One dimensional (1D) serum protein gel electrophoresis (SPE) on agarose gels is a frequently used diagnostic tool for canine diseases; however, little is known regarding the precise composition of the different protein fractions in normal or diseased animals. To analyse the canine serum proteome in more detail we combined conventional 1D SPE with second dimension (2D) polyacrylamide gel electrophoresis (PAGE) and followed by tandem mass spectrometry (MS). One dimensional SPE was performed on the sera of 17 healthy dogs to establish normal reference ranges for the albumin and globulin sub-fractions. Two representative serum samples from the healthy dogs were further separated using a novel method of 2D PAGE, leading to the generation of 26 distinct bands across the six main sub-fractions, which were subjected to MS analysis. Thirty-two individual proteins were identified, most of which were found in both dogs. Twenty proteins belonged specifically to the species Canis lupus familiaris, with the remaining 12 proteins belonging to other mammalian species, likely reflecting incomplete sequencing knowledge of canine proteins. Two dimensional electrophoresis and MS allowed identification of canine serum albumin precursor, serpin peptidase inhibitor, kininogen-1, vitamin D binding protein, hemopexin, complement C4 and a variety of immunoglobulin class molecules and their localisation within their respective serum protein subfractions for the first time. Sera from twenty-one dogs with high grade multicentric lymphoma underwent identical analysis and had significantly elevated α2 globulins on 1D SPE. Further separation of the serum proteins was performed on three patients using a 2D PAGE system. Thirty-eight separate protein bands were submitted for MS and 36 different proteins were identified. Most of the proteins were the same as those previously identified in the serum of healthy dogs, showing reproducibility of this novel proteomic technique. Haptoglobin was found in all three of the lymphoma dogs, having not previously been identified in any of the healthy samples, and could account for the increased α2 globulins. Several other proteins, including α-2 HS glycoprotein, α2 macroglobulin, α1 antichymotrypsin and inter-α-trypsin inhibitor were also present in dogs suffering from lymphoma. Clusterin, an anti-apoptotic protein, was identified for the first time in the serum of one dog suffering from lymphoma. Kininogen, which is present in the serum of healthy dogs, was absent in all three dogs with lymphoma. This 2D electrophoresis technique has identified numerous changes in the serum proteome of dogs suffering from lymphoma and suggests a significant inflammatory component to the pathogenesis of this disease.
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