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Use of high-resolution quantitative proteomic analysis to identify new potential biomarkers for the treatment monitoring of canine leishmaniosis

Background: Proteomics allows identification of new biomarkers for diagnosis, therapeutic intervention of disease and treatment monitoring, Quantitative gel-free proteomic approach using isobaric labeling reagents such as Tandem Mass Tags (TMT® ; Proteome Sciences) enables concurrent identification and quantification of proteins originating from up to ten different samples in a single experiment. Moreover, the use of high resolution mass spectrometry instrumentation for the analysis of differentially TMT-labeled peptide moieties results in high quality data with good sensitivity, excellent signal-to- noise ratio and a broad dynamic range. However, TMT technology has not been previously applied to detect novel biomarkers for treatment monitoring in canine leishmaniosis. So, the objective of this study was to use the TMT labeling approach to generate comparative protein profiles of serum samples from dogs with leishmaniosis before and after treatment in order to identify new potential biomarkers. Methods: Serum samples from 5 clinically diseased dogs collected before and 1 month after the treatment with meglumine antimoniate (100 mg/kg, SC, q24h) and allopurinol (15 mg/kg, PO, q 12h) were used for the proteomic study. Non-depleted serum samples were subjected to reduction, alkylation, digestion and labeling with the 6-plex TMT reagents according to manufacturer procedure (Thermo Scientific) with some modifications. To obtain the information about protein identities and relative quantification, LC–MS analysis of TMT- labeled peptides was performed. Thermo raw files were used for Mascot search against NCBInr database mining Canis lupus familiaris proteins. Results: Gel-free label-based proteomic approach enabled identification of 117 canine proteins. Among these, 23 showed significant difference (p<0.05) in expression (two downregulated and 21 upregulated ranging from 1.25x– 2.5x). Most of these proteins are involved in two main physiopathological mechanisms: (1) immune response, such as IgA, inter-alpha-trypsin inhibitor heavy chain H4, serotransferrin, histidine rich glycoprotein, alpha 2 macroglobulin, paraoxonase and fibronectin ; and (2) coagulation cascade, such as kininogen-1 isoform X1 and X2, plasminogen precursor and beta-2-glycoprotein 1 precursor. Conclusions: TMT-based proteomic approach allowed identification of novel serum proteins that could be potentially suitable biomarkers for treatment monitoring of canine leishmaniosis.

Proteomics, Tandem Mass Tags, canine leishmaniosis

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