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Northwestern Team Develops System for Simplifying Sample Cleanup in Top-Down Proteomics

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NEW YORK (GenomeWeb) – The lab of Northwestern University researcher Neil Kelleher has developed an on-line system for clean up of top-down proteomics samples following gel eluted liquid fraction entrapment electrophoresis (GELFrEE) fractionation.

The system, detailed in a paper published this month in the Journal of Proteome Research, uses asymmetrical flow field-flow fractionation (AF4) to address what is a key challenge in top-down proteomic workflows, and could make top-down approaches more widely shareable as well as improve their applicability to clinical work, Philip Compton, director of instrumentation at NorthWestern's Proteomics Center of Excellence and an author on the paper, told GenomeWeb.

Sample complexity is a major challenge of top-down proteomics and, as such, top-down workflows typically use extensive fractionation and separation. Kelleher's lab — one of the leading developers of top-down methodologies — has adopted GELFrEE fractionation as part of its standard approach.

Using the method, Kelleher and his colleagues have managed several of the largest top-down proteomics studies to date, including a 2013 paper in Molecular & Cellular Proteomics in which he and his team identified 1,220 proteins and more than 5,000 proteoforms in H1299 cells.

GELFrEE fractionation, however, requires use of SDS, which is not compatible with electrospray mass spec and so must be removed prior to mass spec analysis. And while a variety of methods ranging from spin columns to precipitation approaches exist for SDS removal, all have certain disadvantages within top-down workflows.

Spin-columns, the authors noted, can lack reproducibility as they are prone to unpredictable protein losses or modifications.

Precipitation, meanwhile, requires "a very practiced hand" to get good recovery and reproducible results, Compton said. "You have to have someone who is very good at the bench to do it effectively."

This is particularly an issue given the Northwestern researchers' goal of popularizing use of top-down methods in other labs, he noted.

"We've been trying to export this workflow to other labs, and it is this exact [SDS cleanup] step that has been the barrier every time," he said. "Getting somebody practiced and knowledgeable about how to do that cleanup is a real barrier."

Additionally, neither method is easily amenable to automation or incorporation as a continuous part of top-down workflows, which makes the heavy fractionation involved even more time consuming.

"You have the classic problem of doing multidimensional separations," Compton said. "If you aren't doing them in a continuously coupled fashion, your analysis time goes through the roof. Every sample we [fractionate] into 12 molecular weight bins, and then we have to run an analysis on each one of those, and that means that for every sample you have to do that clean up 12 different times, so it becomes very time intensive."

To address this cleanup problem, Compton and his colleagues developed an AF4 device adapted for proteomic workflows. In AF4, molecules are elevated above a semi-permeable membrane while a laminar flow is applied parallel to this membrane. This flow is concentrated at the center of the vessel and analytes move through the device at different speeds depending on their characteristics.

The approach has often been used for separating analytes, but, Compton noted, the Northwestern researchers were interested instead in using it to clean up their samples. To modify it for this purpose, they used a membrane with pore sizes large enough to allow SDS molecules to pass through but not proteins. The protein portion of the sample is focused in a band within the middle of the channel and the smaller SDS molecules pass through the membrane. The protein can then be eluted out the channel and into the next dimension of separation or directly into the mass spec instrument.

The approach enables automated, on-line removal of SDS without the sort of sample handling required by precipitation or spin column-based approaches. In the JPR paper, the researchers used samples containing four protein standards to test the device, finding that depending on the protein, the AF4 approach improved mass spec signal intensities by between two-fold and 10-fold compared to a precipitation approach.

They also tested the system on an extract of nuclei from HeLa S3 cells, finding that they were able to identify all four core histones and to isotopically resolve a variety of histone proteoforms featuring methylations and acetylations while observing no SDS or salt adducts.

Compton noted that in addition to making sample cleanup in top-down proteomics simpler, the approach could also help lower sample volume requirements, which is of great importance as he and his colleagues seek to apply top-down proteomics to clinical research.

"A lot of what our lab is doing is trying to implement top-down in clinical settings," he said. "So we're trying to get a hold of patient tissue samples and things like that, and when you start getting into that world, clinicians can only let you have so much [sample]. So you need to get your workflows compatible with the amount of material they can give you."

In their precipitation-based workflows, the Northwestern researchers typically load around 300 micrograms of total protein onto the GELFrEE device. For clinical samples, though, they need to bring this down to around 50 micrograms, Compton said.

"We have people who are really good at the bench and they have done 50 micrograms total load [in precipitation workflows], but it is not easy," he said, adding that this limited the method's portability to other labs.

The hope, he said, is that the AF4 approach will allow for smaller sample amounts.

"We haven't gone down and tested the floor of this yet, that's our next step," Compton said. "But the recovery from this device is exceptionally good, so I believe that we will be able to get to those very small amounts."

Beyond GELFrEE cleanup, Compton said the AF4 device might also prove useful for cleaning up samples fractionated using isoelectric focusing prior to digestion for bottom-up proteomic experiments. It might also find a place in clean up of intact proteins prior to mass spec analysis in biopharma development, he said.

The Northwestern researchers have a patent on the technology and, Compton said, would be open to partnering with commercial firms to scale up production.

"We want people to do top-down [proteomics], so whatever tools we can give them to make that happen, that is our primary mission," he said.

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