Department of Biomedical and Clinical Sciences, Cell Biology, Faculty of Medicine, Linköping University, Linköping 581 85, Sweden.
Department of Physiology, Faculty of Medicine, and Nursing, University of the Basque Country UPV/EHU, Leioa 489 40, Spain.
Anal Chem. 2023 Feb 28;95(8):4033-4042. doi: 10.1021/acs.analchem.2c04500. Epub 2023 Feb 13.
The membrane proteins are essential targets for understanding cellular function. The unbiased identification of membrane protein targets is still the bottleneck for a system-level understanding of cellular response to stimuli or perturbations. It has been suggested to enrich the soluble proteome with membrane proteins by introducing nonionic surfactants in the solubilization solution. This strategy aimed to simultaneously identify the globular and membrane protein targets by thermal proteome profiling principles. However, the thermal shift assay would surpass the cloud point temperature from the nonionic surfactants frequently utilized for membrane protein solubilization. It is expected that around the cloud point temperature, the surfactant micelles would suffer structural modifications altering protein solubility. Here, we show that the presence of nonionic surfactants can alter protein thermal stability from a mixed, globular, and membrane proteome. In the presence of surfactant micelles, the changes in protein solubility analyzed after the thermal shift assay was affected by the thermally dependent modification of the micellar size and its interaction with proteins. We demonstrate that the introduction of nonionic surfactants for the solubilization of membrane proteins is not compatible with the principles of target identification by thermal proteome profiling methodologies. Our results lead to exploring thermally independent strategies for membrane protein solubilization to assure confident membrane protein target identification. The proteome-wide thermal shift methods have already shown their capability to elucidate mechanisms of action from pharma, biomedicine, analytical chemistry, or toxicology, and finding strategies, free from surfactants, to identify membrane protein targets would be the next challenge.
膜蛋白是理解细胞功能的重要靶标。非偏性地鉴定膜蛋白靶标仍然是系统理解细胞对刺激或干扰的反应的瓶颈。已经有人建议通过在溶解溶液中引入非离子表面活性剂来丰富可溶性蛋白质组中的膜蛋白。该策略旨在通过热蛋白质组学分析原理同时鉴定球状和膜蛋白靶标。然而，热移位测定通常会超过非离子表面活性剂经常用于膜蛋白溶解的浊点温度。预计在浊点温度附近，表面活性剂胶束会发生结构修饰，从而改变蛋白质的溶解度。在这里，我们表明非离子表面活性剂的存在可以改变混合的、球状的和膜蛋白组的蛋白质热稳定性。在表面活性剂胶束存在的情况下，热移位测定后分析的蛋白质溶解度变化受胶束大小的热依赖性修饰及其与蛋白质的相互作用的影响。我们证明，为了溶解膜蛋白而引入非离子表面活性剂与热蛋白质组学分析方法的靶标鉴定原理不兼容。我们的结果导致探索热独立的膜蛋白溶解策略，以确保膜蛋白靶标的可靠鉴定。全蛋白质组的热移位方法已经显示出它们从制药、生物医学、分析化学或毒理学中阐明作用机制的能力，并且寻找无表面活性剂的策略来鉴定膜蛋白靶标将是下一个挑战。
