Background: Domestic dogs share the same environment as their owners and represent a valuable animal model to study naturally‐occurring human disease. Proteomics holds promise for discovery of cancer biomarkers, however comparative platelet proteomics are lacking.

Aims: To establish a protocol for shotgun proteomic identification and quantification of proteins released from agonist‐activated canine platelets.

Methods: Washed platelets were isolated from ACD‐A anticoagulated blood from a hound mixed‐breed dog by serial centrifugation. Stirred, washed platelets (1.0×109/mL) were stimulated with saline or 50 nM gamma thrombin at 37°C for 6 minutes. Protease inhibitors were added and followed by centrifugation to remove cells and debris. The supernatant was spun at 50,000× g to yield soluble and pellet (microparticle) fractions. The former was concentrated. Protein concentration was measured in both fractions. SDS‐PAGE was used to separate proteins present in soluble and pellet fractions (Figure 1). For shotgun proteomic analysis, the gel was divided into sections and in‐gel trypsin digested. Tryptic peptides were separated by nano‐LC (liquid chromatography) followed by tandem mass‐spectrometry (MS/MS). Proteins were identified with Sequest software searching a canine database. Identified proteins had minimally 1 unique peptide and were sorted based on +/‐ ≥2 peptides. A ratio of ≥2 for MS1 abundance (activated/control) was used to identify proteins released.