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Cedars Sinai Team Develops Method for Quantitative Label-Free Proteomics Without LC Separation

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NEW YORK – A team led by scientists at Cedars Sinai Medical Center has developed a label-free quantitative proteomics workflow capable of measuring thousands of proteins without upfront liquid chromatography separation.

Using the method, which was detailed in a study published last month in Analytical Chemistry, the researchers identified more than 2,000 proteins and quantified roughly 1,000 proteins in HeLa and 293T cell lysate samples with a throughput of around 300 samples per day.

Such direct infusion mass spectrometry workflows are still niche within proteomics, with only a small number of labs exploring such approaches, but emerging technologies in sample handling and ion mobility as well as advances in mass spec instrumentation could drive future adoption.

The Cedars Sinai team now plans to explore applications of the method including high-throughput drug screening and protein biomarker assays, said Jesse Meyer, assistant professor at Cedars Sinai and senior author on the Analytical Chemistry paper.

The work builds on previous research by Meyer and colleagues published in Nature Methods in 2020. In that work, the researchers used light- and heavy-labeled standards to enable protein quantitation. The recent study used a label-free approach, which, Meyer noted, allowed them to quantify roughly double the number of proteins per experiment compared to the original method.

Performing label-free quantitation with a direct infusion approach rested on a few key assumptions, Meyer said, perhaps the most important being that the electrospray device used to generate peptide ions for introduction into the mass spec would produce a stable spray such that the signal produced would be consistent both within and across experiments.

The new paper also used a more refined software package called CsoDIAq that Meyer's lab developed specifically for its direct infusion workflows.

Meyer said that for the original work he "had to kind of hack together a computational pipeline that was very difficult and required multiple programs." His lab presented the CsoDIAq package in a paper published in 2021 and in the recent work expanded it to enable label-free protein quantitation.

Meyer's direct infusion workflow fits into the broader trend within proteomics of decreasing sample separation times in order to increase sample throughput. Typically, this has meant running shorter and shorter LC gradients, which improvements in LC technology, mass spec instrumentation, and informatics have made possible.

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, proteomics researchers have managed to dramatically reduce LC run times while still maintaining substantial proteome coverage. In 2020, for instance, researchers at the Max Planck Institute of Biochemistry developed a workflow on Bruker's timsTOF mass spec in which they quantified more than 3,000 proteins in cell lysate with a throughput of up to 200 samples per day. In 2021, researchers at the Francis Crick institute used Sciex's Scanning Swath method to measure nearly 2,000 proteins in cell lysate in 30-second mass spec experiments.

Researchers are also exploring other approaches to upfront sample separation, such as capillary electrophoresis, which could prove a faster alternative to LC for some experiments.

Direct infusion takes such efforts to their logical extreme, eliminating LC and instead relying on ion mobility for sample separation. Ion mobility separates ions in the gas phase based on their mass and charge and has typically been used after LC to provide an additional level of separation prior to mass spec analysis.

In its direct infusion work thus far, Meyer's lab has used Thermo Fisher Scientific's FAIMS Pro ion mobility device coupled to either an Orbitrap Fusion Lumos or Orbitrap Exploris 240 mass spec. Other ion mobility systems could prove even more effective, though.

One 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. 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 and, therefore, the power of the separation, without increasing the footprint of the device.

SLIM IMS offers other potential advantages for a direct infusion workflow. The FAIMS device works by filtering ions — somewhat like a quadrupole — which means that those ions not selected by the device are discarded, which decreases sensitivity. SLIM, on the other hand, uses a very large proportion of the sample ions — nearly 100 percent in work done by PNNL scientists.

Additionally, SLIM IMS collects collisional cross section information on ions passing through the system. This additional dimension of information could improve the confidence of peptide identification in direct infusion workflows.

"In theory, it would definitely help," Meyer said. "More constraints is always better."

"I'm totally interested in exploring SLIM," he added. "The fact that it can have almost limitless separation power and doesn't lose ions — I'm super excited about trying to interface with that."

He said his lab was exploring how their direction infusion workflow might work on other instrument platforms, but he couldn't disclose more specific information.

Daniel DeBord, VP of research and development at Mobilion, said in an email that the company has been following Meyer's direct infusion work and has "has engaged [with him] directly on this topic for a while now and look forward to further exploring its potential implementation using the SLIM technology."

He noted that one challenge to integrating the technologies is synchronizing the ion mobility separation with the ion fragmentation process required for generation of the MS/MS spectra used to identify peptides.

"The theoretical understanding of coupling these two techniques is relatively straightforward, but getting the instrumentation to operate in concert does represent an engineering challenge," DeBord said.

Richard Smith, director of proteomics research at PNNL and one of the leaders of the SLIM development effort, said that he and his colleagues are using SLIM as part of direct infusion workflows for analyzing samples pre-fractionated by LC offline. Such an approach allows for extremely deep proteomic coverage while also maintaining reasonable throughput, he said.

Smith said that the offline fractionation can be done in parallel prior to mass spec analysis in a way that does not limit throughput. Then each fraction can be run using a direct injection approach that takes one to two minutes per fraction, meaning an hour-long experiment can analyze a sample split into somewhere between 30 and 60 fractions.

"So you can dig incredibly deep into a sample," Smith said.

He noted that the approach was also useful for sample-limited experiments like single-cell work, where “you want to get as much information [from the sample] as possible.”

Speaking of the Meyer lab’s work, Smith said the researchers had done “a really nice job of bringing together pieces of software with the mass spec technology.”

Meyer said that in addition to SLIM IMS, he is interested in exploring faster methods for injecting samples into the mass spectrometer. He cited Sciex’s Echo MS acoustic liquid handling system, which can introduce up to three samples per second into a mass spectrometer.

“I would love to get my hands on one of those and try this,” he said. “We’re trying to do faster throughput, and so devices that can introduce samples more quickly would be useful."

Last year, Sciex, which is owned by life science giant Danaher, presented proof-of-principle data showing that the Echo MS system can analyze peptides using a peptide quantitation workflow that makes use of stable isotope standards and capture by anti-peptide antibodies (SISCAPA).

Meyer's Cedars Sinai colleague Jennifer Van Eyk said that she is working with Sciex on using the Echo MS in protein quantitation workflows. In recent work, her lab used the device to quantify levels of two proteins in 10,000 samples in 4.5 hours.

Jose Castro-Perez, senior director of market development at Sciex, said via email that the company plans to present additional work on use of Echo MS for targeted protein quantitation at the 2023 American Society for Mass Spectrometry annual conference in June.

Castro-Perez said that "there is definitely potential for proteomic analysis" using the Echo MS and added that Sciex is launching an Echo MS Center of Excellence focused on developing applications for the device.

One limitation currently preventing use of the Echo MS for proteomics is a lack of compatible instruments. The system can be paired with the Sciex Triple Quad 6500+ instrument but not with high-resolution instruments like the ZenoTOF 7600 that are commonly used for proteomics research. Castro-Perez said that "future integrations are definitely being considered."

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