In drug discovery, natural product libraries have the distinct advantages of high molecular diversity, complex stereochemistry, and biochemical specificity. Although far less than 10% of the world’s biodiversity has been evaluated for potential biological activity, the exploration of natural product libraries has declined since the 1970s in favour of synthetic libraries that can be more readily screened. One potential approach to substantially improve the screening of natural products is to use native mass spectrometry (MS), owing to its high sensitivity, low sample consumption, and rapid analysis time. However, conventional native MS is limited by its intolerance to non-volatile salts and impurities. In practice, current native MS methodology is usually limited to screening one isolated candidate compound at a time, which substantially limits throughput.
Recent research has demonstrated that up to 11 protein-ligand interactions can be measured directly from a single mass spectrum by using nanoscale ion emitters in native MS.1-2 The use of nanoscale emitters can significantly reduce the adduction non-volatile salts common to complex mixtures such as natural extracts.1-3 Here, we report the application of nanoscale emitters in native MS to achieve highly parallelised screening of complex mixtures for ligands of druggable protein targets. The proposed workflow, entitled multiplexed bioaffinity mass spectrometry (MX-BaMS), involves: (i) incubation of the target protein with a crude natural extract and (ii) native MS detection of intact protein-ligand complexes. As a proof-of-concept, three carbonic anhydrases (CA) were selected as protein targets, and five natural products were screened. A total of 15 hits were detected, corresponding to 11 unique compounds putatively identified using metabolomics data from liquid chromatography tandem MS, and multiple hits were confirmed using authentic standards. Two new CA ligands were identified, and an entirely new CA-binding scaffold was discovered. In addition, recent related research in the application of MX-BaMS to the Papain-like protease from SARS-CoV-2, a promising therapeutic target for COVID-19, will also be discussed. Overall, MX-BaMS is a highly promising approach for rapid and efficient screening of hundreds of compounds simultaneously for ligands that bind to protein targets. We envision that nanoscale ion emitters should also be useful for improving native proteomics and live-single cell MS measurements.
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