At the core of nMS is the ability to preserve non-covalent interactions and perform very accurate mass measurements. This ability has enabled advances in structural biology, including the elucidation of protein homogeneity, oligomeric state, sequence variation and the identity of bound ligands. These advances are made possible by preserving proteins in their native state in solution, followed by careful transfer into the gas phase of the mass spectrometer. As a consequence, nMS is finding applications in increasingly diverse research areas such as high-throughput drug screening, the study of amyloid formation and inhibition, the characterisation of antibody-drug conjugates and the elucidation of the interactions of membrane proteins with lipids and therapeutics. This ability to study the influence of small molecule ligand binding on the cascade of protein interactions that underlie many disease states is therefore providing a new paradigm in drug discovery.
Using native mass spectrometry to inform drug discovery
A better understanding of cellular processes is of paramount importance for the development of new therapeutics. These processes are enabled by synchronisation of multiple protein-protein or protein-ligand interactions and, while they dictate how cells interact in tissues, they are guided by molecular rearrangement at the atomic level. Hence, new technologies that are capable of capturing whole protein complexes, yet providing atomic-level details for their structure and function, hold the potential to unravel the complexity of these interactions. Over the last decade nMS in combination with ion mobility (IM) – which measures the overall conformation of an ion – has emerged as a promising method to study protein interactions at equilibrium.
nMS begins with the isolation of protein complexes from their cellular environment and is followed by exchanging the analyte to an MS-compatible medium, while preserving the protein in its folded, native state. Protein complexes are then ionised and transferred into the gas phase via soft ionisation methods such as electrospray ionisation. By optimising the instrumental parameters, ions are desolvated while their folded structure and non-covalent interactions are maintained. This allows the mass of protein and their bound ligands to be determined with high accuracy and resolution, elucidating not only their binding stoichiometry but also the capability to observe each species present in solution. This is a powerful attribute, since most biophysical methods report only the average ensemble present in solution.
Once inside the mass spectrometer, individual protein-ligand complexes can be isolated and dissociated to provide ligand identification or to quantify the stabilisation that ligands provide upon binding to the proteins.^1 The latter can be achieved by monitoring how proteins unfold in the presence or absence of bound ligands. This experiment is performed in combination with IM, a method that measures the time it takes for ions to pass through a tube filled with inert gas known as a drift tube. This drift time is dependent on a protein’s charge and rotational average collision cross-section, or in more simplistic terms – its globular shape. Utilising this additional dimension of separation, it is possible to differentiate highly complex mixtures composed of species of equivalent mass, known as isobaric species, based on different conformations.
In addition, by increasing the activation in the collision cell, proteins can also be fragmented to provide information regarding a primary sequence and report the presence of post-translational modifications, typically known as top-down proteomics. The information derived from nMS can also be combined with results obtained from x-ray crystallography or electron microscopy to refine the protein structures by reporting the identity of bound ligands that reside in areas with weaker electron density.^2
While nMS has predominantly been applied by academic researchers, its exciting capabilities are now proving ...
[2', 3'-di-TBDMS-U]](https://eimg.pharmasources.com/image/product/20231227/25cff4741616f16c5185db80966ec42ajpg)
[2', 3'-diTBDMS-C(Ac)]](https://eimg.pharmasources.com/image/product/20231227/5569723496509e0380118a109ff72ce1jpg)
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