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PNNL Team Develops Single Cell Proteomics Workflow

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NEW YORK (GenomeWeb) – A team led by researchers at Pacific Northwest National Laboratory has developed a mass spectrometry workflow that can measure more than 600 proteins in a single cell.

The approach combines fluorescence-activated cell sorting, microfluidic sample processing, and nanoLC-MS. In a study published last month in Angewandte Chemie, it was able to identify an average of 669 proteins and quantify 332 proteins in single HeLa cells, making it one of the most comprehensive mass spec-based single-cell proteomic experiments conducted to date.

As researchers have come to appreciate the role of cellular heterogeneity in diseases like cancer and in biological processes in general, interest in single-cell analyses has grown. Single-cell techniques for measuring proteins have lagged behind methods targeting nucleic acids, but in recent years, companies like Nanostring, Fluidigm, IsoPlexis, and Bio-Techne have commercialized single-cell proteomic offerings.

These systems have largely relied on immunoassays, however, which limits the breadth of the proteomic profiles they can generate to tens or hundreds of proteins. Mass spec, on the other hand, could potentially measure thousands of proteins, but the technical complexity and sample requirements of mass spec workflows have made it a difficult technique to apply to single-cell experiments.

Both mass spec sample preparation and the limits of LC-MS present challenges to single-cell proteomics. In a paper published earlier this year in Nature Communications, the PNNL team tackled the sample prep portion, developing a platform called NanoPOTS (nanodroplet processing in one pot for trace samples), designed for use with extremely small proteomic samples.

That platform aimed to address problems like sample loss and the slow kinetics of trypsin digestion with very small sample volumes, said Ryan Kelly, a PNNL researcher and senior author of both studies.

"What was lacking was a way to isolate and prepare very small spatial regions of tissues —single cells or rare cells — in a way that is nearly lossless," he said. "When proteins and peptides contact surfaces, some of them get lost on those surfaces. Also, if you're preparing a very small sample in a large volume, that is a challenge for the kinetics of the [digestion] reaction, so you have to overload [with trypsin] to increase the digestion rate and that causes problems itself."

Given these issues, "working in small volumes and with small surface exposure is key," Kelly said.

The NanoPOTS platform addresses these challenges by shrinking sample processing volumes down to less than 200 nanoliters. In the Nature Communications paper, the researchers combined sample prep on the platform with mass spec to identify more than 3,000 proteins from samples as small as 10 cells and to quantify roughly 2,400 proteins from single human pancreatic islet sections.

In the Angewandte Chemie study, Kelly and his colleagues combined the NanoPOTS system with FACS, which allowed them to select individual cells for analysis. Running the single cells using 60-minute nanoLC gradients and a Thermo Fisher Scientific Orbitrap Fusion Lumos instrument, the researchers identified an average of 669 proteins in single HeLa cells with protein concentrations spanning three orders of magnitude.

They also tested whether the approach could distinguish between different human cell types based on their protein expression, running human lung epithelial and mesenchymal cells. They quantified 328 proteins in these cells and, using principal component analysis, were able to sort the two cell types based on their protein expression patterns.

Last year, researchers at Northeastern University and Harvard University developed an approach called Single Cell Proteomics by Mass Spectrometry (SCOPE-MS), which they used to quantify around 500 proteins in single Jurkat and U-937 cells and then distinguish between the two cell types based on their protein expression.

Kelly said that a variety of advances are pushing single-cell mass spec towards feasibility, ranging from sample prep through to more efficient ion sources to improved interfaces between the LC and mass spec to better performance from the mass spec instrumentation itself.

He added that he and his colleagues continue to look at all steps of the process as they work to further optimize their approach. In particular, he said, they are focused on further miniaturizing the LC separation.

"By further miniaturizing the separation, you're exposing your sample to less surface [area], and then you're electrospraying at a lower flow rate, too," Kelly said. "And that boosts your sensitivity, improves your ionization efficiency."

He also noted that the researchers are interested in trying Bruker's recently released timsTOF Pro instrument, which has shown promise for proteomic analysis of small samples. For instance, in a presentation at last year's annual meeting of the Human Proteome Organization, Max Planck Institute of Biochemistry researcher Matthias Mann showed data in which his lab identified 1,803 proteins in a sample consisting of 15 HeLa cells in an analysis using the timsTOF.

"We wouldn't say that we are done in any step of this process yet," Kelly said. "We hope to be, within a year, identifying more like 3,000 proteins from single cells."

In addition to increasing the number of proteins identified in single-cell experiments, researchers are also working to increase the throughput of such experiments, which will likely be necessary to collect data on a sufficient number of cells to make useful observations.

Kelly said that he and his colleagues are currently looking at a set of lung epithelial cells at the single-cell level, sorting them based on antibody markers and then using mass spec to profile those populations more deeply.

"We think that with around 100 cells, we'll be able to start to piece together that population," he said, noting that for this study, the researchers are running around eight single cell samples per day, which means several weeks of mass spec time to run the full 100 samples. 

He said he has a National Institutes of Health grant funding work on upping the throughput of single-cell techniques, with the goal to get to around 100 samples per day.

The bottleneck, Kelly said, is all around the LC-MS portion of the workflow, as the sample prep can be done in parallel. He said he sees two main routes to increasing the throughput of the LC-MS process: using multiplexing approaches like isobaric labeling, and cutting down or eliminating the LC step using approaches like ion mobility.

"Right now, there are big tradeoffs to sacrificing a high-quality [LC] separation, but hopefully, over time we can minimize those," he said.

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