Proximity Sequencing Enables Measurement of Protein Complexes in Single Cells.

Abstract

Advances in single-cell sequencing technology have transformed our understanding of cellular heterogeneity and identity, providing insight on topics ranging from the diversity of microbial ecosystems to tumor biological behaviors. However, accurate measurement of protein complexes in single cells remains challenging as relatively few proteins can form large numbers of complexes that must be distinguished accurately. The inability to accurately differentiate protein complexes hinders in-depth study of critical biological functions such as immune signaling and cellular differentiation. In a recent article in Nature Methods, Vistain et al. (1) present a solution to this problem by combining proximity ligation assay (PLA) with single-cell RNA sequencing (scRNA-seq). This method, which they call proximity sequencing (Prox-seq), can measure hundreds of protein complexes and mRNAs in single cells simultaneously. Prox-seq introduces pairs of DNA-conjugated antibodies to single-cell sequencing methods. When proteins enter a complex, the DNA oligomers on the antibodies come into proximity and are ligated. These ligated DNA oligomers (PLA products) are then sequenced to retrieve protein complex information. Prox-seq also generates transcriptome data through mRNA sequencing, allowing multimodal analyses of single cells. To validate this assay, the authors first demonstrated Prox-seq’s ability to differentiate T and B cells in a mixed population using probes designed for 11 cell-specific protein targets. Prox-seq measurements showed that T and B cells could be accurately differentiated using mRNAs, proteins, or total PLA products. An orthogonal method of flow cytometry was used to confirm that the enriched PLA products in each cell type were cell-specific proteins. This confirmed that PLA products can be measured using scRNA-seq, and PLA data displays cell type-specific differences in protein expression. Quantification of protein expression analysis in the same cell types with a different panel of 13 Prox-seq probes showed high correlation with flow cytometry which confirmed accurate protein characterization and quantification by Prox-seq in single cells. The authors also tested Prox-seq’s ability to measure large numbers of protein complexes in 8700 human peripheral blood mononuclear cells with a panel of probe pairs targeting 38 immune cell markers, and up to 741 unique protein complexes. The data showed 37 protein complexes and measurements identifying the expected cell types. In addition, they identified a novel CD8 and CD9 interaction in naïve CD8 T cells. Lastly, authors demonstrated that the assay can identify variability of receptor components in individual human macrophages after activation of an innate immunity signaling pathway by measuring PLA products detected within a panel designed for 15 pathway-specific protein targets.