Data independent acquisition mass spectrometry, and in particular, SWATH-MS strategy, has revolutionized human biomedical research and is slowly finding its way in clinical veterinary applications. SWATH-MS relies on comprehensive peptide spectral libraries to ease data interpretation and minimise the number of false positives. Plasma is an easily accessed source of protein biomarkers in clinical studies and yet, generating peptide spectral libraries for plasma is not trivial due to a large dynamic range of protein concentrations. In our study in which we employ SWATH-MS based protein quantitation to study a canine model of pain and inflammation, we are therefore employing various fractionation strategies to increase the depth of plasma spectral library.
Fractionation of the dog plasma has been carried out using SDS-PAGE, acetonitrile precipitation and ProteoMiner Protein Enrichment kit. The fractionated samples were digested either in gel or using FASP protocol desalted using StageTip and analysed on TripleTOF 5600 in data dependent acquisition mode. The use of a combination of ProteoMiner and SDS-PAGE resulted in the highest number of proteins followed by a combination of acetonitrile precipitation and SDS-PAGE.
To our knowledge this is the most comprehensive dog spectral library collected to date. Canine models are now increasingly used in translational biomedical research, and hence the generation and availability of a canine plasma proteome would aid advancements in both human and veterinary biomedicine.