In Vivo Assessment of Protease Dynamics in Cutaneous Wound Healing by Degradomics Analysis of Porcine Wound Exudates

As optimal biospecimens, exudates contain an ideal proteome to extracellular proteolytic events, are non-invasively 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 have deposited the ProteomeXchange Consortium with identifier PXD001198. Selected reaction monitoring (SRM) experiments. A spectral library was built based on the Mascot search results describe above using the BiblioSpec implementation contained in Skyline (47). For each spectrum, the five most intense y- ions were selected as target transitions for SRM measurements. Declustering potential (DP) and collision energy (CE) were predicted according to the mass-to-charge ratio of the precursor. SRM measurements were performed on a hybrid triple quadrupole/linear ion trap mass spectrometer (QTRAP 5500, AB Sciex, Concord, Canada), operated in-line with a nano liquid chromatography system (Eksigent nanoLC-Ultra 1D plus, AB Sciex, Zug, Switzerland). In brief, peptides were loaded onto in-house made fused silica columns (75 µ m ID x 100 mm) packed with Magic C18 AQ 200 Å, 3 µ m particle size reverse-phase material The column was maintained at 50 °C using a butterfly heater Wil Wil, Switzerland). Bound peptides were eluted by applying a linear gradient from 3% to 40% acetonitrile over 30 min at a flow rate of 300 nl/min and electrosprayed into the mass spectrometer via a 10 µ m ID spray tip (PicoTip emitter, New Objective, MS Wil GmbH, Switzerland). During data acquisition the MS was operated in unscheduled SRM mode at a dwell time of 40 ms per transition, resulting in a total cycle time of 3.2 s. Data analysis was performed using Skyline (version 2.6) (47). iRT standard peptides (Biognosys, Zurich, Switzerland) (48) were mixed with the samples according to manufacturer’s instructions and likewise recorded to control LC and MS performance.