Anal Chem. 2018 Sep 4;90(17):10501-10509. doi: 10.1021/acs.analchem.8b02557. Epub 2018 Aug 23.
Knowledge about the functions of individual proteins on a system-wide level is crucial to fully understand molecular mechanisms underlying cellular processes. A considerable part of the proteome across all organisms is still poorly characterized. Mass spectrometry is an efficient technology for the global study of proteins. One of the most prominent methods for accurate proteome-wide comparative quantification is stable isotope labeling by amino acids in cell culture (SILAC). However, application of SILAC to prototrophic organisms such as Saccharomyces cerevisiae, also known as baker's yeast, is compromised since they are able to synthesize all amino acids on their own. Here, we describe an advanced strategy, termed 2nSILAC, that allows for in vivo labeling of prototrophic baker's yeast using heavy arginine and lysine under fermentable and respiratory growth conditions, making it a suitable tool for the global study of protein functions. This generic 2nSILAC strategy allows for directly using and systematically screening yeast mutant strain collections available to the scientific community. We exemplarily demonstrate its high potential by analyzing the effects of mitochondrial gene deletions in mitochondrial fractions using quantitative mass spectrometry revealing the role of Coi1 for the assembly of cytochrome c oxidase (respiratory chain complex IV).
关于系统范围内单个蛋白质功能的知识对于充分理解细胞过程背后的分子机制至关重要。所有生物体的蛋白质组中仍有相当一部分功能特征描述不足。质谱是一种用于全局研究蛋白质的有效技术。准确进行蛋白质组范围内比较定量的最突出方法之一是稳定同位素标记的氨基酸在细胞培养中的应用（SILAC）。然而，由于酵母（也称为面包酵母）能够自行合成所有氨基酸，因此 SILAC 应用于原养型生物会受到限制。在这里，我们描述了一种先进的策略，称为 2nSILAC，该策略允许在可发酵和呼吸生长条件下使用重精氨酸和赖氨酸对原养型面包酵母进行体内标记，使其成为研究蛋白质功能的全局工具。这种通用的 2nSILAC 策略允许直接使用和系统筛选科学界提供的酵母突变株文库。我们通过使用定量质谱分析线粒体基因缺失对线粒体部分的影响来说明其高潜力，该分析揭示了 Coi1 对于细胞色素 c 氧化酶（呼吸链复合物 IV）组装的作用。
