Mass spectrometry measures the mass-to-charge ratio of ions, providing information about a sample’s molecular weight and composition. Native mass spectrometry (native MS) is a specialized variation that analyzes biological molecules in their natural, folded, and intact state. This method provides direct detection of intact proteins and protein complexes, offering insights into their higher-order structures.
Native MS is a high-resolution approach that avoids the fragmentation of proteins into peptides, common in traditional methods. It maintains the structural integrity of complex biomolecules, making it suitable for studying their native conformations and interactions. This capability helps understand how these molecules function in biological systems.
Preserving Molecular Structure
The term “native” in native mass spectrometry refers to maintaining the natural, three-dimensional structure and non-covalent interactions of biological molecules. Traditional mass spectrometry often employs denaturing conditions, such as high heat or harsh solvents, which disrupt these fragile non-covalent bonds. Such conditions can lead to the unfolding of proteins or the dissociation of molecular complexes, altering their natural shape and function.
Native MS, in contrast, preserves these delicate interactions, including hydrogen bonds, electrostatic interactions, and hydrophobic forces. This allows scientists to study a wide range of biological molecules, such as proteins, DNA, and their complex assemblies, in their biologically relevant states. For instance, it can analyze oligomeric proteins, protein-nucleic acid assemblies, and protein-ligand interactions without breaking them apart. Understanding these intact structures is fundamental to comprehending how biological molecules perform their specific functions.
The Process of Native Mass Spectrometry
Native mass spectrometry relies on gentle techniques to transfer biological molecules from solution into the gas phase while preserving their native structures. Samples are prepared in volatile buffers, often using aqueous volatile salts like ammonium acetate at a near-neutral pH, to avoid denaturation.
Gentle ionization of the molecules is achieved through electrospray ionization (ESI). In ESI, a high voltage is applied to the sample solution, creating a fine mist of highly charged droplets. As the solvent evaporates, these droplets shrink and release intact, multiply charged ions into the gas phase without causing fragmentation. These ions then enter the mass spectrometer. Inside the instrument, ions are separated based on their mass-to-charge ratio using high-resolution mass analyzers like quadrupole time-of-flight (Q-TOF) or Orbitrap instruments. This separation allows for precise measurement of the mass of the intact molecules and their complexes.
Unlocking Biological Insights
Native mass spectrometry provides diverse insights into biological systems by allowing the study of molecules in their functional states. It determines the stoichiometry, or the precise number of subunits, within protein complexes, helping researchers understand their assembly and component interactions. The technique also analyzes protein-ligand interactions, such as drug binding to target proteins, by providing quantitative data on binding affinities.
Native MS characterizes biopharmaceuticals, including monoclonal antibodies and vaccines, by assessing their structural integrity, stability, and post-translational modifications (PTMs). It can reveal how phosphorylation, acetylation, or glycosylation affect a protein’s structure and function. The method also studies the assembly and disassembly of viruses and identifies novel biomarkers for diseases like cancer by detecting structural alterations in intact proteins. The data obtained from native MS, such as mass, stoichiometry, and binding information, advances our understanding of molecular function, disease mechanisms, and drug discovery processes.