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A Dream of Single-Cell Proteomics As Single-Cell Proteomics Emerges, Perhaps Labs Can Avoid the Need to Infer Protein Levels from Mrna Abundances

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A Dream of Single-Cell Proteomics As Single-Cell Proteomics Emerges, Perhaps Labs Can Avoid the Need to Infer Protein Levels from Mrna Abundances technology feature A dream of single-cell proteomics As single-cell proteomics emerges, perhaps labs can avoid the need to infer protein levels from mRNA abundances. Vivien Marx owadays, labs can generate massive sets of single-cell genomic and Nsingle-cell transcriptomic data. In proteomics, high-throughput single-cell methods have not yet arrived. The nascent field of single-cell proteomics (sc-proteomics) is bringing change, and perhaps helping to ,251...1,252...1, avoid the need to infer proteins from cellular ...2,374, 2,375... mRNA levels. It’s early days, but it’s not 3,554...3,555...3,556 a distant dream to be able to tally the 4,020...4,021...4,022 3,577...5,978...5,97..6,789...6,799 proteins in single cells, says Ruedi Aebersold, 20 a proteomics researcher at ETH Zurich and the University of Zurich. To make that dream an everyday reality, labs push hurdles out of the way. Proteins are tougher to work with than RNA or DNA, for example they’re stickier, but eventually researchers might be able to integrate single-cell mRNA and single-cell proteomic measurements. Over 20 years ago, says Aebersold, Richard Smith at Pacific Northwest National Laboratory and his colleagues characterized hemoglobin from a single red blood cell with a technique called capillary electrophoresis-electrospray ionization Fourier transform ion cyclotron resonance 1 mass spectrometry . The red blood cell It’s not a faraway dream to be able to tally proteins in single cells. Credit: S. Larochelle, E. Dewalt, was a special case, says Aebersold, since Springer Nature it mainly consists of hemoglobin. But this was single-cell analysis. Fast forward to a recent approach that Aebersold and his colleague Ben Collins call note the approach is “inherently single Single molecules a “marriage across the ages”2. Developed in molecule” and thus “there are reasonable It’s hard to speculate how fast technology the labs of Edward Marcotte, Eric Anslyn prospects for decreasing sample volumes for sc-proteomics will develop, says and colleagues, at the University of Texas at and protein abundance requirements.” Harvard Medical School researcher Austin, the technique yields the amino acid They state that once fluorescent labeling Peter Kharchenko, but “I am most excited sequence of individual proteins in a highly of low-abundance proteins with fluorescent about the ability to quantify phosphorylation parallelized fashion3. A spinout company, tags can be achieved, this method has and other modifications.” This would Erisyon, has been launched to commercialize application potential, such as for single-cell move the field “beyond simple abundance- a single-molecule protein sequencer. proteomics experiments. based models to more accurate dynamic The approach involves Edman sequencing, It’s far from single-cell analysis, says descriptions.” Humboldt University with which proteins were sequenced in the Aebersold, but “the method certainly has researcher, Rune Linding, hopes such days before mass spec. Proteins are cleaved potential to do single cells, potentially much approaches might open up new ways to and the peptides are labeled with identifying faster and in ways that the mass spectrometer analyze phosphorylation dynamics. fluorescent tags; the tagged peptides are then would have a hard time doing.” As a student, Proteomics and genomics labs pursue immobilized on a glass cover-slip. Successive he used Edman sequencing and he is different questions “but they clearly provide rounds of Edman degradation chemistry intrigued to see its revival for single-molecule different views on the same system,” says remove one amino acid at a time and the fluorescence. The method piggybacks on Aebersold. “In proteomics, we’re always kind peptides are imaged at each round. The team flow-cell technology used in high-throughput of limping a while behind the genomics found dyes that handled the process but some genome sequencers, he says. It will take work field,” he says. Single-cell genomics and mishaps occurred: dyes fell off, didn’t attach to make this a routine application, he says, transcriptomics can capture “a kind of well or provided inadequate fluorescence. and other labs have such approaches in genealogy of the cell,” and track cells as In their published study, the team analyzed their sights, too. This study is an important they evolve and change through mutations, a zeptomolar mixture of proteins, but they proof of principle. he says. Proteomics labs can now analyze NATURE METHODS | VOL 16 | SEPTEMBER 2019 | 809–812 | www.nature.com/naturemethods 809 technology feature cells with liquid chromatography and tandem sonication by lysing cells with a freeze–heat mass spectrometry (LC-MS/MS). They cycle in pure water5. The team is testing rethought sample preparation: cell lysis, the efficiency of this sample preparation protein purification, digestion and clean-up. technique and “it appears to be at least as And there’s mass spec instruments’ aversion good, if not better than, urea lysis,” he says. to the clean-up chemicals to consider, says He and his team used this method to Slavov. In standard mass spectroscopy, sample quantify 2,000 proteins in 356 cells — a is always lost as it can, for example, stick to sample containing both monocytes the sides of chromatography columns. Bulk and macrophages. sample analysis helps labs cope with that. Vogel says that pure water might avoid But, when assessing single mammalian cells artifacts from chemicals, but she wonders and their few hundred picograms of proteins how soluble proteins without ion content are each, little if any cargo should go missing. in pure water. Speaking more generally about sample preparation for sc-proteomics, she small numbers of cells. “It’s a beginning,” he says that proteins are trickier than RNA and Freeze–heat Protein is digested Water says. This is how single-cell transcriptomics cycle to peptides DNA: they cannot be amplified, they’re sticky started, followed by massive multiplexing and and they degrade easily. “Until someone barcoding strategies that allowed resolution invents something to ‘amplify proteins’ that’ll of large numbers of cells. Such trends will always be the problem,” she says. Many take a while to develop in proteomics. methods, including hers and others, address Labs are exploring cytometry-related ways sample preparation by using techniques such to reap single-cell data. A team that includes 96 or 384-well plate as hydrostatic pressure or engineered surfaces researchers at the University of Ottawa and for peptide enrichment. the University of Oxford, used single-cell The Slavov lab is testing their mass spec mass cytometry (CyTOF) to capture the sample-prep method to lyse cells in pure water, Multiplexing cell-fate decisions during hematopoiesis, and using a freeze–heat cycle. Credit: Slavov lab, With TMT, around 20 barcodes can be tracked how transcription factor expression Northeastern Univ. used. But labeling a protein or peptide changed during a cell’s lineage commitment introduces complexities, says Aebersold: at 13 time-points. They measured 27 proteins the reaction must be just right, excess simultaneously in single cells. Another single- Slavov, his graduate student Harrison has to be removed and there’s clean-up. cell CyTOF effort by a team at the University Specht, and the team, hunted a different way Multiplexing is a scale-up that makes of Zurich, along with colleagues at other to extract proteins efficiently for LC-MS/MS workflow more complex. Transcriptome institutions, profiled tumor and immune cells analysis. “We kept trying different analysis is readily available to biologists with from 144 human breast tumor samples. approaches, most of which didn’t work,” commercially well-supported techniques, In mass cytometry/CyTOF, cells are says Slavov. Sonication to lyse cells with while proteome analysis is mainly done in prepped for analysis with isotope-conjugated focused acoustic waves led them to develop expert labs and “cannot easily reach the antibodies. In sc-proteomics, it will likely be SCoPE-MS. When they validated the throughput, robustness and reproducibility key to integrate different technologies and method, a student in the lab held the tube in of transcriptome analysis,” as Aebersold physical principles, as with the combination the sonicator to lyse cells one at-a-time. The and his colleague Ben Collins point out. of Edman sequencing and microscopy, says team mixed labeled peptides with labeled But it doesn’t have to stay that way. New York University researcher Christine ‘carrier’ peptides to avoid the “never-ending The 200–300 picograms of protein Vogel, who was a postdoctoral fellow in chase” to quantify sample losses from the in one mammalian cell cannot yet be tallied, the Marcotte lab. clean-up process. They used isobaric tandem says Aebersold. A properly tuned and mass tags (TMT), which bind to all peptides, optimized mass spec instrument can detect Rethinking sample prep including the carrier peptides. The TMT tags all or most proteins expressed in a cell only “My ideology is to make this as accessible all have the same molecular weight so “when when many cells are analyzed concurrently. as possible,” says Nikolai Slavov, at they enter the instrument, they’re going to His lab has detected 500 to 1,000 distinct Northeastern University, about his approach correspond to a single peak in m/z space,” proteins in a single cell from a sample to sc-proteomics methods development. In says Slavov. equivalent to a single cell using SWATH-MS, keeping with his training in genetics and Vogel likes SCoPE-MS and says it’s still a type of data-independent analysis. biology, and interests in math, chemistry necessary to test new buffers and optimize They have not yet ‘processed’ a single and physics, Slavov runs cross-disciplinary the approach, which her lab is currently cell but they injected a proportional sc-proteomics meetings: mass spec veterans doing.
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