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

Importance of the topic

Native mass spectrometry of intact protein complexes enables the study of biomolecular assemblies while retaining their noncovalent interactions. This approach is critical in proteomics, structural biology and pharmaceutical research for characterizing composition, stoichiometry and topology of soluble enzymes, membrane channels and large chaperonins without denaturing the complexes.

Objectives and study overview

This work presents the adaptation of a static nanoelectrospray (nESI) interface on an unmodified Agilent 6545XT AdvanceBio LC/Q-TOF mass spectrometer. The goal was to demonstrate high-resolution analysis of native protein assemblies across a wide mass range, including soluble tetramers, membrane trimers and large chaperonin complexes, using only micrograms of material.

Methodology and instrumentation used

Sample preparation and nESI interface were optimized to preserve native interactions and maximize sensitivity:

• Buffer exchange to 200 mM aqueous ammonium acetate (plus 0.5 % C8E4 for membrane proteins) using spin columns.
• Borosilicate capillaries pulled in-house and fitted with a platinum grounding wire in a custom tip holder for static nESI.
• Analysis performed on Agilent Dual Nanospray Ion Source (G3253A) coupled to the 6545XT AdvanceBio LC/Q-TOF.
• Source and collision cell voltages tuned to balance ion transmission and minimize in-source dissociation.
• Data processed with Agilent MassHunter Qualitative Analysis and BioConfirm software.

Main results and discussion

The static nESI approach yielded well-resolved spectra for multiple native complexes:

• Alcohol dehydrogenase tetramer (147 kDa) observed centered at charge state 27+, with measured mass 147.5 kDa matching theory within adduct tolerance.
• Pyruvate kinase tetramer (232 kDa) detected at charge state 36+; minor truncated forms were identified by shifted masses.
• Bacterial ammonium transporter AmtB trimer (127 kDa) showed up to three covalent β-mercaptoethanol adducts; collision-induced dissociation confirmed monomer mass and adduction pattern.
• GroEL chaperonin tetradecamer (800 kDa) displayed charge states centered at 74+; in-source dissociation released monomer, dimer and trimer subunits. Collision cell activation enabled charge stripping to 13-mer and revealed a putative octamer species.

Benefits and practical applications of the method

This configuration allows high-resolution native MS of large assemblies using minimal sample quantities. It supports studies of membrane proteins and megadalton complexes, facilitates detection of modifications and contaminants, and enables downstream applications in structural proteomics, biopharmaceutical quality control and fundamental research.

Future trends and potential applications

Ongoing developments include coupling native MS with ion mobility for conformational analysis, extending mass range and resolution in Q-TOF platforms, integrating top-down fragmentation for sequence mapping of intact complexes and deploying these methods in automated high-throughput workflows for drug discovery and biomarker validation.

Conclusion

The custom static nESI source on the unmodified Agilent 6545XT AdvanceBio LC/Q-TOF provides robust, high-resolution analysis of intact native protein complexes up to and above 800 kDa. This approach enhances sensitivity for diverse assemblies and opens new avenues for detailed structural and functional investigations in proteomics.

Reference

1. Allison, T.; et al. Native Protein Mass Spectrometry. Methods Mol. Biol. 2019, 2073, 287–299.
2. Patiny, L.; Borel, A.; Ozil, M.; Kast, M.; Froidevaux, P.; Christ, C. ChemCalc: A building block for tomorrow’s chemical infrastructure. J. Chem. Inf. Model. 2013, 53(5), 1223–1228.
3. Liao, Y.; Pan, Y.; Hu, Z.; Wong, P.; Lescar, J.; Lu, Y.; Wang, L. Removal of N-terminal Methionine from Recombinant Proteins by Engineered E. coli Methionine Aminopeptidase. Protein Sci. 2004, 13(7), 1802–1810.
4. McCabe, J. W.; et al. First-Principles Collision Cross Section Measurement of Large Proteins and Protein Complexes. Anal. Chem. 2020, 92(16), 11155–11163.