Malarial cases are rising across the globe, reported by the World Health Organization. However, the number of Plasmodium vivax cases have raised gradually in the past few years as an annual disease due to its relapsing life cycle. Vivax is not well understood due to lack of continuous culturing, low parasitemia, lack of diagnostic marker, improper proteome database and poorly understood severity worsens the situation. In our study, we have focused on the problem of lack of diagnostic marker for vivax and parasite proteome database, we used the proteomics approach for the comprehensive analysis of parasite and plasma proteomics of Indian vivax infected population in search of potential biomarker candidates. We employed high throughput MS-based technology which reproducibly provided 230 proteins in plasma and 792 proteins in extracted parasite analysis.
Proteomic analysis of plasma and extracted parasite was done using a quadrupole-Orbitrap mass spectrometer. Data were analyzed with a high stringency level of 0.01% error acceptance using trans-proteomic pipeline, leading us to 5 recurring parasite proteins, PVX_090265, PVX_083555, PVX_094303, PVX_003545, and PVX_101520. These five proteins were further analyzed using bioinformatics studies, which provided proteins unique to the parasite and P. vivax tryptophan-rich antigen (PvTRAg), PVX_090265 was further analyzed for antigenicity with HLA repertoire using NetMHCpan for predicting epitopes and HLA binding affinity. The target gene was then cloned in the E. coli system, expressed, and purified. The protein was devoid of tag hence, the purification was based on the physical and chemical properties of the protein. PvTRAg is hydrophobic, hence hydrophobic interaction chromatography followed by size exclusion chromatography provided us pure band of the parasite protein. The purified protein can be used with one or more host or parasite proteins to make an antibody-antigen interaction-based multiplex assay to diagnose and prognose the malaria infection in clinics for social welfare.