title: Proteoform-Specific Protein Binding of Small Molecules in Complex Matrices.
abstract: Characterizing the specific binding between protein targets and small molecules is critically important for drug discovery. Conventional assays require isolation and purification of small molecules from complex matrices through multistep chromatographic fractionation, which may alter their original bioactivity. Most proteins undergo posttranslational modification, and only certain proteoforms have the right conformation with accessible domains and available residues for small molecule binding. We developed a top-down mass spectrometry (MS) centric workflow for rapid evaluation of the bioactivity of crude botanical extracts after a one-step reaction. Our assay distinguished covalent from noncovalent binding and mapped the residue for covalent binding between bioactive constituents and specific proteoforms of the target protein. We augmented our approach with a nanoflow liquid chromatography-selected reaction monitoring (SRM)-MS assay for simultaneous identification and label-free multiplex quantitation of small molecules in the crude botanical extracts. Our assay was validated for various proteoforms of human serum albumin, which plays a key role in pharmacokinetics of small molecules in vivo. We demonstrated the utility of our proteoform-specific assay for evaluating thymoquinone in crude botanical extracts, studying its pharmacokinetics in human blood, and interpreting its toxicity to human breast cancer cells in tissue culture.
authors.affiliation: Newomics Inc. , Emeryville, California 94608, United States.
authors.forename: Geuncheol
authors.initials: G
authors.lastname: Gil
authors.affiliation: Newomics Inc. , Emeryville, California 94608, United States.
authors.forename: Pan
authors.initials: P
authors.lastname: Mao
authors.forename: Bharathi
authors.initials: B
authors.lastname: Avula
authors.forename: Zulfiqar
authors.initials: Z
authors.lastname: Ali
authors.forename: Amar G
authors.initials: AG
authors.lastname: Chittiboyina
authors.forename: Ikhlas A
authors.initials: IA
authors.lastname: Khan
authors.forename: Larry A
authors.initials: LA
authors.lastname: Walker
authors.affiliation: Newomics Inc. , Emeryville, California 94608, United States.
authors.forename: Daojing
authors.identifier: 0000-0002-4542-5067
authors.initials: D
authors.lastname: Wang
mesh_terms: D001688:Biological Products; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D055808:Drug Discovery; D006801:Humans; D031338:Monarda / Q000737:chemistry; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry*; D054852:Small Molecule Libraries; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry
chemical_list: D001688:Biological Products; D011506:Proteins; D054852:Small Molecule Libraries