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Singapore Researchers Develop Single-Cell Method Offering Data on Secreted Proteins, RNA


NEW YORK – A new single-cell sequencing-based method offers a way to capture data on proteins secreted by cells, such as cytokines, as well as corresponding transcriptome data.

Time-resolved assessment of single-cell protein secretion with sequencing (TRAPS-seq), developed by researchers at the National University of Singapore, uses antibodies to capture secreted proteins near the cell surface, enabling them to be analyzed with 10x Genomics' feature barcoding assay, along with RNAs.

The method offers the potential to add another dimension to immune cell profiling. Currently, cytokine and chemokine profiling are performed in bulk, which precludes the ability to identify immune cell subpopulations with anomalous protein secretion patterns, according to Lih Feng Cheow, a single-cell researcher at NUS and the senior author of a proof-of-concept study published Monday in Nature Methods.

"Existing methods like CITE-seq can be used to jointly profile single-cell transcriptomes and levels of surface or intracellular proteins using barcoded antibodies," said Hao Wu, a researcher at the University of Pennsylvania who was not involved in the study. "This new multiomics method extends this capability to secreted proteins, which are important for understanding the functions of many types of immune cells in response to environmental stimuli."

Though the method is not quantitative, it can identify high, medium, and low levels of secretion, providing opportunities to discover new cell types and identify therapeutic targets.

The paper also provides additional evidence that protein and mRNA levels are "not perfectly correlated," Cheow said.

Cheow and first author Tongjin Wu, also of NUS, are co-inventors on a patent application covering the method. Their study coauthors include researchers who were, at the time, affiliated with Proteona, which was acquired in March 2022 by China's Singleron Biotechnologies. Cheow said he is open to commercializing the assay but has not yet begun discussions with any single-cell technology providers.

Work on TRAPS-seq began about two years ago and consisted largely of optimizing antibody conjugation as well as developing the idea to look at secretion dynamics by sequential probing with detection antibodies.

At its core, TRAPS-seq uses 10x's feature barcoding technology, which offers the ability to detect cell-surface proteins through oligo-conjugated antibodies. Those oligo barcodes are captured and included in the 10x single-cell sequencing library and associated with a particular cell and its transcriptome.

To allow the 10x assay to capture secreted proteins, which are not normally kept at the cell surface, the researchers developed so-called capture antibodies (CapAbs), which attach to both the protein of interest as well as anchor proteins found on a cell's surface. In the study, they used CD45 immune cell markers often found on T cells as anchor proteins. Control experiments in the study showed that the CapAbs do not perturb the cell's transcriptome, the authors noted.

The capture antibody array also allows for the detection of protein secretion over time. By adding different fluorescent markers to the detection antibodies and spreading their introduction over two time points, the researchers were able to use flow cytometry to see differences in the levels of secreted proteins.

The method may compete with existing antibody-based cytokine and chemokine capture assays, including from providers such as Miltenyi Biotec; however, those technologies can be limited in multiplexing capability and don't also include transcriptome data, nor do they offer the ability to interrogate secretion over time. PhenomeX, formed when Berkeley Lights acquired IsoPlexis in December, also offers a secreted cytokine assay for isolated single cells.

Mass spectrometry can analyze secreted proteins in bulk and with unbiased coverage, but cannot provide information on cell origin, Wu noted.

In their study, the researchers demonstrated the ability to profile three secreted proteins at a time, but Cheow said his team is working to increase that number. "We have shown the capacity is more than that," he said. "Minimally, it can go up to 10, although it depends on the amount of protein [secreted]."

Once the secreted proteins are captured near the cell surface, the 10x assay is run as it normally would be. The cost to produce antibodies to capture secreted proteins is cheap, Cheow added, on the order of $10 per protein per assay.

The assay works for any secreted protein, although immediate applications are likely in immune profiling. For cells without CD45 proteins, they developed a universal method based on cell-surface biotinylation. "The extensive catalogs of verified antibody pairs for sandwich ELISA could be adapted to TRAPS-seq to detect a broad range of secreted proteins for various applications," the authors wrote.