Proteins derived by recombinant technologies must be characterized to ensure quality, consistency and optimal production. These properties are usually assayed by liquid chromatography-mass spectrometry (LC-MS) that combines the separation power of ultra-performance liquid chromatography with the characterization capability of high-resolution Orbitrap or QTOF-based mass spectroscopy platforms. LC-MS analysis is typically carried out under denaturing conditions at the end of the manufacturing process following purification procedures that are time consuming and labor intensive. Recently, a native mass spectrometry (nMS) approach for the characterization of intact over-expressed proteins directly from culture supernatants was described (Vimer et al., Nature Protocols 2020). However, the requirement for manual off-line desalting of samples and the need for pulled and gold-coated sub-micron emitters limits this method to academic labs with access to a micropipette puller and a sputter coater. The development of online buffer exchange (OBE) and use of novel multi-nozzle nano-electron spray emitters has enabled the rapid screening of intact proteins or protein complexes by native mass spectrometry (VanAernum et al., Nature Protocols 2020). We are benchmarking different commercially available size exclusion columns and emitters for use in OBE-nMS and testing their suitability for the structural characterization of recombinant protein complexes (Newman et al., 2019), unbiased antibody-drug conjugate drug level quantitation, and the rapid screening of protein-small molecule binding interactions.
CSIRO has a very successful track record in fragment-based drug discovery (FBDD) and provides a range of drug development capabilities, from hit identification by surface-plasmon resonance (SPR) to structure-based lead optimisation by X-ray crystallography (XRD). Access to the ARC LIEF-funded hydrogen deuterium exchange-mass spectrometry (HDX-MS) platform at the Bio21 Mass Spectrometry and Proteomics Facility has allowed us to demonstrate the utility of this technology to validate small molecule binding interaction interfaces of a novel class of carbonic anhydrase inhibitors (Mueller et al., Molecules 2021), and confirm drug-induced conformational changes in ligand-controlled protein complex assembly (Guo et al., Nature Comms in press). The application of HDX-MS as an orthogonal tool to SPR and XRD in validating the mechanism of action of small molecule drugs and/or guiding the design of protein biosensors is discussed.