Reproducible and Consistent Quantification of the Saccharomyces cerevisiae Proteome by SWATH-mass spectrometry
Reproducible and Consistent Quantification of the Saccharomyces cerevisiae Proteome by SWATH-mass spectrometry
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DOI：
10.1074/mcp.m113.035550
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发表时间：
2015-03-01
影响因子：
7
通讯作者：
Aebersold, Ruedi
中科院分区：
文献类型：
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作者：
Selevsek, Nathalie;Chang, Ching-Yun;Aebersold, Ruedi
通过选择反应监测的靶向质谱法（S/MRM）已被证明是一种适用于在多个生物样品和宽动态范围内对蛋白质进行一致和可重现定量的技术。这种性能是系统生物学和生物医学研究的重要先决条件。然而，该方法仅限于每次LC-MS分析测量几百个肽。最近，我们推出了SWATH-MS，这是一种数据独立采集和目标数据分析的组合，极大地扩展了每个样品定量的肽/蛋白质数量，同时保持了S/MRM的良好性能。在这里，我们应用SWATH-MS技术来量化的变化随着时间的推移，在酿酒酵母中表达的蛋白质组的大部分在响应渗透stress.We采样细胞培养物中的生物三重在6个时间点后，应用渗透stress和收购单次注射数据独立的采集数据集上的高分辨率5600 tripleTOF仪器在SWATH模式下操作。通过对来自SWATH-MS数据集的转换信号组进行靶向提取和整合，对特定酵母蛋白的蛋白质型肽进行定量。我们在18个样品中一致地鉴定和定量了超过15，000种肽和2500种蛋白质。我们证明了在所有时间点和蛋白质丰度的技术和生物重复之间的高重现性。此外，我们发现，丰富的数以百计的蛋白质显着调节后，渗透压休克，和途径富集分析表明，蛋白质反应渗透压休克主要是参与碳水化合物和氨基酸代谢。总的来说，这项研究证明了SWATH-MS能够有效地生成跨多个样品的大部分蛋白质组的可重复的、一致的和定量准确的测量结果。
Targeted mass spectrometry by selected reaction monitoring (S/MRM) has proven to be a suitable technique for the consistent and reproducible quantification of proteins across multiple biological samples and a wide dynamic range. This performance profile is an important prerequisite for systems biology and biomedical research. However, the method is limited to the measurements of a few hundred peptides per LC-MS analysis. Recently, we introduced SWATH-MS, a combination of data independent acquisition and targeted data analysis that vastly extends the number of peptides/proteins quantified per sample, while maintaining the favorable performance profile of S/MRM. Here we applied the SWATH-MS technique to quantify changes over time in a large fraction of the proteome expressed in Saccharomyces cerevisiae in response to osmotic stress.We sampled cell cultures in biological triplicates at six time points following the application of osmotic stress and acquired single injection data independent acquisition data sets on a high-resolution 5600 tripleTOF instrument operated in SWATH mode. Proteins were quantified by the targeted extraction and integration of transition signal groups from the SWATH-MS datasets for peptides that are proteotypic for specific yeast proteins. We consistently identified and quantified more than 15,000 peptides and 2500 proteins across the 18 samples. We demonstrate high reproducibility between technical and biological replicates across all time points and protein abundances. In addition, we show that the abundance of hundreds of proteins was significantly regulated upon osmotic shock, and pathway enrichment analysis revealed that the proteins reacting to osmotic shock are mainly involved in the carbohydrate and amino acid metabolism. Overall, this study demonstrates the ability of SWATH-MS to efficiently generate reproducible, consistent, and quantitatively accurate measurements of a large fraction of a proteome across multiple samples.