Assessment of 2nSILAC for proteome quantification

Knowledge about the functions of individual proteins on a systems-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 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).系统层面解析单个蛋白质的功能，对于全面阐明细胞进程背后的分子机制至关重要。目前，各类生物的蛋白质组中仍有相当比例的组分未得到充分表征。质谱技术是开展全局蛋白质组研究的高效手段。精准的全蛋白质组定量分析中，最具代表性的方法之一是细胞培养氨基酸稳定同位素标记（stable isotope labeling by amino acids in cell culture, SILAC）。然而，对于酿酒酵母（Saccharomyces cerevisiae, 又称面包酵母）这类原养型生物而言，由于其可自主合成全部氨基酸，SILAC技术的应用受到极大限制。本文报道一种命名为2nSILAC的改进策略，该方法可在发酵型与呼吸型生长条件下，利用重同位素标记的精氨酸与赖氨酸对原养型面包酵母开展体内标记，使其成为可用于蛋白质功能全局研究的可靠工具。该通用型2nSILAC策略可直接使用科研领域已公开的酵母突变株库并开展系统性筛选。我们通过定量质谱技术分析线粒体组分中线粒体基因缺失的表型效应，验证了该方法的优异性能，并揭示了Coi1在细胞色素c氧化酶（respiratory chain complex IV, 呼吸链复合物IV）组装过程中的关键作用。