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Single-Cell Proteomics Could Benefit From New Sample Prep Technologies

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NEW YORK – Interest in single-cell proteomics has ticked upward in recent years as technical advances have made the required workflows more feasible.

Now, automation and commercialization of sample preparation methods are poised to grow adoption of the technique and help it move beyond the few specialists labs it has been confined to thus far.

Sample prep is a key challenge in single-cell proteomics, as methods are required to manipulate the extremely small amounts of sample used in such experiments with good reproducibility and minimal loss. Throughput is also a consideration given the large number of single-cell experiments required to generate enough data to address biological questions.

Labs specializing in single-cell proteomics have developed various approaches for optimizing sample preparation. For instance, researchers at Northeastern University recently published an updated version of their Single-Cell Proteomics by Mass Spectrometry (SCOPE-MS) method called SCOPE2 that uses PCR thermocyclers and automated liquid handling instrumentation to enable more streamlined, higher-throughput sample prep that can be done in smaller sample volumes than the original assay.

According to Nikolai Slavov, director of the single-cell proteomics center at Northeastern and senior author on the preprint presenting the method, the researchers have developed the protocol for use on Cellenion's CellenOne single-cell handling system, with the aim of making it more widely accessible.

Specializing in single-cell isolation and manipulation, Lyon, France-based Cellenion, a spinoff from Scienion in Germany, has emerged as perhaps the central commercial player in sample prep for single-cell proteomics. In addition to the SCOPE2 approach, the company's technology has been used to automate the NanoPOTS workflow developed by researchers at Pacific Northwest National Laboratory. Designed for use with extremely small proteomic samples, NanoPOTS (nanodroplet processing in one pot for trace samples) shrinks sample processing volumes down to less than 200 nanoliters to limit sample loss and speed trypsin digestion, which has slow kinetics at small sample volumes.

Cellenion is also involved in a project with Karl Mechtler, chair of the proteomics work group at the Vienna BioCenter, to commercialize an automated single-cell proteomics sample prep workflow using the company's technology.

The workflow uses a chip featuring 12 sets of 16 wells, each of which holds a single cell, so 192 single-cell samples can be processed in a volume of 400 nanoliters or less.

Mechtler said the method eliminates all vial changes to reduce sample loss and allows for direct connection to the autosampler while fully automating the sample handling steps. He said Cellenion aims to begin making the method available for use on its systems at the end of April. He presented a single-cell workflow using the approach during the US Human Proteome Organization annual meeting this month.

Mechtler and his colleagues also published a BioRxiv preprint in February that used the sample prep system in combination with liquid chromatography from Pharmafluidics and analysis on a Thermo Fisher Orbitrap Exploris 480 mass spectrometer with FAIMS Pro to profile single-cell levels of cell HeLa lysate. The researchers were able to identify 2,855 proteins from 1 nanogram of lysate and 1,486 proteins from 250 picograms of HeLa trypic digest.

He suggested that having a complete sample prep workflow available as a commercial product would help drive adoption of single-cell proteomic approaches.

Sample prep "was the biggest trouble for our lab, because we are a mass spec lab, and we didn't have experience in miniaturization of pipetting and microfluidics," he said. "But you have to do microfluidics in single-cell because you are working with volumes lower than 1 microliter."

"Cellenion will bring this chip on the market and they will train scientists in how to handle it," he said, adding that his lab is organizing an online workshop to accompany the launch of the system.

Guilhem Tourniaire, managing director at Cellenion, said that the company became interested in applying its technology to single-cell proteomics after being approached by Slavov at a conference several years ago.

"We started to look at single-cell proteomics, and we noticed that there was a need for miniaturization, a need for being able to dispense not only aqueous reagents but also organic ones, which is something we can do, and we thought that we had something unique to offer in that field," he said.

Cellenion began working with Slavov on single-cell sample prep technology, however the two parties ultimately decided to go in different directions, Tourniaire said (though, as Slavov noted, he and his colleagues have developed the SCOPE2 method to work with the CellenOne system).

Cellenion then began its collaboration with Mechtler, leading to the development of the single-cell prep chip the company plans to launch in April. The product will consist of the chip and reagents and will work with the CellenOne platform.

Tourniaire said Cellenion plans to target large proteomic centers and core labs with the product, noting that the company is seeing significant interest from such institutions to move into single-cell proteomics.

"Our goal is to make it easier for people to enter the field by providing an all-in-one solution," he said. "Everything can be done without intervention from the user."

He added that the sort of push-button solution it is developing for single-cell proteomics was a departure from much of its previous work offering flexible methods for single-cell isolation and workflows that include DNA and RNA sequencing.

Tourniaire said he could imagine that the company’s customers in areas like transcriptomics will become interested in the technology after it rolls out to a range of proteomics centers, though he said the need for mass spec instrumentation and expertise would remain a barrier for such labs. He noted, though, that they could possibly prepare samples using the company's platform and then send them to a proteomics core for analysis.

In February, a team led by Ying Zhu, a senior biological scientist at PNNL and one of the inventors of the NanoPOTS method, published a BioRxiv preprint using a nanowell chip with the CellenOne platform to automate the NanoPOTS process.

Tourniaire said that while the CellenOne system can run the NanoPOTS workflow, the company is not offering a commercial version of the NanoPOTS method, and he declined to comment on whether the company is working on one with PNNL.

Ryan Kelly, an associate professor of chemistry and biochemistry at Brigham Young University and a former PNNL researcher who co-developed the NanoPOTS approach, also recently developed an automated version of the workflow, describing it in a January study in Analytical Chemistry.

He noted that while the CellenOne platform has worked well for single-cell proteomic sample prep, including the NanoPOTS method, it is expensive, costing $300,000. In their recent autoPOTS work, Kelly and his colleagues opted instead for the Opentrons OT2 automated pipetting system, which costs just $5,000.

The Opentrons OT2 isn't able to work with sample volumes of less than 1 microliter, which could impact the performance of the autoPOTS method. Kelly said that his team "observed about a 25-percent decrease in proteome coverage when going from NanoPOTS to the OT2-based autoPOTS due to these larger volumes."

However, he said, he hoped the lower price point of the Opentrons system would broaden the approach's accessibility.

"There are also a couple of other commercial solutions in the works, so hopefully sample preparation won't be the limiting factor [for single-cell proteomics] for much longer," Kelly said.

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