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Microparticle assisted precipitation screening method for robust drug target identification

ABSTRACT: While thermal proteome profiling (TPP) shines in the field of drug target screening by analyzing the soluble fraction of the proteome samples treated with high temperature, the counterpart, insoluble precipitate has been overlooked for a long time. The analysis of precipitate is hampered by the inefficient sample processing procedure. Herein, we propose a novel method, termed Microparticle Assisted Precipitation Screening (MAPS), for drug target identification. The MAPS method exploits the principle that drug bound proteins will be more resistant to thermal unfolding similar as in classic TPP method, but the process of protein precipitation is assisted by microparticles. Upon heating, proteins will unfold and aggregate on the surface of microparticles, which benefits the following proteomic analysis of the precipitate. The introduction of microparticle simplifies the whole sample preparation workflow. The proteins that precipitate on the surface of microparticles will be subjected to wash, alkylation, and digestion. The whole sample preparation is processed conveniently on the surface of microparticles without any transferring. With the assistance of microparticles, sample loss is minimized. As a result, MAPS method is compatible with minute amount of initial proteins. MAPS was applied to screen the targets of several well-studied drugs and the known target proteins were successfully with high confidence and specificity. To investigate the specificity of MAPS method, it was applied it to screen the targets of the pan-kinase inhibitor, staurosporine, and 32 protein kinases (specificity of 80%) were identified by using only 20 µg initial proteins of each sample. MAPS is an unbiased robust method for identifying drug targets at the level of proteome, filling the vacancy of stability-based target screening using precipitate.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Thermal Proteome

PROVIDER: PXD021399 | JPOST Repository | Fri Sep 10 00:00:00 BST 2021

REPOSITORIES: jPOST

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Project description:Toxoplasma gondii is an obligate intracellular parasite which can cause toxoplasmosis. Surface and secreted proteins of T. gondii play important roles in infection and immunity, and also are antigen targets in immunological diagnosis and vaccine development. However, it is difficult to investigate identities and antigenicities of surface and secreted proteins due to limitation of surface protein extraction methods. In this study, a total of 785 potential surface and secreted proteins of T. gondii RH tachyzoites were identified using a method combination of biotin labeling, avidin chromatography isolation, and high flux proteomics (LC-MS/MS). Among the highly-expressed 65 proteins, 43 proteins (66%) were originally annotated as surface or secreted proteins in the released T. gondii genomes, which proved the method combination to be a credible strategy. Furthermore, the transcriptomic responses and cytokine secretions induced by the isolated proteins, the live T. gondii RH tachyzoites infection, and the live pathogenic E. coli infection, in the human peripheral blood monocyte THP-1 cell lines, were comparatively analyzed to reveal antigenicities and immunobiological properties of T. gondii surface and secreted proteins. The transciptomic profiles induced by the isolated proteins were similar to those induced by the live bacterium infection, but were different from those induced by the live parasite infection. Contrary to the low cytokine secretion induced by the live parasite infection, the isolated proteins induced significant cytokine and chemokines secretion. Especially, the secretions of several chemokines induced by the isolated proteins were even higher than those induced by the live bacterium infection. These data suggested that T. gondii surface and secreted proteins were effective antigens, while the live parasite could evade the host innate immunity. This study comprehensively revealed the identities and antigenicities of T. gondii surface and secreted proteins, which laid foundation for further screening new T. gondii antigens, developing immunological diagnosis methods, and studying host immune response to T. gondii infection.

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Microparticle assisted precipitation screening method for robust drug target identification

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