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UW-Madison Team Devises Workflow For LC-Free Proteomics

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NEW YORK – Researchers at the University of Wisconsin-Madison have developed a data-independent acquisition mass spectrometry workflow that eliminates upfront liquid chromatography.

Detailed in a paper published this week in Nature Methods, the approach, called direct infusion-shotgun proteome analysis (DISPA), is still in the early stages of development, but it could enable more streamlined and higher-throughput proteomics experiments.

LC is a key part of most proteomic workflows, providing the fractionation of complex samples like plasma or cell lysate that is required to achieve broad and deep coverage across the proteome. LC has also been one of the more unwieldy and time-consuming portions of proteomic experiments, with LC separations often taking hours to run and LC systems subject to instability that can present challenges to experimental reproducibility.

In recent years, improvements in methods and instrumentation have allowed researchers to bring LC run times down to under an hour, and for some analyses, into the five to 10 minute range. Eliminating LC entirely would cut experiment run times even further, potentially allowing labs to run hundreds of samples per day.

"One of my overarching goals is to have much higher-throughput proteomics and higher-throughput omics analysis in general," said Jesse Meyer, first author on the Nature Methods study and a postdoc in the lab of senior author and UW-Madison professor Joshua Coon at the time the research was done. Meyer has since taken a post as an assistant professor of biochemistry at the Medical College of Wisconsin.

"Thinking through the slowest parts, it is pretty obvious that LC is pretty slow compared to the scan speed of the mass spec," he said. "In general, that is a rate-limiting factor."

Key to LC-free proteomics is ion mobility spectrometry (IMS), which separates ions in the gas phase based on their size and charge. Typically, IMS has been used after LC to provide an additional level of separation prior to mass spec analysis. The UW-Madison researchers, though, used it as the sole source of sample separation.

In the Nature Methods paper, Meyer and his colleagues used Thermo Fisher's FAIMS Pro IMS device coupled to the company's Orbitrap Fusion Lumos mass spec. They directly injected the sample of interest into the system's nanospray emitter, which produced ions that passed through the FAIMS system and into the mass spec. For making peptide identifications, the UW-Madison team used the MSPLIT-DIA approach developed by researchers at the University of California, San Diego.

In a set of 100 replicates run over the course of one night, the researchers found that an average of 1,542 peptides were identified per experiment, 869 of them identified in all 100 experiments and 1,264 identified in at least 80 of the 100 experiments, demonstrating both the technique's throughput and potential for good reproducibility.

The researchers also implemented a quantitative version of the approach using heavy and light labeled peptides, quantifying 525 proteins in A549 cells. They also used the approach to measure proteome changes in wild-type and mutant 293T cells in response to treatment with nine mitochondrial toxins. Analyzing 132 samples with an experimental time of around two minutes per sample, the UW-Madison team was able to quantify 451 proteins, 341 of them across all 132 samples, and to identify changes linked to known biology such as expected upregulations of glycolysis and degradation of mitochondrial function.

Meyer said that while the initial analyses measured a relatively limited number of proteins, he believes the approach can be optimized to improve proteome coverage.

"I imagine that eventually you will be able to get, like, the full yeast proteome," he said.

Meyer said he was also working to develop a quantitative approach that did not require labeled standards. He noted that further development of the DISPA method will be a major area of focus for his lab at the Medical College of Wisconsin.

In addition to boosting throughput, eliminating LC greatly simplifies the experiment setup, which Meyer highlighted as another key advantage of the approach.

"Easily the biggest source of problems [in a proteomic experiment] is the LC," he said. "So I think it will make things a lot more robust and more accessible."

"It's a really interesting paper, and it is also very timely," said Lukas Reiter, chief technology officer at Swiss proteomics firm Biognosys. Reiter, who was not involved in the Nature Methods study, added that his company believes that large-scale, high-throughput proteomics experiments are the direction the field is moving.

"And of course if you want to go large scale and parallelize over many platforms, then the robustness of the LC-MS setup is very crucial, and the LC is known to be not the most robust [component]," he said. "So this is an interesting alternative."

Reiter noted that with the field driving LC gradient times down as low as several minutes, the throughput advantages of an LC-free approach might not be so dramatic, but that the simplicity and robustness of a direct injection method like DISPA would remain a compelling advantage.

While Meyer and his colleagues used Thermo Fisher's FAIMS system in developing DISPA, other IMS technologies might also work with the method.

One interesting possibility is the structures for lossless ion manipulations (SLIM) IMS system developed by researchers at Pacific Northwest National Laboratory (PNNL) and licensed to Chadds Ford, Pennsylvania-based separations firm MobiLion.

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The PNNL device uses SLIM technology to extend ion mobility path lengths beyond that allowed by conventional IMS systems, potentially enabling much more extensive separations. SLIM systems use arrays of printed electrodes to confine ions within the ion mobility field. They also make it possible to route ions around turns without losses, meaning that an IMS drift path can be designed to run along a serpentine path, greatly increasing the length of the IMS path without increasing the footprint of the device.

Richard Smith, director of proteomics research at PNNL and one of the leaders of the IMS development effort, has said in the past that the SLIM system could potentially enable LC-free proteomics experiments.

Smith said in an email that while he and his colleagues have not yet explored using SLIM for LC-free mass spec, he believed that "there are significant benefits of using ion mobility spectrometry for such purposes, and specifically getting around the need for LC."

He suggested that the SLIM device could prove more effective than the FAIMS system for this purpose due to its higher separation power, which would reduce the complexity of the sample the mass spec had to deal with, and due to the high ion utilization of the SLIM system, which in recent work the PNNL team has pushed to almost 100 percent.

Additionally, Smith noted that unlike the FAIMS device, the SLIM system collects collision cross section information on ions passing through the system, which can be used to help with peptide identification.

Meyer said that he has set up his lab with a FAIMS system and Thermo Fisher mass spec but mentioned that he is interested in exploring how the method would work with a drift tube-style IMS system like SLIM combined with a QTOF instrument.

"I think the physics of it theoretically make more sense on a drift tube system," he said, adding that he is working with several vendors to test out the approach on these kinds of systems.

He is also beginning to use the approach on clinical samples he was able to access through the Medical College of Wisconsin's biobank.

"I can get de-identified samples and [clinical] data without needing an IRB, so I'm really excited about getting a ton of samples and cranking through them with this method," he said.

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