LAUSR

Significance Next-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. Human cells express thousands of different surface proteins that can be used for cell classification, or to distinguish healthy and disease conditions. A method capable of profiling a substantial fraction of the surface proteome simultaneously and inexpensively would enable more accurate and complete classification of cell states. We present a highly multiplexed and quantitative surface proteomic method using genetically barcoded antibodies called phage-antibody next-generation sequencing (PhaNGS). Using 144 preselected antibodies displayed on filamentous phage (Fab-phage) against 44 receptor targets, we assess changes in B cell surface proteins after the development of drug resistance in a patient with acute lymphoblastic leukemia (ALL) and in adaptation to oncogene expression in a Myc-inducible Burkitt lymphoma model. We further show PhaNGS can be applied at the single-cell level. Our results reveal that a common set of proteins including FLT3, NCR3LG1, and ROR1 dominate the response to similar oncogenic perturbations in B cells. Linking high-affinity, selective, genetically encoded binders to NGS enables direct and highly multiplexed protein detection, comparable to RNA-sequencing for mRNA. PhaNGS has the potential to profile a substantial fraction of the surface proteome simultaneously and inexpensively to enable more accurate and complete classification of cell states.