Department of Chemistry, University of Washington, Seattle, WA, USA.
Department of Genome Sciences, University of Washington, Seattle, WA, USA.
Nat Methods. 2024 Nov;21(11):2094-2106. doi: 10.1038/s41592-024-02456-7. Epub 2024 Oct 21.
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.
在这里，我们描述了一种通过测序进行生化分析的标记和富集（LABEL-seq）方法，该方法可用于大规模平行分析人细胞中混合的蛋白质变体。通过利用 RNA 结合域 (RBD) 与稳定的、编码变体的 RNA 条码的细胞内自组装，LABEL-seq 可以通过简单的 RBD 蛋白融合亲和富集，然后对共富集的条码进行高通量测序，从而直接测量蛋白质的性质和功能。对大约 1600 个 BRaf 变体的大约 20000 个变体效应的测量表明，在癌症中经常发生突变的位置的变异对细胞内丰度的影响最小，但可以极大地改变活性、蛋白质-蛋白质相互作用和药物开发性。综合分析确定了具有相似生化作用的位置网络，并能够对变体对细胞增殖和小分子促进降解的影响进行建模。因此，LABEL-seq 可以在天然细胞环境中直接测量多种生化性质，为蛋白质功能、疾病机制和药物开发性提供了深入的了解。
