Development of a Method for Absolute Quantification of Equine Acute Phase Proteins Using Concatenated Peptide Standards and Selected Reaction Monitoring

The
aim of this study was the development of a quantitative assay
that could support future studies of a panel of acute phase proteins
(APPs) in the horse. The assay was based on a quantification concatamer
(QconCAT) coupled to selected reaction monitoring methodology. Thirty-two
peptides, corresponding to 13 putative or confirmed APPs for the Equus caballus (equine) species were selected for the design
of a QconCAT construct. The gene encoding the QconCAT was synthesized
and expressed as an isotope-labeled chimaeric protein in Escherichia
coli. The QconCAT tryptic peptides were analyzed on a triple-quadrupole
instrument, and the quantotypic properties were assessed in equine
serum, wound tissue, and wound interstitial fluid. Reasonable quantotypic
performance was found for 12, 14, and 14 peptides in serum, wound
tissue, and interstitial fluid, respectively. Seven proteins were
quantified in absolute terms in serum collected from a horse before
and after the onset of a systemic inflammatory condition, and the
observed protein concentrations were in close agreement with previous
data. We conclude, that this QconCAT is applicable for concurrent
quantitative analysis of multiple APPs in serum and may also support
future studies of these proteins in other types of tissues and body
fluids from the horse.