Native Capillary Electrophoresis–Mass Spectrometry Enables Quantitative Profiling of Endogenous Protein Complexes

Researchers from the Department of Chemistry at Michigan State University (East Lansing, Michigan) report that they have developed a label-free quantitative native proteomics workflow using native capillary zone electrophoresis–mass spectrometry (nCZE-MS) to profile endogenous protein assemblies, or complexoforms, in a complex biological sample (1). By leveraging high-resolution native mass spectrometry (MS), the authors quantify differential complexoform expression in Escherichia coli during the transition from logarithmic to stationary growth phases.

The nCZE-MS approach enables sensitive, proteome-scale detection of intact proteoforms and higher-order assemblies under near-physiological conditions, revealing dynamic reorganization of protein complexes associated with stress adaptation. These results highlight native spectrometry as a powerful platform for quantitative analysis of complexoform regulation in living systems. LCGC International spoke to the paper’s authors, Fei Fang and Liangliang Sun, about this work.

How does native size-exclusion chromatography (SEC) contribute to preserving noncovalent complexoforms during MS analysis, and what limitations does SEC introduce when analyzing high-mass protein assemblies?

Native SEC can preserve noncovalent complexoforms because of its unique characteristics. First, SEC columns are composed of porous silica particles with highly hydrophilic surfaces, such as diol coatings, which minimize interactions between analytes and the stationary phase. Second, unlike chromatographic methods such as reversed-phase liquid chromatography (RPLC) that rely on chemical interactions, SEC separates molecules based solely on their hydrodynamic size in solution. Consequently, neutral-pH, volatile buffers—such as ammonium acetate (pH ~7.0–7.4)—could be used in SEC to maintain physiological conditions and reduce the risk of disrupting fragile noncovalent interactions.

Limitation: To run SEC in a truly “native” mode, several critical factors must be optimized to preserve non-covalent complexes while maintaining adequate separation resolution under MS-compatible conditions.

• Choice of mobile-phase compositions: Although SEC is generally considered a non-retentive chromatographic technique, non-specific interactions can still occur between protein analytes and the stationary-phase material. These interactions may cause protein adsorption, shifts in elution time, peak tailing, and band broadening. More importantly, such interactions can alter the native conformational or functional state of the proteins. Volatile salts such as ammonium acetate or ammonium formate at high ionic strengths (100–200 mM) have been shown to sufficiently suppress interactions between biomolecules and the packing material, enabling characterization of higher-order structure, proteoform variants, aggregates, and stable protein complexes. However, operating at these high salt concentrations introduces substantial ion suppression in mass spectrometry, reducing sensitivity to low-abundance and high-molecular-weight species. Furthermore, the evaporation of aqueous SEC solvents during sample introduction requires elevated source temperatures and high desolvation-gas flow, which can induce gas-phase structural disruption of protein complexes.
• Choice of column pore size: The pore size of the SEC column must be carefully selected so that smaller analytes can enter the pores while larger complexes are appropriately excluded. An incorrect pore size can result in large complexes being completely excluded or smaller analytes experiencing undesired retention, both of which compromise separation quality and structural preservation.
• Size of the SEC column and flow considerations: Most current SEC separations for native mass spectrometry (MS) are performed at microflow rates, which generate shear forces capable of mechanically disrupting fragile, non-covalent assemblies. Reducing the flow rate—such as through nanoflow SEC—can mitigate these shear-related effects, which is especially important for weakly bound complexes susceptible to flow-induced dissociation. However, to the best of our knowledge, only one study to date has integrated a home-built nanoflow SEC system for native MS analysis (2).

What are the advantages and limitations of using mixed-bed ion-exchange chromatography coupled online to mass spectrometry for separating native protein complexes?

Compared with other chromatographic methods, ion exchange chromatography (IEC) stands out for its high resolving power, as it separates protein complexes based on their distinct surface charge distributions. Under nondenaturing conditions, IEC separates proteins according to their surface charge characteristics, which are largely predictable from their isoelectric points (pI). Because the proteome exhibits a wide, bimodal pI distribution, complex biological samples contain large numbers of both acidic and basic proteins, making IEC method development challenging. To overcome the recovery issue, a mixed-bed ion exchange column—containing both cation- and anion-exchange materials—operated at neutral pH is expected to retain both acidic and basic proteins and thus represents a promising approach for native analyses of complex endogenous mixtures.

The core challenges with using IEC include:

• Limited selection of volatile additives for IEC–MS: Direct coupling of IEC to MS requires volatile salts, with the narrow selection of volatile salts (mainly ammonium acetate, ammonium formate, and ammonium bicarbonate) restricting both the usable buffer range and buffering capacity. As a result, the resulting pH gradients are often nonlinear, which can cause unexpected protein retention behavior or coelution. For native IEC, salt-mediated pH gradient elution mode is mostly employed, where proteins are typically released using high salt concentrations—often up to 1 M ammonium acetate—which necessitates harsh desolvation conditions during electrospray ionization. These conditions lead to severe ion suppression (where the analyte signal decreases as salt concentration increases) and can induce protein denaturation, resulting in poor MS sensitivity.
• Limited analytical sensitivity compared with low flow LC–MS: IEC–MS, including mixed-bed IEC, is typically performed at analytical-scale flow rates, which limits sensitivity relative to low flow LC–MS. This presents challenges for proteomics applications, where endogenous samples contain proteins spanning a wide dynamic concentration range.
• Difficulty in preparing homogeneous mixed-bed columns: Fabricating a uniform mixed-bed IEC column remains technically challenging, and such columns are not commercially available to the best of our knowledge.

What chromatographic principles allownative capillary zone electrophoresis (nCZE) to resolve intact complexoforms, and how does protein adsorption to the capillary wall impact this process?

CZE employs open tubular capillaries to separate analytes at a nanoliter-per-minute flow rate under a strong electric field without a stationary phase. These features of CZE provide fundamental benefits for large biomolecules. CZE minimizes peak broadening and sample carry-over issues in LC for large biomolecules (such as intact complexoforms) because it does not use a stationary phase for separation. In addition, CZE separates complexoforms based on their electrophoretic mobility, which correlates to their charge-to-size ratios. Therefore, various volatile buffers could be performed in the native CZE experiment.

In an ideal situation, the separation efficiency of CZE, represented by the number of theoretical plates (N), only depends on the electrophoretic mobility of analytes (μ), the voltage applied across the capillary (V), and the diffusion coefficient (D) of analytes in the equation below:

When separating large biomolecules (for example, intact complexoforms), CZE can achieve high separation efficiency because complexoforms are large biomolecules with a low D. When a high voltage (for example, 30 kV) is applied for CZE separation of complexoforms, a high N can be achieved.

When proteins adsorb non-specifically onto the capillary inner wall due to electrostatic and hydrophobic interactions between the protein and the capillary surface, the separation efficiency and reproducibility can be significantly reduced. Therefore, a proper coating on the capillary inner wall is essential for CZE-MS-based native proteomics.

How does buffer exchange using centrifugal filters (such as Amicon 10 kDa units) mimic a chromatographic desalting step, and why is matching volatility and ionic strength critical for downstream native MS analysis?

The SEC chromatographic desalting step uses a column packed with porous resin beads. Large molecules cannot enter the pores and travel quickly through the column. Small molecules, such as salts, enter the pores and take a longer, slower path. The SEC desalting is helpful for native MS and has been online coupled to native MS for complexoform analysis (3). Buffer exchange with centrifugal filters mimics a chromatographic desalting step by separating macromolecules from smaller moleculesbased on size exclusion, using a semi-permeable membrane with a molecular weight cut-off (MWCO) (for example, 10 kDa units) and centrifugal force. The SEC approach can be operated online with native MS, and the centrifugal filter approach is usually operated offline.

High salt concentrations are often necessary to maintain protein stability in solution. However, the sample buffer with a higher salt concentration than the background electrolyte of CZE will cause peak broadening during CZE separation. Therefore, matching volatility and ionic strength is critical for native CZE-MS.

What chromatographic considerations govern the selection of background electrolytes (BGEs) in nCZE-MS for native proteomics, and how might altering pH or ionic strength affect complexoform stability and resolution?

The key chromatographic considerations for selecting BGEs in nCZE-MS are preserving protein structure, compatibility with electrospray ionization, and achieving sufficient separation resolution. BGEs must be MS-compatible (such as ammonium acetate), and their conductivity and viscosity will affect separation efficiency, while their volatility will affect MS performance.

The pH of BGE affects complexoform stability and resolution by influencing the charge of the molecules involved, which alters intermolecular forces like electrostatic repulsion and hydrogen bonding, and can cause changes in solubility or precipitation. Therefore, optimal pH conditions are necessary to maintain the desired complex's structural integrity and prevent its dissociation or precipitation. In addition, with pH influencing the charge of the complexes, the migration rates of complexoforms could be affected, further reducing separation resolution.

An appropriate ionic strength of BGE helps to maintain the protein's native structure and prevents unfolding or dissociation. High salt concentrations are often necessary to maintain protein stability in solution by promoting favorable intermolecular interactions or reducing electrostatic repulsion. However, using buffers containing such high concentrations of volatile salts leads to significant ion suppression in MS, compromising the detection of low-abundant and high-molecular-weight species. Moreover, evaporation of the aqueous solvents requires high temperatures during sample introduction into the ion source. The higher desolvation-gas flow and source temperature often result in gas-phase events that disrupt the protein’s higher-order structure. In addition, high ionic strength decreases the effective mobility of complexoform due to increased ion-pairing, thereby reducing separation speed and affecting separation resolution.

From a separation-science perspective, how does orthogonality between chromatographic and gas-phase separation steps enhance complexoform characterization?

When dealing with a complex biological sample, multi-dimensional separations are essential to provide a deep view of the biomolecules in the sample. The separation principles in the different dimensions need to be different enough to allow orthogonal separations to maximize the separation peak capacity. The liquid-phase separation method, CZE, can be coupled with ion mobility separations in the gas phase, for an online two-dimensional separation of complexoforms in a complex system. The approach has been used for denaturing top-down proteomics of proteoforms in our previous studies (4,5).

Migration-time drift was observed across repeated nCZE runs. What chromatographic mechanisms could explain this drift, and what strategies—instrumental or chemical—can be implemented to stabilize migration behavior for quantitative studies?

When proteins adsorb non-specifically to the capillary inner wall, the inner wall chemistry will change, leading to inconsistent flow in the capillary and a migration time shift. To reduce protein nonspecific adsorption and migration time shifts, neutral and hydrophilic capillary coatings (such as LPA) can be employed. It is worth pointing out that the polymers having the nitrogen element can lead to significant protein adsorption (6). Therefore, the LPA coating may not be ideal. Alternative capillary coating might help further reduce protein adsorption. For example, carbohydrate-based polymers, which were reported to have excellent resistance to protein non-specific adsorption (7,8).

Compared to classical chromatography-based bottom-up proteomics, how does chromatographic peak width and dispersion in native CZE-MS influence quantification accuracy and the ability to detect low-abundance protein complexes?

In comparison with peptides under denaturing chromatography conditions, which yield sharp, well-resolved peaks, the intact protein complexes in native CZE-MS often have broader peaks due to their size, heterogeneity in conformations, and potential interactions with the capillary inner wall. Therefore, the proteome coverage from native proteomics is usually limited. To improve proteome coverage, a multi-dimensional approach that combines LC fractionation (SEC) with CZE-MS will be essential. In terms of protein complex quantification, this study represents the first example of label-free quantitative native proteomics of complex samples. One advantage of CZE-MS-based quantitative native proteomics is that CZE uses a consistent background electrolyte (BGE) composition for separation, and the ionization efficiency of complexoforms is insensitive to migration time shifts from run to run, making CZE-MS a valuable tool for quantitative native proteomics.

Given the wide mass range of detected complexoforms (20–320 kDa), how would you design a chromatographic method—SEC, IEC, CZE, or hybrid approaches—to maximize separation capacity while maintaining native-state integrity for complex bacterial lysates?

To maximize separation capacity while preserving native complexoform integrity, a hybrid approach that balances resolving power, gentle sample handling, and MS compatibility is needed. In our opinion, a combination of SEC and CZE is a good option since CZE has high separation efficiency for complexoforms and CZE-MS offers excellent sensitivity for complexoform detection (3,9,10). SEC separates complexoforms by size and allows the use of optimized CZE-MS conditions for a specific size range of complexoforms in different SEC fractions. In addition, SEC and CZE-MS can both maintain the structural integrity and topology of complexoforms because they both avoid strong interactions between complexoforms and solid surfaces (for example, LC beads) (8,11,12). In our recent work, we employed this strategy for bacterial lysate and detected native endogenous protein complexes with masses up to 1.5 MDa from SEC fractions of a whole cell lysate.

References

1. Fang, F.; Sun, L. Quantitative Native Proteomics by Capillary Zone Electrophoresis-Mass Spectrometry. Anal. Chem. 2025.DOI: 10.1021/acs.analchem.5c06099
2. Zhai, Z.; Holmark, T.; van der Zon, A. A. M. et al. Nanoflow Size Exclusion Chromatography-Native Mass Spectrometry of Intact Proteoforms and Protein Complexes. Anal Chem. 2025,97 (23), 12241-12250.DOI: 10.1021/acs.analchem.5c01019
3. VanAernum, Z. L.; Busch, F.; Jones, B. J. et al. Rapid Online Buffer Exchange for Screening of Proteins, Protein Complexes and Cell Lysates by Native Mass Spectrometry. Nat. Protoc. 2020, 15 (3),1132-1157. DOI: 10.1038/s41596-019-0281-0
4. Xu, T.; Wang, Q.; Wang, Q. et al. Coupling High-Field Asymmetric Waveform Ion Mobility Spectrometry with Capillary Zone Electrophoresis-Tandem Mass Spectrometry for Top-Down Proteomics. Anal. Chem. 2023, 95 (25), 9497-9504.DOI: 10.1021/acs.analchem.3c00551
5. Wang, Q.; Fang, F.; Wang, Q. et al. Capillary Zone Electrophoresis-High Field Asymmetric Ion Mobility Spectrometry-Tandem Mass Spectrometry for Top-Down Characterization of Histone Proteoforms. Proteomics 2024, 24 (3-4), e2200389.DOI: 10.1002/pmic.202200389
6. Metzke, M.; Guan, Z. Structure-Property Studies on Carbohydrate-Derived Polymers for Use as Protein-Resistant Biomaterials. Biomacromolecules 2008, 9 (1), 208-15. DOI: 10.1021/bm701013y
7. Metzke, M.; Bai, J. Z.; Guan, Z. A Novel Carbohydrate-Derived Side-Chain Polyether with Excellent Protein Resistance. J. Am. Chem. Soc. 2003, 125 (26), 7760-7761. DOI: 10.1021/ja0349507
8. Wang, Q.; Wang, Q.; Qi, Z. et al. Native Proteomics by Capillary Zone Electrophoresis-Mass Spectrometry. Angew Chem. Int. Ed. Engl. 2024, 63 (48), e202408370.DOI: 10.1002/anie.202408370
9. Belov, A. M.; Viner, R.; Santos ,M. R. et al. Analysis of Proteins, Protein Complexes, and Organellar Proteomes Using Sheathless Capillary Zone Electrophoresis - Native Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2017, 28 (12), 2614-2634. DOI: 10.1007/s13361-017-1781-1
10. Jooß, K.; McGee, J. P.; Melani, R. D. et al. Standard Procedures for Native CZE-MS of Proteins and Protein Complexes up to 800 kDa. Electrophoresis 2021, 42 (9-10), 1050-1059. DOI: 10.1002/elps.202000317
11. Moeller, W. J.; Qi, Z.; Wang, Q. et al. Does Native Capillary Zone Electrophoresis-Mass Spectrometry Maintain the Structural Topology of Protein Complexes? Anal. Chem. 2025, 97 (14), 7616-7621. DOI: 10.1021/acs.analchem.4c06949
12. Shen, X.; Kou, Q.; Guo, R. et al. Native Proteomics in Discovery Mode Using Size-Exclusion Chromatography-Capillary Zone Electrophoresis-Tandem Mass Spectrometry. Anal. Chem. 2018, 90 (17), 10095-10099. DOI: 10.1021/acs.analchem.8b02725