Comprehensive Approaches to Higher-Order Structure of Intact Proteins by Native Mass Spectrometry

Importance of the Topic

Native mass spectrometry (nMS) has become a vital tool in analytical chemistry for studying proteins and their noncovalent assemblies in near-native states. By preserving higher-order structures and enabling sensitive mass confirmation with minimal sample consumption, nMS supports biopharmaceutical characterization, structural biology research, and quality control of monoclonal antibodies (mAbs), antibody–drug conjugates, and large protein complexes.

Objectives and Study Overview

This application note outlines a suite of complementary techniques for the analysis of intact proteins and their noncovalent complexes using Agilent high-resolution mass spectrometers. It evaluates offline and online buffer exchange workflows, varying flow rates from nanoelectrospray infusion to conventional microflow, capillary electrophoresis (CE), and high-throughput capillary flow with JetStream electrospray. The study also integrates ion mobility–mass spectrometry (IM-MS) for probing conformational features of proteins.

Methodology

Sample preparation combined offline buffer exchange with Micro Bio-Spin columns and online desalting via size exclusion chromatography (SEC) using ammonium acetate buffers at neutral pH. Analytical approaches included:

• NanoESI infusion of buffer-exchanged samples on Q-TOF instruments for sensitive detection of low-abundance complexes.
• Online microflow SEC with capillary and standard nebulizers to remove salts and introduce proteins directly to the mass spectrometer.
• High-throughput SEC workflows using Agilent AdvanceBio guard columns and JetStream ESI for rapid desalting.
• Capillary electrophoresis–ESI for both native and denaturing separations, employing sheath liquid interfaces and isocratic pumps.
• Ion mobility separations on the Agilent 6560 IM-Q-TOF to monitor collision-induced unfolding and conformational dynamics.

Used Instrumentation

• Agilent 1290 Infinity II LC system (binary pump, multisampler, thermostatted column compartment).
• Agilent 6500 and 6200 Series accurate-mass Q-TOF MS with dual ESI and JetStream sources.
• Agilent 7100 Capillary Electrophoresis system with CE-ESI sprayer and splitter assemblies.
• Agilent 6545XT AdvanceBio LC/Q-TOF with extended mass range (m/z up to 30,000) and SWARM autotune for high-mass transmission.
• MassHunter software suite (Acquisition, Qualitative Analysis, BioConfirm) and UniDec for deconvolution.

Main Results and Discussion

Optimizing source conditions—lower gas temperature, reduced drying gas flow, and moderate nebulizer pressure—preserved noncovalent assemblies such as glutamate dehydrogenase (hexamer) and GroEL (14-mer) without subunit dissociation. High-throughput JetStream ESI increased sensitivity for alcohol dehydrogenase by 50% compared to standard nebulization. The 6545XT instrument enabled detection of complexes up to 801 kDa in native mode and supported tandem MS (MS/MS) and collision-induced unfolding experiments in the IM-MS format. NanoESI infusion of NIST mAb reference material demonstrated robust detection at on-column loads as low as 1 µg, with consistent mass accuracy and minimal adduct formation.

Benefits and Practical Applications

The described workflows offer flexible solutions for routine and advanced applications:

• Rapid online desalting of intact proteins for mass confirmation and heterogeneity assessment.
• High-sensitivity nanoESI for low-abundance samples in pharmacokinetics and drug-metabolism studies.
• CE-ESI to separate proteoforms and monitor structural differences under native versus denaturing conditions.
• IM-MS to investigate conformational stability, folding intermediates, and collision-induced unfolding profiles.

Future Trends and Applications

Continued development of high-mass transmission technologies and software deconvolution will extend nMS to even larger assemblies, including virus capsids and membrane protein complexes. Integration with hydrogen–deuterium exchange, cross-linking, and top-down fragmentation promises deeper insights into protein dynamics. Automation of buffer exchange and microfluidic interfaces will further increase throughput for biopharmaceutical screening.

Conclusion

The combination of SEC-based desalting, nanoESI infusion, CE-ESI separation, and ion mobility on Agilent high-resolution MS platforms delivers a versatile toolkit for characterizing intact proteins and their higher-order structures. Optimized source conditions and extended mass range capabilities enable reliable detection of large noncovalent complexes with high sensitivity, making these methods well suited for both research and quality-control environments.

References

1. Valliere-Douglass, J. F.; McFee, W. A.; Salas-Solano, O. Native Intact Mass Determination of Antibodies Conjugated with Monomethyl Auristatin E and F at Interchain Cysteine Residues. Anal. Chem. 2012, 84, 2843–2849.
2. Freeke, J.; Robinson, C. V.; Ruotolo, B. T. Residual Counter Ions Can Stabilise a Large Protein Complex in the Gas Phase. Int. J. Mass Spectrom. 2010, 298, 91–98.
3. Campuzano, I.; Giles, K. Nanospray Ion Mobility Mass Spectrometry of Selected High Mass Species. In Nanoproteomics: Methods and Protocols; 2011; pp 57–70.
4. Marty, M. T.; et al. Bayesian Deconvolution of Mass and Ion Mobility Spectra: From Binary Interactions to Polydisperse Ensembles. Anal. Chem. 2015, 87, 4370–4376.