
Proteases control complex tissue responses by modulating inflammation, cell proliferation and migration, and matrix remodeling. All these processes are orchestrated in cutaneous wound healing to restore the skin's barrier function upon injury. Altered protease activity has been implicated in the pathogenesis of healing impairments, and proteases are important targets in diagnosis and therapy of this pathology. Global assessment of proteolysis at critical turning points after injury will define crucial events in acute healing that might be disturbed in healing disorders. As optimal biospecimens, wound exudates contain an ideal proteome to detect extracellular proteolytic events, are noninvasively accessible, and can be collected at multiple time points along the healing process from the same wound in the clinics. In this study, we applied multiplexed Terminal Amine Isotopic Labeling of Substrates (TAILS) to globally assess proteolysis in early phases of cutaneous wound healing. By quantitative analysis of proteins and protein N termini in wound fluids from a clinically relevant pig wound model, we identified more than 650 proteins and discerned major healing phases through distinctive abundance clustering of markers of inflammation, granulation tissue formation, and re-epithelialization. TAILS revealed a high degree of proteolysis at all time points after injury by detecting almost 1300 N-terminal peptides in ∼450 proteins. Quantitative positional proteomics mapped pivotal interdependent processing events in the blood coagulation and complement cascades, temporally discerned clotting and fibrinolysis during the healing process, and detected processing of complement C3 at distinct time points after wounding and by different proteases. Exploiting data on primary cleavage specificities, we related candidate proteases to cleavage events and revealed processing of the integrin adapter protein kindlin-3 by caspase-3, generating new hypotheses for protease-substrate relations in the healing skin wound in vivo. The data have been deposited to the ProteomeXchange Consortium with identifier PXD001198.
Highlights
From the ‡ETH Zurich, Department of Biology, Institute of Molecular Health Sciences, Otto-Stern-Weg 7, 8093 Zurich, Switzerland; §Paul Hartmann AG, Paul Hartmann Strasse 12, 89522 Heidenheim, Germany; ¶University of British Columbia, Department of Pathology and Laboratory Medicine and Department of Chemistry, Centre for Blood Research, 4.401 Life Sciences Institute, 2350 Health Sciences Mall, Vancouver, British Columbia, Canada V6T 1Z3
To facilitate statistically robust analyses, we extracted wound fluids from foams derived from five different pigs per time point and determined their proteomes and N-terminomes in five separate 4plexiTRAQ-Terminal Amine Isotopic Labeling of Substrates (TAILS) experiments, applying a labeling scheme that included a pooled reference sample for interexperimental normalization (Fig. 1C)
Using a clinically relevant pig wound healing model and our highly sensitive multiplex isobaric tags for relative and absolute quantitation (iTRAQ)-TAILS approach, we identified more than 650 proteins including ϳ50 proteases and spanning a concentration range of more than six orders of magnitude
Summary
Pig Wound Healing Model—15 female pigs (Sus scrofa, Seghers Hybrid) with a weight of 87.0 kg Ϯ 3.86 kg were acclimatized and prepared for surgery by intramuscular injection of atropine and tiletamine-zolazepam and intravenous application of thiopental-pentobarbital. Solid particles were removed by centrifugation (10 min, 13,000 ϫ g, 4 °C), the protein concentration determined by the Bradford assay (BioRad, Hercules, CA) and samples either directly further processed or stored at Ϫ80 °C. 4plex-iTRAQ-TAILS Procedure—TAILS is a multiplex quantitative proteomics platform for the determination of N-terminomes and the system-wide identification of protease substrates and cleavage sites in complex biological samples [26]. In this method, all natural and cleaved protein N termini and lysines in a proteome are blocked by differential isotopic labeling.
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