QconCATs in equine wound healing

Bundgaard L, Bendixen E, Sørensen MA, Harman VM, Beynon RJ, Petersen LJ, Jacobsen S. (2016) A selected reaction monitoring based analysis of acute phase proteins in interstitial fluids from experimental equine wounds healing by secondary intention. Wound Repair Regen. 2016 Feb 22. doi: 10.1111/wrr.12425.

In horses pathological healing with formation of exuberant granulation tissue
(EGT) is a particular problem in limb wounds, whereas body wounds tend to heal
without complications. Chronic inflammation has been proposed to be central to
the pathogenesis of EGT. This study aimed to investigate levels of inflammatory
acute phase proteins (APPs) in interstitial fluid from wounds in horses. A novel
approach for absolute quantification of proteins, selected reaction monitoring
(SRM)-based mass spectrometry in combination with a quantification concatamer
(QconCAT), was used for the quantification of 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).
Wound interstitial fluid was recovered by large pore microdialysis from
experimental body and limb wounds from five horses at days 1, 2, 7, and 14 after
wounding and healing without (body) and with (limb) the formation of EGT. The
QconCAT included proteotypic peptides representing each of the protein targets
and was used to direct the design of a gene, which was expressed in Escherichia
coli in a media supplemented with stable isotopes for metabolically labeling of
standard peptides. Co-analysis of wound interstitial fluid samples with the
stable isotope-labelled QconCAT tryptic peptides in known amounts enabled
quantification of the APPs in absolute terms. The concentrations of fibrinogen,
haptoglobin, ceruloplasmin, prothrombin, and α-1-antitrypsin in dialysate from
limb wounds were significantly higher than in dialysate from body wounds. This is
the first report of simultaneous analysis of a panel of APPs using the
QconCAT-SRM technology. The microdialysis technique in combination with the
QconCAT-SRM based approach proved useful for quantification of the investigated
proteins in the wound interstitial fluid, and the results indicated that there is
a state of sustained inflammation in equine wounds healing with formation of EGT.