For years, the relative abundance of particular proteins has been estimated from mRNAs in bulk tissue samples. Single-cell transcriptomics allowed a more pinpointed inference of protein levels based on detected RNA transcripts. But now the nascent field of single-cell proteomics promises direct measurements of the proteins within or secreted from individual cells. Here is a look at the emerging field of single-cell proteomics and recent applications of its technologies to combating SARS-CoV-2, the virus responsible for the COVID-19 pandemic.
Proteomics by mass spectrometry
The Thermo Scientific™ Orbitrap Eclipse™ Tribrid™ mass spectrometer, along with the Thermo Scientific™ FAIMS Pro™ Interface, provides the high resolution and selectivity needed to analyze proteins at the single-cell level. “We now can identify over 1000 proteins from a single HeLa cell, which improved the protein coverage by 5x versus previous studies,” says Khatereh Motamedchaboki, vertical marketing specialist at Thermo Fisher Scientific.
Increasing throughput is important because of the nature of single-cell proteomics. “There are no technical replicates when studying single cells,” says Motamedchaboki. “Each cell is unique; therefore, you need to analyze many cells to make sure that we can get meaningful biological data to overcome the large variability from cell to cell.”
Thermo Fisher Scientific combines mass spectrometry instrumentation with tandem mass tag (TMT) labeling to boost detection in single cells. They also increase analysis throughput by using multiplexing reagents that enable the analysis of several individual cells in a single mass spectrometry analysis. The Thermo Scientific TMTpro™ 16plex Label Reagent, along with Real-Time Search, an intelligent data acquisition strategy, helps to increase single-cell analysis throughput by up to 16 cells per analysis run. This method has been optimized to use in conjunction with the main single-cell sample preparation protocols (SCoPE-MS and NanoPOTS).
Future solutions will require a wider adaptation of TMT labeling to increase throughput and quantitation accuracy. “Sample preparation, analysis throughput, and optimized single-cell data analysis workflows are all good challenges to have, because if we solve them, I see lots of opportunities,” says Motamedchaboki. “I believe it’s going to be an exciting time for the field of single-cell proteomics, which is still in its infancy.”
Proteomics by antibody detection
BioLegend’s TotalSeq™ reagents are oligo-conjugated monoclonal antibodies that provide a protein detection signal that is read by next-generation sequencing (NGS). These can be combined with RNA detection for multi-omic analysis of single cells. “The oligonucleotide contains a unique barcode sequence that identifies epitope specificity,” says Iván Godinez, product manager at BioLegend. “This allows the combination of protein expression data with either targeted or whole-transcriptome analysis.”
TotalSeq reagents are available in three formats denoted as A, B, and C. TotalSeq-A antibodies have a poly-A sequence and work with any platform using poly-dT-based mRNA capture, while TotalSeq-B and -C conjugates are compatible with the 10x Genomics Solutions. “Single-cell proteogenomics is a rapidly growing and evolving field with tremendous potential,” says Godinez. “I envision that single-cell proteogenomics will revolutionize vaccine development, oncology research, pharmaceutical development, and personalized medicine.”
IsoPlexis offers the IsoLight platform for highly multiplexed single-cell proteomics of over 30 cytokines released from individual cells. The IsoLight System analyzes cells that are loaded onto an IsoCode chip containing microchambers for single cells. In each microchamber, which contains a unique proteomic barcode, multiplexed ELISA reactions using highly sensitive antibodies occur to detect cytokines secreted by the cell. Known as functional phenotyping, the results of IsoPlexis’s Single-Cell Secretome solution allow researchers to take proteomic snapshots of individual immune cells by measuring the many cytokines they secrete.
Applications of their technology are helping to predict which patients might respond to particular therapies for blood and solid tumor cancers as well as infectious disease, says Sean Mackay, co-founder and CEO of IsoPlexis. “Our technology allows clinical researchers to uniquely pinpoint each single cell’s functional phenotype that influences the course of therapy, and identifying these cell subsets has helped predict durable and quality immune response correlating to outcome preclinically and in the clinic,” he says.
Future plans include greater integration of multi-omics tools, like the IsoPlexis Single-Cell Intracellular Proteome and Metabolome solutions. “This will unlock the next level of resolution in cellular and immune research,” says Mackay.
Applying single-cell proteomics tools to COVID-19
Using MS-based single-cell proteomics to combat COVID-19 begins with learning more about the structure of the SARS-CoV-2 virus. “For example, experts are using mass spectrometry to look at the virus spike protein involved in the infection of human cells,” says Motamedchaboki. “Researchers are using MS strategy tools such as glycoproteomics and glycomics to map out the glycans on the virus spike protein.” MS-based methods to monitor the virus or the progression of COVID-19 may also be in the offing. “Once markers are validated, new MS targeted methodologies such as SureQuant and internal standard triggered MS analysis could be implemented for high-throughput, sensitive, and accurate detection of these biomarkers from biological samples,” she says.
In the antibody-based realm, BioLegend is partnering with researchers using TotalSeq reagents “to analyze immune responses to SARS-CoV-2 at a single-cell RNA and protein level in the lungs of over 100 infected individuals,” says Godinez. IsoPlexis is collaborating with Shelli Farhadian of Yale University to study the role of cytokines and neuroinflammation in COVID-19 patients with neurological disorders using IsoPlexis’s CodePlex Secretome. In addition, IsoPlexis is collaborating with the Institute for Systems Biology (ISB), Swedish Medical Center, Merck, and others to identify immune biomarkers for SARS-CoV-2. For example, researchers at ISB are using IsoPlexis’s IsoLight platform to analyze samples from COVID-19 patients collected at different timepoints in the disease progression: initial infection, acute disease, and convalescence. The goal is to map the functional immune response in patients, both ill and recovered.
ISB President James Heath is co-leading the ISB-Swedish COVID-19 trial, and he says that a patient’s immune profile may help to understand the trajectory of the disease. “The IsoPlexis assay may be especially helpful in resolving the underlying mechanisms of the cytokine storms observed in severely ill COVID-19 patients,” says Heath. “For example, what are the dominant cell types that have participated in those cytokine storms?” Immune analysis of serum reflects the population response, so you can’t tell which cell types are involved. “With the IsoPlexis assay, you can separate out just the macrophages or just the cytotoxic T cells, for example, and look at what’s being secreted by those cell types,” he says.
While the study isn’t yet complete, their results indicate that macrophages are dominating the cytokine storms. Heath says they are also researching the effects of the IL-6 inhibitor drug tocilizumab. “Tocilizumab appears to promote the formation of a novel population of protective memory T cells,” he says. “Whether patients can benefit from this drug is a question that will only be cleanly resolved through double-blind clinical trials, but any such benefit will almost certainly involve exactly when the drug is administered with respect to disease symptoms.”
Stay tuned for more exciting developments in the newly emerging field of single-cell proteomics—both for fighting COVID-19 and for a wide range of other applications.