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UC Berkeley Team Develops Western Blotting-based Approach for Single Cell Proteomics

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NEW YORK (GenomeWeb News) – Researchers at the University of California, Berkeley have developed a method for performing single-cell western blotting.

Detailed in a study published this week in Nature Methods, the technique offers multiplexing capabilities and high throughput as well as potential advantages in terms of specificity compared to other single-cell immunoasssays, Amy Herr, a UC Berkeley researcher and author on the paper, told ProteoMonitor.

Like conventional western blots, the approach uses electrophoresis-based separation combined with antibody detection, which, Herr noted, provides "an extra layer of specificity."

"If you see signal on weights that you didn't expect, you're alerted to the fact that maybe there is something not quite [right] with your antibody, or maybe there is something going on about the biology that is worth investigating," she said.

Single-cell proteomics has recently emerged as an area of growing interest, with several companies traditionally focused on single-cell genomics moving into protein analysis.

Fluidigm, for instance, this year purchased mass cytometry firm DVS Sciences, giving it a foothold in both the high-end flow cytometry and single-cell proteomics markets. This followed a co-marketing deal the company signed in July 2013 with Olink Biosciences that combined the two firms' tools to create a high-throughput proteomics platform.

Also this year, NanoString obtained an exclusive option to license intellectual property to a proteomic assay developed by researchers at Massachusetts General Hospital that uses DNA-barcoded antibodies to simultaneously measure in the range of 100 proteins at single-cell sensitivity.

The UC Berkeley scientists have also received commercial interest in their single-cell western platform, which they've named scWestern, though Herr declined to give specifics regarding any plans to bring the method to market. She said that they were in the process of patenting the technique.

As presented in the Nature Methods paper, the approach uses microscope slides coated with a thin layer of photoactive polyacrylamide gel divided into 6,720 microwells. These wells are then seeded with the cells of interest, with each well collecting up to four cells.

They then lysed the cells and performed electrophoresis, achieving separation of molecular mass differences of 51 percent in separation lengths of 500 μm and a separation time of 30 seconds, making the approach significantly faster and more compact than existing microwestern arrays. Following separation, they exposed the gel to UV light to immobilize the proteins and allow for probing with antibodies.

Herr and her colleagues have been developing the materials used in the scWestern for several years, she said. "These photoactive hydrogels act as a molecular sieving mechanism for the separation and then upon UV light stimulation they become, essentially, a blotting membrane, but in situ, so you're not doing any handling of the sample."

In their earlier efforts, they focused on integrating the assay into microfluidic channels. However, Herr said, the limited capacity of such devices led them to shift to a microwell-based approach.

"As we were thinking about ways to apply this to single cells, we realized that microchannels aren't a great way to do it," she said, noting that if, for instance, "you want to make 2,000 single-cell measurements, you are starting to talk about a lot of microchannels."

And so, inspired by the success other groups have had in compartmentalizing single cells in microwells, the group decided to give that format a try.

In the Nature Methods study, Herr and her colleagues applied the technique to an analysis of stem cell signaling, looking at the heterogeneity of signaling in rat neural stem cells in response to stimulation with fibroblast growth factor 2. Measuring total and phosophorylated levels of the kinases ERK1/2 and MEK1/2, the researchers tracked variations in stem cells' responses to the stimulus.

They also used the technique to track stem cell differentiation to both astrocytes and neurons, measuring levels of the proteins NESTα, NESTβ, SOX2, βIIITUB, and GFAP, which are used as markers of differentiation. The method, they reported, identified high cell-to-cell variability in terms of marker expression.

The analysis also determined that while NESTα was variably expressed between cells and downregulated during differentiation, NESTβ was consistently present throughout differentiation – a finding that, they wrote, might account for past observations of NEST expression in mature neural cells. The two isoforms could not be distinguished between using conventional single-cell antibody-based assays such as flow cytometry or immunocytochemistry, they noted.

The method is able to multiplex protein measurement via an iterative process in which antibodies are applied and then stripped to allow for probing of the next target. Using this approach, the UC Berkeley team multiplexed 11 proteins per single cell. However, Herr said, she believes they will be able to multiplex larger numbers in the future.

"We actually just stopped working on [the multiplexing] part because our time was up in terms of responding to [journal] comments," she said. "I'm pretty sure we can go further."

She noted that, ultimately, from a quantitation perspective, the method's multiplexing will be limited by the fact that a bit of sample is lost in each round of stripping and probing.

"So at some point it will break down," she said. "We just don't know if it will be at 12 [proteins] or 25 or 50."

One possible guide to the technique's multiplexing potential might be GE Healthcare's MultiOmyx platform, an antibody-based system that similarly uses iterative rounds of stripping and probing. In studies, GE researchers have multiplexed as many as 65 proteins on the system and found that they can perform as many as 100 staining cycles without added background.

Looking forward, Herr said she and her colleagues plan to apply the scWestern approach for continued work on stem cell differentiation, using it to complement data from single-cell genomics experiments.

They are also using it to study rare cell populations like circulating cancer cells, she said.

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