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New Initiative Aims to Drive Development, Use of Capillary Electrophoresis in Top-Down Proteomics

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NEW YORK – With the launch last month of a new initiative, a group of top-down proteomics researchers is looking to drive technology development and increase use of online capillary electrophoresis as a separation technique upfront of mass spec analysis.

Sponsored by the Consortium for Top-Down Proteomics, the effort aims to develop guidelines for the use of CE in top-down research as well as to characterize the capabilities of current CE systems across a variety of labs and CE-MS systems.

The initiative comes as CE has seen significant uptake within the biopharma industry as a tool for characterization of intact proteins and has made inroads into both bottom-up and top-down proteomics research.

In August, for instance, Boston-based CE and mass spec firm 908 Devices announced the formation of a dedicated proteomics panel within its scientific advisory board, with Chris Brown, the company's chief technology officer, noting that it has in recent years consistently received inquiries from current and potential customers about the applications of its ZipChip CE platform within proteomics.

In June, Thermo Fisher Scientific signed an agreement with Ontario, Canada-based Advanced Electrophoresis Solutions, under which the firms will jointly promote Thermo Fisher's mass spectrometry systems for biopharma and proteomics applications and AES' whole-column imaging detection capillary electrophoresis systems for protein separation, quantification, and characterization.

Two forms of CE, capillary zone electrophoresis, which separates molecules based on their mass and charge, and capillary isoelectric focusing, which separates molecules based on their isoelectric points (the pH at which they have no net charge), are most commonly used for proteomics, though both techniques are far less common in proteomics than liquid chromatography.

Limited uptake of CE by proteomics researchers is due to a variety of factors, said Liangliang Sun, an assistant professor at Michigan State University and one of the leaders of the consortium's CE initiative.

One major issue, he noted, has been the poor stability of interfaces between CE devices and mass spectrometers. Another significant challenge has been the small loading capacity of CE systems, which has limited the amount of sample researchers can separate and analyze using CE, which in turn limits the sensitivity of their mass spec analyses.

These loading capacity limitations have been further exacerbated by the fact that in addition to the sample itself, a "sheath liquid" is in many setups required to establish the electrical connection used to separate the sample molecules. This sheath liquid further dilutes the sample, noted Kevin Jooss, a researcher at Northwestern University and one of the leaders of the CE initiative.

In the past, Jooss said, these limitations meant researchers couldn't with CE reach deep enough into samples to analyze the portions of the proteome that most interested them.

"There were applications for CE-MS and proteins, but there were always sensitivity issues or complexity issues," he said.

Now, though, improvements in CE technology have enabled researchers to reach similar levels of sensitivity with CE as they could with nanoLC, Jooss said. "We're reaching now the stuff that people are really interested in, like the low-abundance proteoforms of cancer-related proteins, for example."

He said that companies like Sciex and 908 Devices have improved the performance and ease of use of their CE technologies in response to demand from the biopharma industry.

Advantages sustain interest

Interest in CE has endured despite the limitations of earlier iterations of the technology due to its several potential advantages, particularly for top-down and intact protein separations.

One advantage is what Alexander Ivanov, associate professor of chemistry at Northeastern University and a leader of the initiative, described as CE's "open and tubular configuration," which allows it to separate proteins without denaturing them, making it a good fit for analysis of native proteins and protein complexes.

CE is also able to work with very small sample amount with little sample loss. Additionally, Jooss noted, CE systems are very flexible in that the capillaries can be coated with different compounds to optimize separations of different kinds of samples.

"Usually you have your one LC column that has these properties, and if I want to analyze a different type of sample I need to switch to a different column," he said. "With dynamic coating that is something you can avoid with CE because you can start with the same basic capillary and adapt it for your needs."

CE also provides sharper separation peaks than LC. Sun noted that it was particularly useful for separating larger molecules like proteins and protein complexes.

"If the analyte diffuses more slowly, your separation efficiency will be higher," he said. "So as we go from small molecules to peptides to proteins to protein complexes, we are getting bigger and bigger, and that means they diffuse slower, which means in theory we get better separation efficiency."

In the case of LC, this slower diffusion leads to wider peaks and lower-quality data.

Sun said that studies by his group and others have also demonstrated that CE offers significantly higher sensitivity than LC, noting that CE-MS can generate data comparable to that generated using LC-MS with tenfold to one hundredfold less sample.

He cited as another potential advantage the relative ease of predicting the behavior of a protein in a CE device, which he said meant data on, for instance, a proteoform's movement through a CE system could be used as additional information to confirm its identification.

That said, "there is still a long way to go to bring [the technology] to the next stage," Ivanov noted.

Development of more stable and user-friendly interfaces between the CE and the mass spectrometer remain an area in need of improvement. Reproducibility of CE separations is also an issue as even small changes can alter results.

"We are applying a high voltage to generate a current and move ions across the channel or capillary, and the composition [of the reagents used] changes," Ivanov said. "If the run is too long, or if you use the same buffer for multiple rounds and multiple injections and we are depleting certain ions in the buffer, that can contribute to certain reproducibility issues."

Jooss said that while CE has advanced to where it is possible to hit levels of reproducibility comparable to nanoLC, "there are just more factors of variation [in CE]. For instance, if your background electrolyte pH is 4.0 or 4.1 can make a slight difference in your separation. So you have to be very rigid in how you prepare your samples."

The consortium's initiative aims to develop and promulgate standards and practices that will help researchers use CE most effectively. One question, for instance, is whether the inclusion of standards within CE runs can help with the reproducibility question by enabling researchers to normalize CE data across runs and labs.

The participants also hope that raising the profile of CE will help drive uptake and technology development.

"If someone was never taught CE properly or is even aware of its capabilities or existence, it's unlikely they [will] decide on their own to venture into the field, which consequently has an influence on how it is perceived in industry and academia," Jooss said.

To start the consortium has lined up 12 labs around the world that will analyze standardized protein mixtures using their in-house CE-MS setups to get a sense of what kind of data is generated using the various mixes of instrumentation and protocols in place.

"We're trying to demonstrate the CE-MS techniques using the different expertise in these different labs," Sun said. "That will give us a solid foundation for the initiative, and then we will move on to more complex protein samples."

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