Development of a target identification approach using native mass spectrometry

Miaomiao Liu,Ronald J Quinn,Wesley C Van Voorhis

doi:10.1038/s41598-021-81859-4

Miaomiao Liu, Ronald J Quinn + Show 1 more

Open Access

https://doi.org/10.1038/s41598-021-81859-4

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Abstract

A key step in the development of new pharmaceutical drugs is the identification of the molecular target and distinguishing this from all other gene products that respond indirectly to the drug. Target identification remains a crucial process and a current bottleneck for advancing hits through the discovery pipeline. Here we report a method, that takes advantage of the specific detection of protein–ligand complexes by native mass spectrometry (MS) to probe the protein partner of a ligand in an untargeted method. The key advantage is that it uses unmodified small molecules for binding and, thereby, it does not require labelled ligands and is not limited by the chemistry required to tag the molecule. We demonstrate the use of native MS to identify known ligand–protein interactions in a protein mixture under various experimental conditions. A protein–ligand complex was successfully detected between parthenolide and thioredoxin (PfTrx) in a five-protein mixture, as well as when parthenolide was mixed in a bacterial cell lysate spiked with PfTrx. We provide preliminary data that native MS could be used to identify binding targets for any small molecule.

Highlights

Because of limited compound supply and relative poor structure–activity relationship information a vailable[12,13]
We recently reported the use of native Mass spectrometry (MS) with high-resolution electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FT-ICR-MS) for direct protein–ligand detection between individual proteins and compound libraries or natural product extracts[29,30,31,32,33]
The liquid sample desorption electrospray ionization–mass spectrometry (DESI-MS) has been successfully applied to detect intact protein−ligand complexes formed between ribonuclease A with cytidine nucleotide ligands and lysozyme with acetyl chitose ligands[40,41]

Summary

Introduction

Because of limited compound supply and relative poor structure–activity relationship information a vailable[12,13]. We investigated effects of different concentrations and pH values of ammonium acetate on the total MS intensities of the protein mixture (Fig. 3). In our study, we found amongst the four investigated concentrations of ammonium acetate (10 mM, 50 mM, 100 mM, and 200 mM), that the highest protein total signal intensities were observed in 200 mM ammonium acetate, showing an increase of about 1.37 times in signal intensities of the protein mixture compared to 100 mM ammonium acetate under the same instrument tuning condition (Fig. 3A).

Results

Conclusion