Proteoform-Specific Protein Binding of Small Molecules in Complex Matrices.

Area of Science:

• Biochemistry
• Analytical Chemistry
• Pharmacology

Background:

• Characterizing small molecule-protein interactions is crucial for drug discovery.
• Conventional methods for analyzing bioactive compounds in complex mixtures are often laborious and can affect compound integrity.
• Proteins exist in various proteoforms, and only specific ones are relevant for drug binding.

Purpose of the Study:

• To develop a rapid, mass spectrometry (MS)-centric workflow for evaluating the bioactivity of crude botanical extracts.
• To distinguish between covalent and noncovalent binding and map binding sites.
• To enable simultaneous identification and quantitation of small molecules in complex matrices.

Main Methods:

• A top-down mass spectrometry (MS) workflow was developed for analyzing crude botanical extracts after a one-step reaction.
• Nanoflow liquid chromatography-selected reaction monitoring (SRM)-MS was used for label-free multiplex quantitation.
• The assay was validated using various proteoforms of human serum albumin (HSA).

Main Results:

• The MS assay successfully distinguished covalent from noncovalent binding and identified the specific residue involved in covalent interactions.
• The workflow enabled rapid evaluation of bioactive constituents in crude botanical extracts.
• The assay was validated for HSA proteoforms and demonstrated utility in evaluating thymoquinone's pharmacokinetics and toxicity.

Conclusions:

• The developed proteoform-specific MS assay offers a rapid and efficient method for evaluating small molecule bioactivity in complex mixtures.
• This approach aids in understanding drug pharmacokinetics, toxicity, and facilitates drug discovery from natural products.
• The workflow provides valuable insights into small molecule-protein interactions at the proteoform level.