Multiplexed, Proteome-Wide Protein Expression Profiling: Yeast Deubiquitylating Enzyme Knockout Strains.
Multiplexed, Proteome-Wide Protein Expression Profiling: Yeast Deubiquitylating Enzyme Knockout Strains.
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DOI：
10.1021/acs.jproteome.5b00802
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发表时间：
2015-12-04
影响因子：
4.4
通讯作者：
Gygi SP
中科院分区：
文献类型：
--
作者：
Isasa M;Rose CM;Elsasser S;Navarrete-Perea J;Paulo JA;Finley DJ;Gygi SP
描述蛋白质的功能通常需要描述缺乏蛋白质的细胞状态。基于质谱的蛋白质组学多路复用技术现在已经实现了同时在酵母10种细胞状态下全球测量蛋白质表达水平的能力。我们应用这种方法来量化野生型和9种去泛素化酶（DUB）敲除菌株的表达差异，目的是创建“信息网络”，这可能为蛋白质的生物学作用提供更深入的机制见解。总共有超过3700种蛋白质在三个生物重复（总共30个样本）中进行了高重复性的定量分析。DUB突变体表现出不同的蛋白质组学特征，与每个家庭成员的不同角色一致。这些差异包括总泛素水平和特定链连接的差异。此外，特定的表达变化表明一些DUB家族成员具有新的功能。例如，ubp3Δ突变体显示细胞色素C氧化酶复合物成员的大量表达变化，与Ubp3在线粒体调节中的作用一致。一些dub在磷酸盐转运体和无机磷酸盐信号通路的其他组分中也表现出广泛的表达变化，这表明这些dub在调节磷酸盐代谢中起作用。这些数据突出了多重蛋白质组分析在生物学研究中的潜力，并为进一步研究DUB家族提供了一个框架。我们的方法很容易适用于酵母缺失突变体的全部收集，并可能有助于酵母和其他生物的系统分析。
Characterizing a protein’s function often requires a description of the cellular state in its absence. Multiplexing in mass spectrometry-based proteomics has now achieved the ability to globally measure protein expression levels in yeast from 10 cell states simultaneously. We applied this approach to quantify expression differences in wild type and nine deubiquitylating enzyme (DUB) knockout strains with the goal of creating “information networks” that might provide deeper, mechanistic insights into a protein’s biological role. In total, more than 3700 proteins were quantified with high reproducibility across three biological replicates (30 samples in all). DUB mutants demonstrated different proteomics profiles, consistent with distinct roles for each family member. These included differences in total ubiquitin levels and specific chain linkages. Moreover, specific expression changes suggested novel functions for several DUB family members. For instance, the ubp3Δ mutant showed large expression changes for members of the cytochrome C oxidase complex, consistent with a role for Ubp3 in mitochondrial regulation. Several DUBs also showed broad expression changes for phosphate transporters as well as other components of the inorganic phosphate signaling pathway, suggesting a role for these DUBs in regulating phosphate metabolism. These data highlight the potential of multiplexed proteome-wide analyses for biological investigation and provide a framework for further study of the DUB family. Our methods are readily applicable to the entire collection of yeast deletion mutants and may help facilitate systematic analysis of yeast and other organisms.