Proteoform-specific protein binding of small molecules in complex matrices

Authors: GIL, GEUNCHEOL - Newomics, Inc; MAO, PAN - Newomics, Inc; AVULA, BHARATHI - University Of Mississippi; ALI, ZULFIQAR - University Of Mississippi; CHITTIBOYINA, AMAR - University Of Mississippi; KHAN, IKHLAS - University Of Mississippi; WALKER, LARRY - University Of Mississippi; WANG, DAOJING - University Of Mississippi

Submitted to: ACS Chemical Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/12/2016
Publication Date: 12/21/2016
Citation: Gil, G., Mao, P., Avula, B., Ali, Z., Chittiboyina, A.G., Khan, I.A., Walker, L.A., Wang, D. 2017. Proteoform-specific protein binding of small molecules in complex matrices. ACS Chemical Biology. 12:389-397.

Interpretive Summary: The identification of the specific binding between protein targets and small molecules is critically important for drug discovery. Most proteins undergo post-translational modification, and only certain proteoforms have the right conformation with accessible domains and available residues for small molecule binding. A top-down mass spectrometry (MS) centric workflow for evaluation of the bioactivity of crude botanical extracts after a one-step reaction was developed. This approach was augmented 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. The assay was validated for various proteoforms of human serum albumin, which plays a key role in pharmacokinetics of small molecules in vivo. The utility was demonstrated in evaluating thymoquinone in crude botanical extracts, studying its pharmacokinetics in human blood, and interpreting its toxicity to human breast cancer cells in tissue culture.

Technical 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.