engleski

Insights from quantitative proteome profiling of serum in canine leishmaniosis using different proteomic strategies

Proteomic investigations in veterinary medicine are slowly gaining importance but are still lagging behind investigations in human medicine, as well as in their clinical research application. In most of the shotgun proteomic studies in veterinary medicine, mass spectrometers have been operated in data- dependent acquisition (DDA) mode addressing new biomarkers discovery in biological fluids, especially in serum. Recently, data-independent acquisition (DIA) has been introduced into this field, providing insights into physiology and pathophysiology in animals. Our interest is focused to canine leishmaniosis, a vector-borne zoonotic disease, which is caused by Leishmania infantum. Phlebotomine sand flies are the biological vectors and dogs are the main reservoir for human infection. Due to its zoonotic potential, it is of a great importance to diagnose this disease and treat infected animals. The aim of our study was to apply both proteomic strategies, e.g. DDA and DIA, for proteome profiling of serum from leishmania infected dogs in order to understand the pathophysiology of the diseases, as well as find proteins with diagnostic potential and for monitoring the success of treatment. Serum samples of healhy dogs and Leishmania- seropositive dogs, as well as serum of clinically diseased dogs before and one month after the treatment were collected and analysed using Ultimate 3000 RSLCnano system combined with Q Exactive Plus and Orbitrap Elite mass spectrometers, respectively. DDA-based quantitative proteomic approach using tandem mass tags (TMT) followed by statistical analysis showed that from 117 identified serum proteins, 27 were significantly deregulated (p<0.05) after the treatment. Bioinformatic analysis using Cytoscape revealed their functions in immune response (e.g. IgA, ITIH4, fibronectin) and coagulation cascade (e.g. kininogen-1 isoform X1 and X2). Unlike DDA- based approach, DIA strategy using Spectronaut software and sample specific spectral libraries built from DDA runs enabled significant increase in number of identified proteins in shorter LC gradient time. A number of 15 statistically significant deregulated proteins (p<0.05) were detected in serum of Leishmania- seropositive dogs compared to healthy dogs. In conclusion, DIA-based strategy showed greater potential for detailed proteomic profiling of serum in canine leishmaniosis compared to DDA- based strategy. Deregulated proteins, some of which were reported for the first time in canine leishmaniosis, were involved in inflammatory, coagulation or defence mechanisms and could be potentially suitable for the disease progression and treatment monitoring of this parasitic disease.

data dependent analysis, data independent analysis, mass spectrometry, canine serum, proteomics, leishmaniasis

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