engleski

LC-MS/MS analysis of dog serum phosphoproteome reveals novel phosphorylation sites and differential phosphoprotein patterns in babesiosis caused by B. canis

Phosphorylation is the most commonly studied protein post-translational modification (PTM) in biological systems due to its importance in controlling division, survival, cell growth etc. During the last several years, many large- scale profiling studies of phosphoproteins and mapping of phosphorylation sites of proteins from human and animal cell lines or tissues have been published. Nevertheless, there is little information on general phosphoproteomic characterization and description of the content of circulating phosphoproteins (present in serum, plasma and other biofluids). We have performed a gel-free analysis of dog serum samples using LC-MS/MS supported by phosphopeptide enrichment. We compared serum from healthy dogs and dogs affected by B. canis-caused babesiosis. After digestion, phosphopeptides present in samples were enriched using TiO 2 magnetic beads. Our approach was both qualitative and quantitative using TMT-labeled serum pool samples in combination with LC-MS/MS. Results show a moderate number of phosphorylated proteins (45- 50), with around 85 phosphorylation sites, many of which have homologous phosphorylation sites in databases for other animal species but were never published for dogs. 75.5 % of the detected phosphorylated sites were phosphoserine, 15.9 % were phosphothreonine and only 8.6 % were phosphotyrosine residues. We detected a higher number (300-400) of non- phosphorylated peptides in enriched samples though a high percentage of them are considered to be phosphoproteins in databases. Quantitative analysis of pools containing 10 control samples vs. 10 babesiosis samples confirm the differences observed for individual samples. Differences in phosphorylation of proteins involved in blood coagulation pathways could reflect the adaptation of hemostasis to thrombocytopenia and variable degree of hemolysis observed in babesiosis. This study represents the first haracterization of phosphorylation site mapping of dog biofluids.

canine babesiosis, LC-MS/MS, phosphopeptide enrichment, phosphoproteomics, Tandem Mass Tag

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