Single-cell proteomics takes centre stage


Claudia Ctortecka was each sceptical and intrigued when her thesis adviser advised her in 2018 a couple of new technique that makes use of mass spectrometry to analyse the protein contents of particular person cells. When he stated he was in search of somebody to pursue this single-cell proteomics technique in his laboratory on the Vienna BioCenter analysis institute, she determined to take an opportunity.

“I was always very much interested in mass spectrometry,” she says. “And I thought, ‘why not go for the challenge?’ I wanted to look into that [strategy] a bit deeper and closer.”

With no backup plan, the undertaking was a sink-or-swim proposition, Ctortecka says. “It was basically just: do single cell, make it work, or try harder.” Yet work it did. In April, she and her colleagues detailed a brand new sample-preparation gadget known as proteoCHIP, which they used to map some 2,000 proteins throughout 158 single cells from 2 human cell sorts1.

That research is certainly one of at the very least half a dozen over the previous yr which have described single-cell proteomics methods, instruments and preliminary findings. And extra are coming. In 2018, Nikolai Slavov, a methods biologist at Northeastern University in Boston, Massachusetts, hosted his first annual convention on single-cell proteomics, which attracted about 50 attendees. This yr’s (largely digital) convention had greater than 1,300. “The growth has been exponential,” he says.

Most single-cell research give attention to nucleic acids, particularly the transcriptome — which represents all of the expressed genes in a cell. But proteins, says Neil Kelleher, a biochemist at Northwestern University in Evanston, Illinois, are “the worker bees” of the cell. “The amounts, the post-translational modifications, the proteoform dynamics — this is what is closer to the phenotype,” he explains. “And that means that disease diagnostics, response to drugs, all the human biology we want to engage with — to control, steer, detect — it needs proteomics.”

Proteomics goals to catalogue and characterize the full complement of protein isoforms from a cell, tissue, organ or organism. (These ‘proteoforms’ are encoded by the identical gene however have non-identical amino-acid sequences or post-translational modifications.) However, on the single-cell stage, that’s simpler stated than performed. Each kind of nucleic acid behaves largely in a predictable approach. But the proteome has an enormous array of various chemistries, interactions, dynamics and abundances. And with no protein equal to PCR amplification of DNA, any approach to detect proteins should be delicate sufficient to determine them, nonetheless little materials a cell incorporates.

Using antibodies, that’s comparatively easy. Flow cytometry and mass cytometry, as an example, can every quantify as much as about 50 proteins per cell. And high-resolution microscopy, as used within the Human Protein Atlas project, intrinsically offers single-cell decision.

But not all proteins have corresponding antibodies, and a few antibodies bind to proteins solely weakly or non-specifically. Furthermore, as a result of antibody-based approaches goal particular proteins, researchers can solely see that portion of the proteome. Many within the single-cell proteomics group have as a substitute turned to mass spectrometry, a non-targeted technique that identifies and quantifies molecules on the premise of their mass and cost (see ‘Two paths to the proteome’).

Source: Adapted from N. Slavov Science 367, 512–513 (2020).

The proven fact that mass spectrometry is delicate sufficient to determine at the very least some proteins on the single-cell stage was by no means unsure: some devices can detect attomolar (10–18 moles) portions of fabric, the equal of a number of hundred thousand ions. According to 1 research2, the median mammalian protein is current at 18,000 copies per cell. But manipulating the contents of a single cell and faithfully transferring them right into a mass spectrometer poses challenges.

As lately as 5 years in the past, says Matthias Mann, director of the Max Planck Institute of Biochemistry in Munich, Germany, “the community was so far away from single-cell sensitivity, and also from handling single cells” that he used to assume “it might happen some time, but not in my career”. Yet the sphere has accelerated quicker than he anticipated.

Small samples

According to Mann, that acceleration stems not solely from advances in instrumentation and analytical instruments, but additionally, crucially, in pattern preparation. “You want to have this whole reaction happen in a small volume so that you don’t lose the proteins and they don’t adsorb everywhere,” he explains.

Ctortecka’s proteoCHIP is one such design; one other is nanoPOTS, developed by chemists Ryan Kelly and Ying Zhu on the Pacific Northwest National Laboratory in Richland, Washington.

NanoPOTS is sort of a nanolitre-scale microtiter plate fabricated onto the floor of a microscope slide3. Each ‘well’ is a hydrophobic circle about one millimetre in diameter, with a small hydrophilic ‘pedestal’ on the centre at which cells are deposited and ready. “Think about the mesas in Arizona,” says Kelly, now at Brigham Young University in Provo, Utah, referring to the US state’s iconic, flat-topped hills: “All the stuff is taking place on the top of the mesa.”

The pedestal’s small space means there’s a smaller floor for proteins to stick to — about 99.5% lower than a 0.5-millilitre centrifuge tube, as Kelly and Zhu word of their research. The correspondingly small response volumes (lower than 200  nanolitres) enhance enzyme focus and thus effectivity. Add the truth that the response protocol limits liquid-handling steps, and the result’s an elevated yield of proteins per cell. Kelly’s crew noticed from 2- to 25-fold extra peptides with nanoPOTS than when samples had been ready in 0.5-millilitre centrifuge tubes. Using nanoPOTS, Kelly’s crew has detected a mean of 1,085 and 1,012 proteins for every of two courses of main human neuron4.

How complete that’s depends upon the way you rely — some genes encode a number of proteoforms, as an example, and never all proteins are expressed in all cells. However, that quantity is par for the course for single-cell proteomics: some researchers declare to have improved on it in unpublished work, however most research determine about 1,000 proteins per cell (though the full variety of recognized proteins throughout all cells is increased). In a February preprint5, as an example, Mann’s crew used a brand new instrument design from mass-spectrometry vendor Bruker in Billerica, Massachusetts, to detect proteome variations as cells progress by way of the cell cycle. The median variety of proteins detected per cell-cycle stage ranged from 611 within the development section of cell division to 1,263 within the subsequent section, when DNA is synthesized. Ongoing work has detected extra (952 and 1,773, respectively). But that quantity was sufficient to tease aside organic variations. “Every single cell has quite a stable proteome,” Mann notes, that means that researchers may be capable to analyse fewer cells than different single-cell strategies require. “Conceptually, that is the most exciting result of that paper,” Mann says.

It nonetheless takes a very long time to amass these information, nonetheless. Single-cell proteomics research have a tendency to make use of ‘bottom-up’ methods to determine proteins from a smattering of peptide fragments quite than in search of intact proteins. But these peptides are recognized separately, not in parallel. And the mass spectrometer wants time to build up every ion. For one research, Erwin Schoof, a organic mass spectrometrist on the Technical University of Denmark in Lyngby, allotted half a second per peptide in a 160-minute run. “On a good day we are measuring 4,500 peptides per cell,” Schoof says. As a consequence, his crew might analyse simply eight samples per day.

Sample preparation can also be a bottleneck. With 27 wells, the unique nanoPOTS might course of 27 single cells at a time. Zhu’s second-generation ‘nested nanoPOTS’ (N2) design incorporates a 3 × Three grid of pedestals in every effectively, supporting as much as 243 cells (27 × 9) directly6. According to Zhu, N2 was designed to accommodate one other essential growth in single-cell proteomics: multiplexing, which will increase throughput. 

Close up on the Q Exactive mass spectrometer

Part of the mass spectrometer utilized by methods biologist Nikolai Slavov to review single-cell proteomics.Credit: Northeastern University/Ruby Wallau

In 2018, Slavov’s crew described a technique known as SCoPE-MS (single-cell proteomics by mass spectrometry)7, which blends a mass-spectrometer-friendly cell-lysis protocol with a protein service that will increase the quantity of fabric obtainable for sequencing. “This kind of approach immediately increased our ability to determine peptide sequences without doing anything difficult,” Slavov explains. “We were outsmarting the problem rather than brute-forcing it.”

Barcode breakthrough

Crucially, SCoPE-MS additionally options mass spectrometry’s model of barcoding: isobaric tags. These are molecules with similar lots that fragment into in another way sized ions inside a mass spectrometer. By coupling completely different tags to completely different samples, researchers can work out how a lot of a given protein is current in each. Using tandem mass tag (TMT) reagents, as an example, researchers can differentiate between as much as 18 samples in a single combination8. But to take action, the samples should be labelled individually after which pooled — a technically difficult step, given the small volumes concerned. “The robot has to be very precise to withdraw this nanolitre volume and put them together for mass-spectrometer analysis,” Zhu explains. N2 permits researchers to course of cells individually however then pool them in a single step by including a big sufficient droplet of buffer to cowl all the person pedestals in a single ‘well’, thus circumventing that concern.

At this yr’s Single Cell Proteomics convention in Boston, Slavov’s graduate scholar Andrew Leduc introduced an alternate method. Leduc described how he and his colleagues used a piezo-acoustic meting out gadget to array and put together some 1,500 cells in 20-nanolitre droplets. These had been clustered in teams of 12–14 on microscope slides to simplify pooling, and surrounded by a fringe of water droplets to extend humidity and stop evaporation9. The crew has used that technique to review macrophage stimulation and the cell division cycle.

Meanwhile, different members of Slavov’s crew have revamped SCoPE-MS. SCoPE2 makes use of an easier cell-lysis method and improved evaluation pipeline10, and is broadly accessible and scalable for manufacturing use, Slavov says.

Other researchers try to profit from their devices’ treasured time by altering how they gather information. Most mass spectrometrists run their machines in a ‘data-dependent acquisition’ mode, wherein the instrument identifies and sequences probably the most plentiful ions. As a consequence, these analyses are likely to overlook probably the most fascinating, lower-abundance proteins.

Another possibility is a focused method, wherein the instrument is advised particularly which ions to search for. But some researchers are actually exploring methods that scan every part within the pattern and work out the main points later. These ‘data-independent analysis’ strategies usually are not sometimes appropriate with multiplexing, however in February, Ctortecka and her colleagues reported a method for combining the 211. “So you have a systematic way to look at your peptides in your sample, and this is performed in every single run exactly the same,” she says.

For his half, Schoof says he’s working with distributors to speed up chromatographic separations, and thus velocity up experiments from 160 minutes to an hour. Using different optimizations, he has a roadmap to ramp as much as 20 samples, or 360 multiplexed cells, per day. At that price, he says, “a 10,000-cell experiment like you see in single-cell RNA-seq is, for lack of a better word, ‘only’ one month of runtime. In terms of doing single-cell proteomics, that’s already quite an achievement.”

Another dimension

Most single-cell strategies take away cells from their tissue context. But the place within the tissue a cell resides really issues. By disaggregating cells, researchers lose what Mann calls their “sociology”. So he and different researchers are working so as to add a spatial dimension to single-cell proteomics, though not one of the approaches is but on the single-cell stage.

Last yr, Kelly and his colleagues revealed a method combining nanoPOTS, laser-capture microdissection (which makes use of a laser to excise cells from tissue) and mass spectrometry to element some 2,000 proteins per 100-micrometre pixel12. In May, a crew led by most cancers researcher Thomas Cox of the Garvan Institute of Medical Research in Sydney, Australia, and imaginative and prescient scientist Gus Grey on the University of Auckland, New Zealand, mixed ultra-high-resolution mass spectrometry and an R software program bundle known as HIT-MAP to sequence and determine proteins in intact samples of bovine lens tissue13.

And in January, Mann and his crew reported a method known as Deep Visual Proteomics14, which blends synthetic intelligence, microscopy and laser-capture microdissection to mechanically determine, isolate and characterize as few as 100 cells of a given kind in tissue. His crew used the method to distinguish between cells on the centre and periphery of human melanoma samples. “I think this can be quite a game-changer,” he says.

Others, akin to Kelleher, are pushing for single-molecule, single-cell proteomics — that’s, the power to sequence particular person protein molecules in a cell. At the second, he says, “we’re barely at proof-of-concept for some of these underlying technologies.” But their growth is prone to get a lift. In July, the US National Institutes of Health introduced some US$20 million in funding for know-how growth in single-molecule and single-cell proteomics. And Kelleher estimates that personal traders have poured some $2 billion extra into the subfield.

To profit from these applied sciences, Kelleher and others advocate for a complete atlas of all of the human proteoforms that may very well be current in a pattern. Just because the Human Genome Project offered a reference genome that made next-generation DNA sequencing applied sciences extra highly effective, Kelleher and his colleagues envision a Human Proteoform Project to create what they name “a definitive reference set of the proteoforms produced from the genome”15. Such a useful resource might improve the ability of each single-cell and single-molecule proteomics applied sciences by permitting researchers to pay attention extra on ‘scoring’ proteins than discovering them, Kelleher says.

There’s no assure that such an atlas will come to move. But in the case of ’omics, one ought to by no means wager in opposition to the know-how. When she began her doctoral work, Ctortecka doubted her undertaking would succeed, however thought she would be taught one thing fascinating in any occasion. “I was very much convinced that this would never be possible,” she says. “Look where we are now.”

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