title: Development of a method for absolute quantification of equine acute phase proteins using concatenated peptide standards and selected reaction monitoring.
abstract: 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.
authors.affiliation: Department of Large Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen , Højbakkegaard Allé 5, Taastrup 2630, Denmark.
authors.forename: Louise
authors.initials: L
authors.lastname: Bundgaard
authors.forename: Stine
authors.initials: S
authors.lastname: Jacobsen
authors.forename: Thomas F
authors.initials: TF
authors.lastname: Dyrlund
authors.forename: Mette Aa
authors.initials: MA
authors.lastname: Sørensen
authors.forename: Victoria M
authors.initials: VM
authors.lastname: Harman
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Beynon
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Brownridge
authors.forename: Lars J
authors.initials: LJ
authors.lastname: Petersen
authors.forename: Emøke
authors.initials: E
authors.lastname: Bendixen
mesh_terms: D000209:Acute-Phase Proteins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002138:Calibration; D006736:Horses; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D012015:Reference Standards; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000592:standards; D014945:Wound Healing
chemical_list: D000209:Acute-Phase Proteins; D010446:Peptide Fragments
keywords: QconCAT; acute phase proteins; equine; quantotypic peptides; selected reaction monitoring; validation; veterinary proteomics; wound healing