Analysis of Native Intact Protein Complexes Using the Agilent 6545XT AdvanceBio LC/Q-TOF Mass Spectrometer

Significance of the Topic

Native mass spectrometry preserves noncovalent interactions of protein complexes, enabling detailed analysis of stoichiometry, topology, and dynamics in their physiological state. This approach is crucial for proteomics, structural biology, and biopharmaceutical characterization.

Study Objectives and Overview

This work describes the development of a custom static nanoelectrospray ionization source for the unmodified Agilent 6545XT AdvanceBio LC/Q-TOF mass spectrometer. The study demonstrates high-resolution native MS analysis of diverse protein assemblies, including soluble enzymes, membrane channels, and chaperonin complexes up to 800 kDa, using microgram-scale samples.

Instrumentation Used

• Agilent Dual Nanospray Ion Source (G3253A)
• Agilent 6545XT AdvanceBio LC/Q-TOF mass spectrometer

Methodology

Samples of enzyme multimers (yeast alcohol dehydrogenase, rabbit pyruvate kinase), a bacterial membrane channel (AmtB mutant), and the GroEL chaperonin were buffer exchanged into 200 mM ammonium acetate. Borosilicate capillary tips were pulled in-house, fitted with a platinum wire, and used for static nanoelectrospray. Instrument parameters including capillary exit, skimmer, fragmentor, and collision cell potentials were optimized. Data were acquired in extended mass range modes and processed with Agilent MassHunter Qualitative Analysis and BioConfirm; molecular weights were calculated using ChemCalc.

Key Results and Discussion

• Alcohol dehydrogenase tetramer: zero-charge mass 147.5 kDa vs theoretical 146.9 kDa.
• Pyruvate kinase tetramer: measured 232.0 kDa; minor truncation species at ~229.9 kDa.
• AmtB homotrimer: 126.7 kDa; detection of up to three 77 Da β-mercaptoethanol adducts; monomer at 42.2 kDa.
• GroEL tetradecamer: 801.1 kDa matching 800.8 kDa theoretical; in-source dissociation yielded monomer, dimer, trimer subunits; collision-induced dissociation produced charge-stripped 13-mer and revealed a possible eight-mer species likely corresponding to a monomer-trapped ring.

Optimization of source potentials achieved a balance between ion transmission and minimal dissociation, while high vacuum and extended m/z acquisition enabled clear resolution of large complexes and low-abundance adducts or contaminants.

Benefits and Practical Applications

This platform delivers high sensitivity and resolution for native intact complexes using microgram quantities. It supports stoichiometric and structural characterization of enzymes, membrane proteins, and chaperonins, with applications in quality control, drug discovery, and biopharmaceutical development.

Future Trends and Potential Applications

Emerging directions include integration with ion mobility separation, expansion to megadalton assemblies, coupling with chromatographic techniques, and advanced data analysis for top-down native proteomics. These advances will enhance structural insights and facilitate screening of complex biomolecular interactions.

Conclusion

The Agilent 6545XT AdvanceBio LC/Q-TOF mass spectrometer with a custom static nESI source provides a robust, high-resolution solution for native MS of large protein complexes. This approach extends the capabilities of commercial instruments for detailed analysis of biomolecular assemblies.

References

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3. Liao Y.; et al. Removal of N-terminal Methionine from Recombinant Proteins by Engineered E. coli Methionine Aminopeptidase. Protein Sci., 2004, 13(7), 1802–1810.
4. McCabe J.; et al. First-Principles Collision Cross Section Measurement of Large Proteins and Protein Complexes. Anal. Chem., 2020, 92(16), 11155–11163.