Development of a method for absolute quantification of equine acute phase proteins using concatenated peptide standards and selected reaction monitoring.

This research article outlines the development of an assay – a procedure for measuring the presence or activity – to support future study of a group of proteins known as acute phase proteins (APPs) in horses.

Development of the Quantitative Assay

• The researchers aimed to develop a quantitative assay for future study of a group of proteins (APPs) in horses. These proteins are present during early stages of inflammatory response, serving as helpful markers in diagnosing various inflammatory conditions.
• The researchers opted for a specific methodology called quantitative concatamer (QconCAT) coupled with selected reaction monitoring. This approach involves generating synthetic genes encoding proteins of interest for accurate quantification in complex mixtures.
• A total of 32 peptides, equating to 13 putative or confirmed APPs specific to horses, were identified for the QconCAT construct.

Expression and Analysis of the QconCAT

• The gene encoding the QconCAT was synthesized and expressed as a labeled protein in a strain of Escherichia coli, a well-known bacterium often used for genetic engineering purposes.
• The resulting peptides were then analyzed using a triple-quadrupole instrument, a type of mass spectrometer known for its high level of sensitivity and precision.

Quantotypic Properties and Inflammatory Condition Monitoring

• The quantotypic properties of the QconCAT were assessed in three different horse samples: serum (blood without cells), wound tissue, and wound interstitial fluid (fluid surrounding tissue cells).
• Of the QconCAT peptides, 12, 14, and 14 showed reasonable quantotypic performance in each sample type, respectively.
• The researchers highlighted seven proteins that were quantified in absolute terms in serum collected from a horse before and after the onset of a systemic inflammatory condition.
• The observed protein concentrations paralleled previous data, suggesting that the new assay could provide an effective way to monitor systemic inflammatory conditions in horses.

Conclusion

• The researchers concluded that this new QconCAT is applicable for concurrent quantitative analysis of multiple APPs in serum. Due to its functionality, it may also support future studies of these proteins in other types of tissues and body fluids from the horse.