在这里，我们描述了条形码标记和测序富集生化分析 (LABEL-seq)，这是一种对人类细胞中汇集的蛋白质变体进行大规模并行分析的分析方法。通过利用 RNA 结合结构域 (RBD) 与稳定的变异编码 RNA 条形码的细胞内自组装，LABEL-seq 可以使用 RBD 蛋白融合物的简单亲和力富集，然后进行高通量测序，促进直接测量蛋白质特性和功能。 -共富集条形码的通量测序。对约 1,600 个 BRaf 变体的约 20,000 个变体效应的测量表明，癌症中经常突变的位置的变化对细胞内丰度的影响最小，但可能显着改变活性、蛋白质-蛋白质相互作用和成药性。综合分析确定了具有相似生化作用的位置网络，并能够对细胞增殖和小分子促进的降解的变异效应进行建模。因此，LABEL-seq 能够直接测量天然细胞环境中的多种生化特性，提供对蛋白质功能、疾病机制和药物性的见解。© 2024。作者获得 Springer Nature America, Inc. 的独家许可。

Here we describe labeling with barcodes and enrichment for biochemical analysis by sequencing (LABEL-seq), an assay for massively parallel profiling of pooled protein variants in human cells. By leveraging the intracellular self-assembly of an RNA-binding domain (RBD) with a stable, variant-encoding RNA barcode, LABEL-seq facilitates the direct measurement of protein properties and functions using simple affinity enrichments of RBD protein fusions, 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 frequently mutated in cancer minimally impacted intracellular abundance but could dramatically alter activity, protein-protein interactions and druggability. Integrative analysis identified networks of positions with similar biochemical roles and enabled modeling of variant effects on cell proliferation and small molecule-promoted degradation. Thus, LABEL-seq enables direct measurement of multiple biochemical properties in a native cellular context, providing insights into protein function, disease mechanisms and druggability.© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.