Division of Cell Signaling, Fujii Memorial Institute of Medical Sciences, Institute of Advanced Medical Sciences, Tokushima University, Tokushima 770-8503, Japan.
Kuramoto Division, Technical Support Department, Tokushima University, Tokushima 770-8503, Japan.
J Proteome Res. 2022 Sep 2;21(9):2094-2103. doi: 10.1021/acs.jproteome.2c00130. Epub 2022 Aug 17.
Chemical or enzymatic biotinylation of proteins is widely used in various studies, and proximity-dependent biotinylation coupled to mass spectrometry is a powerful approach for analyzing protein-protein interactions in living cells. We recently developed a simple method to enrich biotinylated peptides using Tamavidin 2-REV, an engineered avidin-like protein with reversible biotin-binding capability. However, the level of biotinylated proteins in cells is low; therefore, large amounts of cellular proteins were required to detect biotinylated peptides. In addition, the enriched biotinylated peptide solution contained many contaminant ions. Here, we optimized the workflow for efficient enrichment of biotinylated peptides and removal of contaminant ions. The efficient recovery of biotinylated peptides with fewer contaminant ions was achieved by heat inactivation of trypsin, prewashing Tamavidin 2-REV beads, clean-up of biotin solution, mock elution, and using optimal temperature and salt concentration for elution. The optimized workflow enabled identification of nearly 4-fold more biotinylated peptides with higher purity from RAW264.7 macrophages expressing TurboID-fused STING (stimulator of interferon genes). In addition, sequential digestion with Glu-C and trypsin revealed biotinylation sites that were not identified by trypsin digestion alone. Furthermore, the combination of this workflow with TMT labeling enabled large-scale quantification of cell surface proteome changes upon epidermal growth factor (EGF) stimulation. This workflow will be useful for BioID and cell surface proteomics and for various other applications based on protein biotinylation.
蛋白质的化学或酶促生物素化在各种研究中被广泛应用，而与质谱联用的邻近依赖性生物素化是分析活细胞中蛋白质-蛋白质相互作用的有力方法。我们最近开发了一种使用 Tamavidin 2-REV 富集生物素化肽的简单方法，Tamavidin 2-REV 是一种具有可逆生物素结合能力的工程化类似抗生物素蛋白。然而，细胞中生物素化蛋白的水平较低；因此，需要大量的细胞蛋白来检测生物素化肽。此外，富集的生物素化肽溶液中含有许多杂质离子。在这里，我们优化了工作流程，以实现生物素化肽的高效富集和杂质离子的去除。通过热失活胰蛋白酶、预洗 Tamavidin 2-REV 珠、清洁生物素溶液、模拟洗脱以及使用最佳温度和盐浓度进行洗脱，有效地回收了带有较少杂质离子的生物素化肽。优化后的工作流程使从表达 TurboID 融合 STING（干扰素基因刺激物）的 RAW264.7 巨噬细胞中鉴定到的生物素化肽数量增加了近 4 倍，且纯度更高。此外，用 Glu-C 和胰蛋白酶进行连续消化揭示了仅用胰蛋白酶消化无法识别的生物素化位点。此外，将该工作流程与 TMT 标记相结合，可实现表皮生长因子 (EGF) 刺激后细胞表面蛋白质组变化的大规模定量。该工作流程将有助于 BioID 和细胞表面蛋白质组学以及基于蛋白质生物素化的各种其他应用。
