Massively parallel profiling of the intracellular activity, interactions, and druggability of protein variants using LABEL-seq

Multiplexed assays of variant effect are powerful tools for assessing the impact of protein sequence variation. Here, we describe LAbeling with Barcodes and Enrichment for biochemicaL analysis by sequencing (LABEL-seq), a platform for massively parallel profiling of thousands of pooled protein variants in cultured human cells. LABEL-seq directly measures protein properties and functions at scale using simple affinity enrichments by leveraging the intracellular self-assembly of an RNA binding domain with a highly stable RNA barcode. Variant effects are quantified by affinity enrichment of RNA binding domain-protein fusions based on a biochemical property or function, followed by high throughput-sequencing of co-enriched barcodes. Measurement of ~20,000 variant effects for ~1,600 BRaf variants revealed that variation at positions that are frequently mutated in cancer minimally impacted folding and intracellular abundance but could dramatically alter activity, protein-protein interactions, and druggability. Integrative analysis identified networks of positions with similar biochemical roles and enabled predictive modeling of variant effects on cell proliferation and small molecule-promoted degradation. LABEL-seq provides a scalable approach for the direct measurement of multiple biochemical effects of protein variants in their native cellular context, yielding insight into protein function, disease mechanisms, and druggability.