Computational protocol: Novel giardicidal compounds bearing proton pump inhibitor scaffold proceeding through triosephosphate isomerase inactivation

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Protocol publication

[…] GlTIM (6 mg/mL; 214 µM) was incubated with 1 mM of compounds (omeprazole, BHO2 and BHO3) during 2 hours in TE buffer at 37 °C. Then the samples were incubated with 2 mM of DTT during 10 min at 25 °C; the samples were later centrifuged at 15,000 × g/15 min at 25 °C and supernatant filtrated by 3 mL Sephadex G-25 column equilibrated with Milli-Q water. After that, the samples were totally desiccated and sending to the CBC-UIC Research Resource Center Mass spectrometry, Metabolomics and Proteomic Facility (University of Chicago, Illinois) for performing an LC-MS/MS analysis. The proteins were digested by trypsin and the resulting peptides were applied to a Dionex Ultimate 3000 HPLC system coupled to a Thermo Fisher Scientific Orbitrap mass spectrometer. The obtained data were analyzed by using MAXQUAT Software.We performed an additional in-house strategy of analysis by using the Peptide Shaker suite (V 1.12.2). To this purpose, the raw data were transformed to Mascot generic format files for the analysis; a target-decoy database was built including mutant GlTIM-C202A and 116 sequences reported as potential proteins contaminants. Three.mgf data containing spectra data of modified GlTIM with omeprazole, BHO2 or BHO3 were submitted to the bundled SearchGUI tool (V3.0.3). We added five new post-translational modifications belonging to the compounds (Fig. ). For omeprazole, adducts “A” (C17H18N3O2S, Δm 329.1197) and “B” (C8H8N2O, Δm 148.0637) were added. For BHO2 and BHO3, their corresponding adducts “A” (C17H18N3OS, Δm 313.1248 and C17H15F3N3OS, Δm 367.0966, respectively) were added. Finally, only one adduct “B” was added since BHO2 and BHO3 generate the same one (C8H7N2, Δm 131.0609). In addition, the variable modifications of methionine oxidation (Δm 15.9949), and asparagine deamidation (Δm 0.9840) were also included. The m/z tolerance values of the precursors and fragments were 10 ppm and 0.9 Da, respectively. From suite, X!Tandem version 2015.12.15, MyriMatch, MS-GF + , OMSSA and Comet were used as search engines. Modifications identified were confirmed by manual analysis by using MS-Product bundled in Protein Prospector Suite (V5.17.1; […]

Pipeline specifications

Software tools SearchGUI, MyriMatch, MS-GF, OMSSA, Protein Prospector
Application MS-based untargeted proteomics
Organisms Homo sapiens
Diseases Giardiasis, Parasitic Diseases
Chemicals Omeprazole