Quantitative proteome profiling in HEK293 to validate RNAi targets

The solute carrier (SLC) transporter superfamily are important coordinators of metabolic and cellular homeostasis. Despite their importance for cellular homeostasis, SLCs remain a largely understudied group of proteins regarding their protein interactome. Thus, we conducted a largescale interactome proteomics study to characterize the interactome of roughly 400 of the 450 superfamily members. Within this study, we validated selected SLC-protein interactions by different assays (e.g.: protein stability, transporter activity assays), in combination with RNA interference (RNAi). For a small subset of the targeted interactors, we used full protein profiling by quantitative mass spectrometry to confirm alleviation of the protein abundance upon RNAi. To validate RNAi downregulation of gene expression on protein level, we assessed protein abundance of ZPFL1, LTN1 and NFXL1 after siRNA treatment.

溶质载体（SLC）转运蛋白超家族是调控代谢与细胞稳态的关键蛋白家族。尽管其对细胞稳态至关重要，但目前针对SLC家族蛋白的蛋白质相互作用组（protein interactome）的研究仍相对匮乏。为此，我们开展了一项大规模相互作用组蛋白质组学研究，对该超家族450个成员中约400个的蛋白质相互作用组进行了系统表征。本研究中，我们借助多种实验方法（如蛋白质稳定性检测、转运活性检测），联合RNA干扰（RNA interference, RNAi）技术，验证了部分筛选得到的SLC-蛋白质相互作用。针对少量靶向相互作用蛋白亚群，我们采用定量质谱法开展全蛋白质谱分析，以证实RNAi处理后蛋白质丰度的下调现象。为验证RNAi在蛋白质层面的基因表达下调效应，我们检测了经siRNA处理后ZPFL1、LTN1及NFXL1的蛋白质丰度。