<b>Background</b>: Serum electrophoretic profiles in cats are poorly characterized with respect to the protein components of the globulin fractions, and interpretation of the electrophoretograms has routinely been done in ignorance of the identity of the proteins found within each fraction. \ud <b>Objectives</b>: To compare the protein fractions from serum protein electrophoresis (SPE) in healthy cats and those with lymphoma and to confirm some component proteins in the major fractions after feline SPE, using tandem mass fingerprinting analysis (TMFA). \ud <b>Methods</b>: Total protein was measured and agarose gel SPE performed on blood collected from 14 healthy cats and 14 with lymphoma. The absolute protein concentration within each fraction was compared between the two groups. Bands corresponding to the SPE fractions were excised from two controls and a lymphoma cat and analysed by liquid chromatography coupled to mass spectrometry. Results were compared to sequences in the NCBI protein database.\ud <b>Results</b>: Median albumin concentrations were significantly decreased in lymphoma cats and median beta globulin concentrations were elevated. Narrow electrophoretic spikes were present in the beta/gamma fraction in 3 lymphoma cats. Following TMFA, multiple proteins were identified from each fraction and their mobility agreed with results from previous studies generated using alternative techniques. Inter–alpha (globulin) inhibitor 4 was identified in feline serum for the first time. \ud <b>Conclusions</b>: Cats with lymphoma had lower median albumin and higher beta globulin concentrations than healthy cats. Despite the limitations of 1D agarose gel SPE, TMFA provided preliminary data to confirm the protein components of the various fractio
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