Use of High-Resolution Quantitative Proteomic Analysis to Identify New Potential Biomarkers

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

Martinez-Subiela S, Horvatic A, Escribano D, Pardo-Marin L, Kocaturck M, Mrljak V,Burchmore R,Ceron JJ, Yilmaz Z.

BACKGROUND

Proteomics allows identification of new biomarkers for treatment monitoring diagnosis and therapeutic intervention of disease.Quantitative gel-free proteomic approach using isobaric labelling reagents such as Tandem MassTags (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 instrumentationfor the analysis of differentially TMT-labeled peptide moietiesresults 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 possible biomarkers for treatment monitoring in canine leishmaniosis.So, the objective of this study was to use the TMT approachto 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 (T0) and 1 month after of treatment (T1) with meglumineantimoniate (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 labeled using 6-plex TMT reagents according to manufacturer procedure (Thermo Scientific) with some modifications. To obtain the information about protein identities and relative quantification, liquid chromatography–MSanalysis 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, in which it can be included IgA, inter-alpha-trypsin inhibitor heavy chain H4, serotransferrin, histidin rich glycoprotein, alpha 2 macroglobulin, paraoxonase and fibronectin; and (2) coagulation cascade such as kininogen-1 isoform X1 and X2, plasminogen precursor andbeta-2-glycoprotein 1 precursor.

CONCLUSIONS

TMT-based proteomic approach allowed identification of novel serum proteins that could be potentially suitable biomarkers for treatment monitoringof canine leishmaniosis.