Highly multiplexed and quantitative cell-surface protein profiling using genetically barcoded antibodies

Abstract

SignificanceNext-generation sequencing (NGS) has allowed the comprehensive study of the genome and transcriptome. However, a similarly broad, highly multiplexed, and inexpensive method for proteomics using NGS remains elusive. Here, we describe a phage display-based method using preselected antibodies that are genetically encoded and capable of simultaneous profiling of hundreds of cell-surface targets on cells in culture or singly at low cost and without the need for chemical conjugation to purified antibodies. We use the method to identify cell-surface proteins that change in cancer cells, some of which are coordinately regulated and could lead to new biomarkers and cancer targets.

Light-mediated discovery of surfaceome nanoscale organization and intercellular receptor interaction networks

“…Depending on the binding specificity of the employed viral particle, LUX-MS enables the spatiotemporal elucidation of viral attachment sites, target receptors, and global surfaceomes of live prokaryotic and eukaryotic cells in complex biological systems. This is of particular interest considering the broad application of advanced phage display assays 77 and related screening platforms 5,78 in basic and translational research. Finally, we employed LUX-MS for the spatiotemporal analysis of intercellular surfaceome signaling domains within immunological synapses.…”

Nucleic Acid‐Barcoding Technologies: Converting DNA Sequencing into a Broad‐Spectrum Molecular Counter

“…Notably,t he Wells lab recently reported as imilar workflow using alibrary of preselected, phage-displayed antibodies for the high-throughput identification of cell surface proteins via NGS. [97] Another common DNA-antibody conjugate technique for the detection of proteins and protein complexes is proximity ligation, [98] which occurs when antigen recognition by two independent antibodies induces proximity between the corresponding barcodes ( Figure 8b). Theh igh effective molarity of the two localized DNAstrands can be exploited to promote barcode ligation, and ligated sequences can be identified in ahigh-throughput format by NGS to determine protein-protein complex identities.…”

“…After incubation of the antibody library with a cell line of interest, the DNA barcodes that remained bound to cells were identified to determine potential pathway dependencies in patient tumor samples. Notably, the Wells lab recently reported a similar workflow using a library of preselected, phage‐displayed antibodies for the high‐throughput identification of cell surface proteins via NGS . Another common DNA–antibody conjugate technique for the detection of proteins and protein complexes is proximity ligation, which occurs when antigen recognition by two independent antibodies induces proximity between the corresponding barcodes (Figure b).…”

CellectSeq: In Silico Discovery of Antibodies Targeting Integral Membrane Proteins Combining In Situ Selections of Phage Displayed Synthetic Antibodies and Next-Generation Sequencing

“…Third, the library was constructed with a single, highly stable human framework resulting in negligible display bias where most library members are presented at similar levels 37 . Also, the abundances of individual clones in pools enriched for target antigens are highly correlated with relative a nities 39 ; this property enhances NGS analysis based on enrichment ranking, allowing for the identi cation of highly selective and high a nity clones. Fourth, the synthetic Abs are diversi ed at only four complementary determining regions (CDRs; H1, H2 and H3, and L3), which permit standard NGS procedures utilizing primers that anneal to common framework regions in a cost-effective manner ( Figure S2 & S3).…”