Advancements in Capillary Zone Electrophoresis for Top-Down Proteomics

Proteoforms are distinct protein variations derived from a single gene that are critical to understanding biological processes and human disease. While top-down proteomics traditionally relies on reversed-phase liquid chromatography (RPLC) to analyze these intact proteins, the technique struggles efficiently separating larger proteoforms. Capillary zone electrophoresis combined with mass spectrometry (CZE-MS) offers a highly efficient alternative, separating proteoforms based on their charge-to-size ratio. Despite historical concerns regarding robustness, recent technological advancements have made CZE-MS highly sensitive and accessible.

A worldwide cross-laboratory study across 12 research groups now proves that diverse CZE-MS platforms provide reproducible, complementary insights to traditional methods. LCGC International spoke to Kevin Jooß of the Vrije Universiteit Amsterdam, one of the authors of a paper outlining the study,¹ about this work.

Why is intact mass measurement by high-resolution mass spectrometry (MS) often insufficient for unambiguous proteoform identification and how does tandem mass spectrometry (MS/MS) in top-down proteomics address this limitation?

Intact mass measurement with high-resolution MS is extremely powerful, but on its own it’s often not enough to unambiguously identify a proteoform. The main reason is that different proteoforms can end up having the same, or nearly the same, overall mass. For example, different post-translational modifications or sequence variants may be isobaric, and intact mass doesn’t tell you where a modification is located on the protein. So, while you get a very accurate molecular weight, you’re still missing important structural information.

That’s where MS/MS in top-down proteomics becomes essential. By fragmenting the intact protein and analyzing the resulting fragment ions, you can map modifications to specific regions or residues and confirm sequence features directly. In other words, MS/MS provides the sequence-level and positional information that intact mass alone can’t deliver.

What are the main physicochemical reasons reversed-phase liquid chromatography (RPLC) struggles to efficiently separate large intact proteoforms (>30 kDa), and how do these limitations manifest in chromatographic performance?

Large intact proteoforms often differ subtly in structure, which can be challenging for any separation techniques. For instance, physicochemically, their very low diffusion coefficients, orders of magnitude lower than small molecules, lead to slow mass transfer effects and an increased C-term contribution in the van Deemter relationship. In addition, full pore accessibility cannot be assumed, requiring wide-pore materials (e.g., 300 Å) to mitigate steric exclusion and restricted diffusion. These combined effects can manifest as reduced efficiency, peak broadening, adsorption artifacts, and limited resolving power for subtle proteoform variants.

How does the separation mechanism of capillary zone electrophoresis (CZE) differ fundamentally from RPLC, and why is this particularly advantageous for resolving closely related proteoforms with charge-altering post-translational modifications (PTMs)?

This is a question I get asked frequently. Fundamentally, RPLC separates based on hydrophobic partitioning between two phases, whereas capillary zone electrophoresis separates analytes in free solution in an electric field according to their electrophoretic mobility, which depends on charge-to-size ratio. Because many proteoforms differ primarily by charge-altering PTMs such as phosphorylation or deamidation, CZE translates even small net charge differences directly into mobility shifts. This makes it particularly powerful for resolving closely related proteoforms that may be poorly differentiated by hydrophobicity in RPLC.

Explain how electrophoretic mobility (µep) governs proteoform separation in CZE and discuss the relative influence of net charge versus hydrodynamic radius for intact proteins.

Electrophoretic mobility governs CZE separation because analytes migrate according to their charge-to-friction ratio in an electric field. For intact proteoforms, net charge is typically the dominant determinant of mobility, as PTMs frequently alter ionizable groups while only minimally affecting hydrodynamic radius. Consequently, even single charge differences can produce measurable mobility shifts, making CZE particularly powerful for resolving closely related charge variants.

CZE can achieve nearly one million theoretical plates for proteoform separations. What factors contribute to this exceptionally high efficiency compared to liquid chromatography (LC)-based separations?

Indeed, under ideal circumstances, CZE achieves exceptionally high efficiency because it eliminates the eddy diffusion and mass transfer terms that fundamentally limit LC. Separation occurs in free solution under a plug-like electroosmotic flow profile, and for large proteoforms, their low diffusion coefficients further suppress longitudinal band broadening. When combined with high electric field strengths, this allows theoretical plate counts approaching one million, exceeding typical LC-based separations. However, in practical proteoform separations, plate numbers exceeding one million are rare. For instance, even when advanced capillary coating strategies are employed, unwanted protein-wall interactions can potentially introduce adsorption kinetics and peak tailing, injection plug dispersion can significantly broaden zones, and Joule heating can create temperature-induced mobility gradients. Thus, while CZE has an exceptionally high theoretical efficiency ceiling, real-world performance depends critically on surface passivation, injection control, and thermal management.

Historically, CZE-MS has been viewed as less robust and sensitive than LC-MS. What were the main technical challenges and how have modern interface designs addressed these concerns?

I think CZE-MS has come a long way since its introduction decades ago. Interfacing CE technology with MS instruments comes with a unique set of challenges. including unstable electrospray at ultra-low flow rates, electrical decoupling difficulties, limited sample loading, and protein adsorption effects. These concerns have certainly been true historically but have been the focus of industrial and academic development and technological advancements over the last decades. Modern (sheathless and nano-sheath flow) interfaces, improved nano-electrospray ionization (ESI) sources, advanced capillary coatings, and on-line preconcentration strategies have largely overcome these issues. As a result, contemporary CZE–MS systems now offer sensitivity and robustness that are competitive with, and in some cases superior to, LC–MS for intact proteoform analysis.

Compare sheath-flow and sheathless CE-MS interfaces in terms of sensitivity, robustness, and compatibility with top-down proteomics workflows.

Traditional sheath-flow interfaces, such as the triple-tube design, offer high operational robustness and spray stability, albeit at the expense of sensitivity due to high CE effluent dilution. In contrast, nano-sheath-flow and sheathless designs substantially reduce or eliminate dilution and operate in the true nano-ESI regime, thereby enhancing sensitivity for intact proteoforms. Although nano-sheath-flow configurations still introduce some dilution of the CZE effluent, spray conditions can be carefully optimized via the sheath liquid, making nano-sheath-flow and sheathless interfaces largely comparable in terms of sensitivity. While these low-flow interfaces may require more deliberate optimization, they are particularly well suited for high-sensitivity top-down proteomics workflows. A related and more specialized approach involves microchip capillary electrophoresis (CE)–MS platforms, in which the nano-electrospray emitter and sheath liquid delivery are integrated directly onto the chip. Ultimately, interface selection depends on the specific application and user priorities; in our work, we aimed to demonstrate the versatility of multiple CE–MS platform configurations.

Why is CZE-MS considered complementary rather than competitive to RPLC-MS in top-down proteomics, and how does this complementarity translate into increased proteoform coverage?

CZE and RPLC are considered largely complementary because they separate proteoforms based on fundamentally different physicochemical principles: charge-to-size ratio in the case of CZE and hydrophobicity in RPLC. Because proteoforms often differ in charge, hydrophobic character, or both, each technique resolves a partially distinct subset of species. Integrating data from both separation strategies therefore expands overall proteoform coverage in top-down proteomics workflows beyond what either method can achieve independently.

What insights does a multi-laboratory, multi-platform benchmarking study provide that single-lab method development studies cannot, particularly in assessing reproducibility and robustness of CZE-MS?

While single-laboratory studies demonstrate what is technically achievable under carefully optimized conditions, multi-laboratory benchmarking evaluates what is reproducibly achievable across diverse platforms, instruments, and operators. Our goal was to benchmark and highlight the diversity of CE–MS implementations, demonstrating that reproducible and high-quality results can be obtained across different interface designs and configurations.

Furthermore, our study provides the community with a practical reference framework, including transferable experimental protocols and performance metrics. This not only improves the collective understanding of CZE–MS capabilities in top-down proteomics but also supports standardization efforts and accelerates broader adoption of CZE–MS for proteoform analysis.

Looking ahead to the Human Proteoform Project, what remaining technical challenges must be addressed for broader adoption of CZE-MS in top-down proteomics (TDP), and what strategies appear most promising to overcome them?

Of course, there is always room for improvement, and I am excited about the future development of CZE–TDMS. While many aspects of top-down proteomics workflows require further optimization, I will focus on a few key challenges specific to CZE.

A central limitation of CZE–MS in the analysis of complex samples is its relatively low sample loading capacity, which can constrain sensitivity and overall proteome coverage. To address this, substantial progress has been made in on-line preconcentration strategies, such as dynamic pH junction and solid-phase microextraction, which significantly enhance effective loading amounts. Nevertheless, continued improvements in capillary coating stability and sample introduction strategies will be essential to enable more routine and robust analyses of complex and low-input samples.

Although CZE provides exceptionally high separation efficiency, its inherently narrow peak widths, often only a few seconds, can become a bottleneck if the MS duty cycle cannot keep pace. In highly complex proteome samples, this may result in undersampling of proteoform peaks within a single or only few runs. However, tandem MS technology is continuously evolving, and we expect that CZE can particularly benefit from advancements in terms of acquisition speed to further unlock the potential of CZE–MS.

Ultimately, no single separation technique will be sufficient to cover the entire spectrum of the human proteome, and we expect that CZE–MS will serve as a critical complementary technology to RPLC-MS in achieving comprehensive proteoform coverage for the Human Proteoform Project.

References

1. Gould, N.; Wang, Q.; Agar, J. N. et al. Intact Proteoform Analysis by Capillary Electrophoresis-Mass Spectrometry. Are We There Yet? Angew Chem. Int. Ed. Engl. 2025, e18366. DOI: 10.1002/anie.202518366