A selected reaction monitoring-based analysis of acute phase proteins in interstitial fluids from experimental equine wounds healing by secondary intention.

The study investigating the presence of inflammatory proteins (acute phase proteins, or APPs) in horse wounds found higher concentrations in limb wounds compared to body wounds. The research used a novel technique, SRM-based mass spectrometry, aided by a quantification concatamer (QconCAT) to measure levels of APPs.

Research Objective and Methodology

• The aim of the study was to assess the levels of inflammatory APPs in the interstitial fluid (fluid that bathes and surrounds the tissue cells) drawn from wounds in horses. This was investigated through the use of a technique called selected reaction monitoring (SRM)-based mass spectrometry in conjunction with a quantification concatamer (QconCAT).
• The research focused on analyzing five established equine APPs (fibrinogen, serum amyloid A, ceruloplasmin, haptoglobin, and plasminogen) and three proposed equine APPs (prothrombin, α-2-macroglobulin, and α-1-antitrypsin).
• Large pore microdialysis, a technique for recovering interstitial fluid, was employed on experimental body and limb wounds from five horses at different stages of healing (days 1, 2, 7, and 14).
• The process of healing was compared between wounds healing normally (body) and those healing with formation of exuberant granulation tissue (EGT), a common issue in horse limb wounds often attributed to chronic inflammation.

Procedure and Results

• The QconCAT approach involved using proteotypic peptides representing each of the target proteins. This guided the design of a gene, expressed in Escherichia coli (a common bacteria) in a medium supplemented with stable isotopes. These isotopes helped in labelling standard peptides and facilitated accurate quantification.
• Quantification of the APPs was achieved by co-analysing the wound interstitial fluid samples with the stable isotope-labelled QconCAT tryptic peptides in known amounts.
• The investigation found significantly higher concentrations of fibrinogen, haptoglobin, ceruloplasmin, prothrombin, and α-1-antitrypsin in interstitial fluid from limb wounds compared to body wounds.

Significance of the Study

• This study is the first of its kind to simultaneously analyse a panel of APPs using the QconCAT-SRM technology.
• The findings suggest a sustained state of inflammation in horse limb wounds healing with EGT, potentially explaining the problematic overgrowth of tissue in these wounds.
• Understanding this aspect of wound-healing in horses could provide crucial insights into better treatment strategies for equine wounds, especially in limbs, and potentially reduce the occurrence of EGT.