NanoPOTS N2 Chip Enables High-Throughput and High-Efficiency Sample Preparation for Single-Cell Proteomics

The Science  

The throughput of single-cell genomics and single-cell transcriptomics techniques has vastly improved over the past few years. However, single-cell proteomics methods have lagged behind. Researchers from the Environmental Molecular Sciences Laboratory and Pacific Northwest National Laboratory recently developed a new high-throughput and high-efficiency sample preparation workflow for single-cell proteomics using a novel nested nanowell chip. They designed the new chip—named the nested nanoPOTS, or N2 chip—as a second-generation chip for use on the commercially available single-cell analysis system, CellenONE. The N2 chip-based workflow features an image-based single-cell isolation system for sorting single cells into the nanowells and facilitating protein digestion, tandem mass tag (TMT) labeling, and integration into a liquid chromatography–mass spectrometry workflow using a nanoPOTS autosampler. 

The Impact 

Analysis of the new workflow shows that this platform can quantify roughly 1,500 proteins from about 100 individual cells from three cell lines. The new workflow eliminates the tedious and time-consuming TMT pooling steps, improves sample recovery and proteomics sensitivity, and provides high reproducibility. The new chip and workflow also cut the processing time from the previous nanoPOTS-TMT workflow by half. This workflow improves the throughput and efficiency of single-cell proteomics. 

Summary 

The nanoPOTS N2 chip is a new high-throughput and high-efficiency sample preparation workflow for single-cell proteomics that uses a novel nested nanowell chip. The N2 chip features a total of 27 nanowell clusters containing nine nanowells each. This chip design reduces reaction volume from 200 to less than 30 nanoliters and increases the capacity from 44 to more than 240 individual cells on a single chip. Each nanowell cluster is surrounded by a hydrophilic ring that facilitates the pooling and retrieval steps of the TMT labeling protocol.  

The new single-cell proteomics workflow featuring the N2 chip first involves the sorting of individual cells into each nanowell. Next, reagents are used to lyse cells and reduce, alkylate, and digest proteins. The CellenONE image-based single-cell isolation system is used for both steps. After this, the TMT reagents are dispensed to label peptides, and reference peptides are added to each nanowell cluster. The N2 chip can then be integrated into the liquid chromatography–mass spectrometry workflow using a nanoPOTS autosampler.  

Contact

Ying Zhu 
Environmental Molecular Sciences Laboratory 
ying.zhu@pnnl.gov 

Funding 

This work was supported by a Laboratory Directed Research and Development award from Pacific Northwest National Laboratory and an Intramural program at the Environmental Molecular Sciences Laboratory, a DOE Office of Science User Facility. Part of this work was also supported by several National Institutes of Health grants. 

Publication

Woo, J., Williams, S. M., Markillie, L. M., Feng, S., Tsai, C.-F., Aguilera-Vazquez, V., Sontag, R. L., Moore, R. J., Hu, D., Mehta, H. S., Cantlon-Bruce, J., Liu, T., Adkins, J. N., Smith, R. D., Clair, G. C., Pasa-Tolic, L., and Zhu, Y. 2021. “High-throughput and high-efficiency sample preparation for single-cell proteomics using a nested nanowell chip.” Nature Communications. [DOI: 10.1038/s41467-021-26514-2]