Mass spectrometry (MS) is an analytical technique used to measure the mass-to-charge ratio ($m/z$) of ionized molecules, acting as a sophisticated molecular scale. This measurement identifies a molecule’s exact mass, revealing its elemental composition or chemical identity. Molecules must first be converted into gas-phase ions for separation and detection.
Native mass spectrometry (Native MS) is a specialized extension designed to analyze large, intact biological molecules, such as proteins and their massive assemblies. Unlike traditional MS, which often fragments molecules, Native MS transfers them into the gas phase while preserving their complete, complex structure. This approach provides a unique window into biological architecture, allowing researchers to study how multiple components fit together.
Preserving Molecular Shape
The central advancement of Native MS is its ability to maintain the natural, three-dimensional shape of protein complexes during analysis. This requires analyzing molecules under “native” conditions, where the solution environment closely mimics physiological conditions. Preservation is achieved using volatile aqueous buffers, such as ammonium acetate, maintained near a neutral pH.
This gentle approach is necessary because the functional state of a protein complex depends on its higher-order structure. This structure is held together by non-covalent interactions, such as hydrogen bonds and electrostatic attractions, which allow subunits to assemble into a functional machine. Traditional MS methods use organic solvents and low pH, which disrupt these weak forces, causing the complex to unfold or dissociate.
Native MS ensures complex assemblies remain folded and associated as they transition into the mass spectrometer’s vacuum. This structural preservation is fundamental because a protein’s biological function, like binding to a drug, is intrinsically linked to its three-dimensional conformation. Analyzing the molecule in this native-like state provides data relevant to its behavior in a biological system.
How It Measures Massive Complexes
Measuring extremely large biological assemblies, which can range from hundreds of kilodaltons to several megadaltons, presents a significant technical challenge for mass spectrometry. To analyze these massive structures successfully, Native MS relies on soft ionization techniques designed to gently lift the molecules from the liquid solution into the gas phase.
The most common technique is nano-electrospray ionization (nano-ESI). This method uses a fine needle and a voltage potential to create a spray of charged droplets. As the solvent evaporates, the protein complexes emerge as intact, multiply charged ions ready for mass analysis. This process is considered soft because it avoids the high energy that would cause the large complex to fragment or lose its native structure.
Molecules must be charged to be manipulated by electric fields. For a massive protein complex, nano-ESI imparts multiple charges, which reduces the overall mass-to-charge ($m/z$) ratio. This reduction brings the signal for the massive complex into the measurable range of modern mass analyzers. The distinct set of multiply charged ions produced can then be mathematically deconvoluted to determine the accurate mass of the neutral, intact biological assembly.
Analyzing Protein Assembly and Stoichiometry
A core strength of Native MS is its ability to precisely determine the composition and architecture of complex biological machines. By measuring the mass of the entire, intact assembly, scientists gain quantitative insight into the exact number of subunits present, a property known as stoichiometry. This is possible because the mass of the complex is the sum of the masses of its individual protein subunits and any bound molecules, such as ligands or cofactors.
Native MS can easily distinguish between a complex formed by three identical subunits (a trimer) and one formed by four (a tetramer) by measuring the mass difference of a single subunit. This level of precision is invaluable for understanding enzyme complexes, where a slight variation in subunit count can alter the entire function of the machine. Furthermore, Native MS can be used to monitor the binding of small-molecule ligands, revealing exactly how many ligand molecules attach to the large protein.
The technique also provides insights into the dynamic nature of these assemblies, which often exist in multiple states or oligomeric forms within a solution. By resolving the mass of each distinct species in a single experiment, researchers can determine the equilibrium between different assembly states. They can also monitor how environmental changes, like pH, shift the composition of the complex. This comprehensive mass-based fingerprinting allows for the detailed mapping of how protein subunits interact and assemble.
Impact on Biomedical Discovery
The precise measurements offered by Native MS have made it a valuable tool in biomedical applications, particularly in drug development. It is frequently used in the biopharmaceutical industry to characterize therapeutic antibodies, known as biologics, which are complex protein-based drugs. Native MS quickly assesses the quality and integrity of these large molecules, checking for correct assembly, post-translational modifications, and the presence of attached sugar molecules (glycans).
For a newer class of drugs, called Antibody-Drug Conjugates (ADCs), Native MS is uniquely suited to determine the Drug-to-Antibody Ratio (DAR). The DAR—the average number of drug molecules attached to the antibody—strongly influences the drug’s effectiveness and safety. Native MS can measure the mass of the entire ADC to confirm this ratio. The technique is also used in early-stage drug discovery to analyze drug-target interactions directly in a native-like state.
Scientists use Native MS to screen potential drug candidates by observing their binding to target proteins, such as those implicated in diseases like cancer or Alzheimer’s. By measuring the mass of the protein before and after a small drug molecule binds, researchers confirm the interaction, determine the number of drug molecules bound, and assess the strength of the binding affinity. This capability speeds up the selection of promising drug leads and provides structural context that is difficult to obtain with other high-throughput methods.