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University of Oklahoma Team Develops Device for Mass Spec Analysis of Living Single Cells


NEW YORK (GenomeWeb) – Scientists at the University of Oklahoma have developed an approach for single cell mass spec analysis.

Detailed in a paper published last month in Analytical Chemistry, the method uses a miniaturized probe for sampling and ionization attached to a conventional LC-MS instrument. According to Zhibo Yang, an Oklahoma researcher and senior author on the study, the technique offers an advantage over other single cell mass spec techniques, such as secondary ion mass spec and MALDI, in that it is able to analyze living cells in real time.

Such methods "are limited by the environment that the sample must be put into," which typically kills the cell, Yang told ProteoMonitor. "In our assay, we culture the cells and bring them to the [instrument], and in that moment [of analysis] they are still alive."

The team's probe consists of a quartz needle for sampling, a silica capillary, and a nano-ESI emitter. In the Analytical Chemistry paper, Yang and his colleagues attached the device to a Thermo Fisher Scientific LTQ Orbitrap XL instrument, though Yang said it should be compatible with any LC-MS system. Using this setup, they detected a variety of metabolites and lipids in individual HeLa cells. They also identified the anticancer drugs doxorubicin (Adriamycin), paclitaxel (Taxol), and OSW-1 in single cells treated with these agents.

To date, Yang said, the researchers have focused on metabolomic and other small molecule measurements with the probe. Protein measurements are possible in principle, he said, but given the small amount of protein present in a typical cell and the complicated matrix of this environment, they could prove challenging for the mass spec to detect. However, the method could prove effective for protein analysis in larger cells like yeast, he said.

The device offers researchers the opportunity to bring experiments previously performed only on the bulk samples down to the single cell level, said Anthony Burgett, Yang's Oklahoma colleague and co-author on the paper.

"My lab and my research is more in cell biology and pharmacology, so from my viewpoint the real power [of the method] is that it is an avenue to bring single cell analysis into cell biology labs," he told ProteoMonitor.

"If you think about how we do basically all biological analysis, it starts with thousands to millions of cells, and every cell population is actually a very heterogeneous population," he said. "And even things like cell lines, if you go in and look at the cells, there are lots of genetic differences."

Additionally, Burgett noted, the cells in these large populations "are undergoing different things at different times – cell division or apoptosis or whatever," which further complicates the analysis.

By moving to the single cell level, researchers can avoid such issues of sample heterogeneity, he said. They will also be able to analyze very small sets of cells from, for instance, patient clinical samples, Yang noted.

"Ultimately, biology is built on the single-cell level, so it would be a very powerful analytical approach to begin to interrogate systems" at that level, Burgett said.

Indeed, the Oklahoma group's study is one of a number of recent efforts in both academia and industry to develop tools for single-cell analysis. Companies such as NanoString and Fluidigm have been active in the single-cell genomics space for years, but more recently these firms and others have demonstrated an interest in single-cell proteomics, as well.

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.

Fluidigm also announced in August plans to launch in 2015 a CyTOF imaging system for single-cell proteomics based on work done by University of Zurich researcher Bernd Bodenmiller and Swiss Federal Institute of Technology Zurich researcher Detlef Günther.

The lab of Stanford University researcher Garry Nolan, who was one of the earliest adopters of mass cytometry, has also developed a technique using mass spectrometry and metal-conjugated antibodies for highly multiplexed protein analyses at the subcellular level.

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.

And, last year, GE Healthcare launched its MultiOmyx protein detection system through its Clarient Diagnostic Services.

Additionally, last month, University of California, Berkeley spinout Zephyrus Biosciences, which is focused on single-cell Western blotting, launched with $1.86 million in funding.

Beyond its ability to operate on a single-cell level, the Oklahoma group's method might also enable new insights due to its ability to analyze living cells, Burgett said.

"If you think about [conventional] mass spec analysis – you have 500,000 HeLa cells and you subject them to very harsh conditions to rupture the cell, destroy all of the intracellular structure, and then this all gets mixed together and sits there before you get to do the analysis." he said. "So there's a good chance that there are biomolecules we haven't identified yet because those molecules did not survive that process."

Their single-cell analyses typically pick up thousands of ions in the small molecule range, Burgett said, "So we're not just getting signals of a few very abundant molecules. We think we're getting a complete snapshot of the small molecules present."

Burgett said he is particularly interested in applying the technique to pharmacology research, adding that he and Yang are developing quantitative assays on the device for this work.

"You can treat populations of cells with the drug and then you can go into the cells and quantify exactly how much compound got into that cell and begin to understand exactly what effect the compound at that concentration had on the cell," he said. "You can begin to look at the molecular pharmacology of compounds in real time on a single-cell level."

The researchers have patented the device and are considering commercialization options, Yang said, noting that several mass spec vendors have expressed interest.