Will Capillary Electrophoresis Move to the Front of the Line in Top-Down Proteomics?

Will Capillary Electrophoresis Move to the Front of the Line in Top-Down Proteomics?

This blog post covers market trends in proteomics research - a field recently profiled by Kalorama Information withProteomics Research Markets: Instruments (Mass Spectrometry, Liquid Chromatography, Electrophoresis), Disease Areas (Oncology, Cardio/Blood, Neurology, Other) Market Share and Trends. The report offers unique insight into a mass spectrometry- and analytical separation-centric life sciences market, including market shares by geography, instrument type, and vendor.

The steadily evolving field of discovery proteomics can be divided roughly between “bottom-up” and “top-down” approaches to the screening and analysis of sample proteomes. The bottom-up shotgun approach has powered over a decade of extensive research in proteome mapping and protein discovery for biomarker and drug development. However, bottom-up approaches do not lend themselves well to the differentiation and discovery of proteoforms, or the different in vivo forms the same protein sequence can take including the fixation of post-translational modifications (PTMs). Top-down proteomics or the analysis of intact proteins is a less mature approach, but could contribute significantly to systems biology understanding and the characterization of drug targets in the drug discovery process. One obstacle to higher efficiency top-down proteomics studies is effective sample separation, which has motivated recent research into capillary electrophoresis (CE) methods.

Increasingly powerful liquid chromatography-tandem mass spectrometry (LC-MS/MS) systems form the basis of bottom-up shotgun proteomics, where digested peptides are scanned for initial mass/charge ratio (m/z value) and select peptides are fragmented for secondary m/z determination.  The approach is capable of identifying thousands of proteins in a single-run using informatics that determine the amino acid sequences of peptides and peptide fragments and match them to proteins. The rise of shotgun proteomics coincided with improvements to mass spectrometry acquisition rates and resolution able to match the rapid separation capabilities of high performance liquid chromatography (HPLC). Much as robust bottom-up proteomics came about through the successful interface of nanoscale LC and electrospray, the future of top-down proteomics may hinge on the demonstration and standardization of CE-electrospray ionization (ESI)-MS systems.

In a sense, electrophoresis embodied proteomics before the latter even became a term. Two-dimensional gel electrophoresis (2DGE) was profound in its ability to resolve up to thousands of sample proteins by separating them by molecular weight (MW) and isoelectric point (pI). Separated protein spots could later be excised from the gel slab and mass analyzed. While robust, 2DGE has lagged and taken a complementary position to LC-MS as its weaknesses were exposed under the mounting complexity and sensitivity demand in research. Gel electrophoresis does not provide adequate reproducibility for protein quantitation, which is increasingly important even in discovery phase activities. The separation technology also has limited dynamic range and imposes significant sample handling requirements with little amenability to high-throughput automation. Chromatography-mass spectrometry has provided superior dynamic range and reproducibility while operating in-line with mass spectrometry.

Widely deployed as an orthogonal or complementary form of separation and fractionation, capillary electrophoresis (CE) saw limited prospects for breakout growth in the proteomics research market until a recent shift in analytical priorities from high-throughput protein identification to protein characterization. Intact protein analysis is increasingly important to proteomics research for the identification of drug targets and elucidation of cell system pathways.

Several factors make CE, particularly capillary zone electrophoresis (CZE), attractive or superior to LC for use in top-down proteomics:

  • CE mitigates weak bonding or analyte interaction with separation media as in the case with LC columns; interaction between proteins or polypeptides and LC separation media impacts sample recovery and separation efficiency
  • Better handles large proteins and polypeptides (+50 kDa); large proteins in reverse-phase LC (RPLC) columns can take longer and are more difficult to elute, produce broad spectrum peaks, and reduce column lifetime
  • Faster separation can be accomplished using CZE than HPLC or within 10-30 minutes for most elutions compared to 50 minutes with HPLC
  • Greater sensitivity with resolution of analytes in the low nanogram range compared to the microgram-high nanogram range of HPLC
  • Higher efficiency separation is possible with theoretical plate counts approaching 100,000
  • Utilizes very small amounts of sample with CZE loading capacity one to three orders of magnitude lower than HPLC

The potential advantages of CE in top-down proteomics applications has not translated to market domination, however, due to the current limitations of the technology in common practice and proteomics MS platforms:

  • The efficiency and speed of CZE separation outstrip the spectra acquisition rate of current MS systems; single separations are not able to thoroughly capture high-complexity sample proteomes
  • Small sample loading capacities in the nanoliter can also be a hindrance by limiting the detectable dynamic range (low-abundance proteins in such a small sample can escape MS sensitivity) and MS instrument sensitivity

Several research programs are focused on optimizing CE technology and analytical practices for top-down proteomics. Much of the published research on top-down proteomics using CE has been in the last three years from researchers at institutes including Notre Dame , Northwestern, and at the Scripps Research Institute. A solution to the minute sample loading capacity of CZE is coupling the separation method with capillary isoelectric focusing (CIEF) or another second dimension of separation, though at a cost of added complexity in sample manipulations. Work also continues on optimizing CZE, as the simplest, most common form of capillary electrophoresis, for top-down proteomics. Importantly, researchers have been able to apply and refine electrospray ionization (ESI) with a sheathless design for on-line CZE-ESI-MS development.

An upcoming title from Kalorama Information will explore the markets for proteomics research instrumentation with a thorough accounting of the capabilities, specific applications, and development of technologies such as capillary electrophoresis, mass spectrometers, and liquid chromatography. A broader market outlook for proteomics, including clinical IVD products, is available with Proteomics Markets for Research and IVD Applications (Mass Spectrometry, Chromatography, Microarrays, Electrophoresis, Immunoassays, Other Technologies).