Native Mass Spectrometry: Core Techniques and Recent Advancements
Native mass spectrometry is a powerful analytical technique that enables the investigation of proteins and their complexes under near-physiological conditions, supporting advancements in proteomics, structural biology, and biomedical research. By maintaining the native conformations of proteins and protein complexes in both solution and gas phases through mild ionization, native mass spectrometry provides critical insights into their assembly states, molecular interactions, and dynamic behavior. In recent years, substantial advancements in mass spectrometer performance, ionization strategies, and data processing methods have driven the continuous evolution of native mass spectrometry core technologies, leading to notable progress in areas such as protein-small molecule interactions, high-throughput analysis, and precision medicine. This review explores the core techniques and recent developments in native mass spectrometry.
Core Techniques of Native Mass Spectrometry
1. Ionization Strategies
The effectiveness of native mass spectrometry hinges on the use of gentle ionization methods to maintain protein complex integrity. Electrospray ionization (ESI) is the predominant technique, as it facilitates the generation of charged ions in solution while preserving the native structure of protein complexes upon transfer to the gas phase. Optimizing ESI parameters (e.g., voltage, flow rate, and capillary temperature) enhances ionization efficiency and minimizes unintended dissociation of complexes. Recently, the introduction of advanced ionization techniques, such as supercritical fluid electrospray ionization (SFC-ESI), has further expanded the capability of native mass spectrometry for analyzing large protein assemblies.
2. Mass Analyzers
Accurate native mass spectrometry measurements rely on high-resolution mass spectrometers, including:
(1) Quadrupole Time-of-Flight (Q-TOF) MS: Combining quadrupole selectivity with the high-resolution capabilities of time-of-flight (TOF) analyzers, Q-TOF MS is well-suited for precise mass measurements of protein complexes.
(2) Orbitrap MS: Featuring ultra-high resolution, Orbitrap MS effectively resolves protein heterogeneity and supports accurate quantitative analyses.
(3) Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR-MS): Recognized for its exceptional mass accuracy, FT-ICR-MS enables in-depth studies of ultra-large protein complexes and detailed characterization of protein-ligand interactions.
3. Integrated Mass Spectrometry Techniques
(1) Ion Mobility-Mass Spectrometry (IM-MS): Separates protein complexes with distinct conformations (e.g., folded vs. partially unfolded states) based on differences in ion mobility through an electric field and buffer gas. When coupled with tandem MS (MS/MS), IM-MS facilitates the characterization of dynamic assembly processes.
(2) Liquid Chromatography-Mass Spectrometry (LC-MS): Enhances the detection of low-abundance biomolecules (e.g., disease biomarkers in serum) by utilizing liquid chromatography for sample separation before mass spectrometric analysis, thereby improving sensitivity.
4. Gas-Phase Dissociation Techniques
(1) Surface-Induced Dissociation (SID): Gently disrupts multi-subunit protein complexes, providing insights into subunit composition and interaction networks.
(2) Electron Transfer Dissociation (ETD): Selectively cleaves proteins via electron transfer, allowing detailed investigation of protein tertiary structures.
Recent Advancements in Native Mass Spectrometry
1. High-Throughput Proteomics
With continuous improvements in mass spectrometry instrumentation, native mass spectrometry is increasingly being adapted for high-throughput applications. By integrating automated liquid chromatography, high-speed data acquisition, and AI-driven data analysis, researchers can now investigate large sets of protein complexes within significantly reduced timeframes, thereby enhancing the efficiency and scalability of proteomic studies.
2. Advanced Characterization of Protein–Small Molecule Interactions
Native mass spectrometry has emerged as a powerful tool for precisely characterizing protein–small molecule interactions. Recent advancements, including isotope labeling, competitive binding assays, and quantitative kinetic analyses, have enabled the accurate determination of binding affinities, binding sites, and conformational changes. These innovations provide key insights into structural biology and facilitate drug discovery efforts.
3. Enhanced Detection of Low-Abundance Proteins and Transient Complexes
Detecting low-abundance proteins and transient interaction complexes remains a challenge in native mass spectrometry. To address this, researchers have developed and integrated novel ionization strategies, such as enhanced electrospray ionization (ESI), alongside enrichment techniques like immunoprecipitation-MS. These advancements have significantly improved detection sensitivity, making native mass spectrometry a valuable tool for investigating complex biological systems.
4. Investigation of Protein Complex Dynamics
The integration of cryo-electron microscopy (Cryo-EM) with native mass spectrometry has provided a more comprehensive approach to studying the structural dynamics of protein complexes. While native mass spectrometry delivers precise mass and assembly state information, Cryo-EM offers high-resolution three-dimensional structural insights. This complementary combination has greatly expanded the scope of protein structural investigations, particularly in understanding conformational transitions under varying physiological conditions.
By leveraging advancements in ionization techniques, high-resolution mass analyzers, and multidimensional coupling strategies, native mass spectrometry has solidified its position as a fundamental tool for elucidating the native conformations of biomacromolecules. Ongoing technological refinements continue to enhance its applications in structural biology, proteomics, and pharmaceutical research. As these developments progress, native mass spectrometry is poised to become an indispensable platform for characterizing protein complex structures and interactions.
MtoZ Biolabs is committed to providing high-quality native mass spectrometry analysis service, including protein complex characterization, protein–ligand interaction studies, and conformational dynamics analysis. With a specialized research team and cutting-edge bioinformatics capabilities, we offer tailored solutions for fundamental research, drug discovery, and precision medicine, empowering scientists to drive innovations in life sciences.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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