Quantification of extracellular proteins, protein complexes and mRNAs in single cells by proximity sequencing

We present proximity sequencing (Prox-seq) for simultaneous measurement of proteins, protein complexes and mRNAs in thousands of single cells. Prox-seq combines proximity ligation assay with single-cell sequencing to measure proteins and their complexes from all pairwise combinations of targeted proteins, providing quadratically scaled multiplexing. We validate Prox-seq and analyze a mixture of T cells and B cells to show that it accurately identifies these cell types and detects well-known protein complexes. Next, by studying human peripheral blood mononuclear cells, we discover that naive CD8(+) T cells display the protein complex CD8-CD9. Finally, we study protein interactions during Toll-like receptor (TLR) signaling in human macrophages. We observe the formation of signal-specific protein complexes, find CD36 co-receptor activity and additive signal integration under lipopolysaccharide (TLR4) and Pam2CSK4 (TLR2) stimulation, and show that quantification of protein complexes identifies signaling inputs received by macrophages. Prox-seq provides access to an untapped measurement modality for single-cell phenotyping and can discover uncharacterized protein interactions in different cell types.

Automated summary

We present Proximity Sequencing (Prox-seq), a method that allows simultaneous measurement of proteins, protein complexes, and mRNAs in thousands of single cells. Prox-seq combines proximity ligation assay with single-cell sequencing and enables the measurement of protein interactions and complexes. Validation experiments on T cells and B cells show that Prox-seq accurately identifies different cell types and detects known protein complexes. Further analysis on human peripheral blood mononuclear cells reveals new protein complexes and interactions, providing insights into cell signaling processes.