System-wide perturbation analysis with nearly complete coverage of the yeast proteome by single-shot ultra HPLC runs on a bench top Orbitrap.
System-wide perturbation analysis with nearly complete coverage of the yeast proteome by single-shot ultra HPLC runs on a bench top Orbitrap.
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DOI：
10.1074/mcp.m111.013722
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发表时间：
2012-03
期刊：
影响因子：
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通讯作者：
Mann M
中科院分区：
文献类型：
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作者：
Nagaraj N;Kulak NA;Cox J;Neuhauser N;Mayr K;Hoerning O;Vorm O;Mann M
酵母仍然是系统生物学和评估蛋白质组学策略的重要模型。深入的shot弹枪蛋白质组学研究已经达到了几乎全面的覆盖范围，并为这种生物体开发了快速，有针对性的方法。最近，我们证明了使用长色谱柱和耦合到线性离子陷阱轨道仪器的单个LC-MS/MS分析具有出乎意料的大型动态蛋白质鉴定范围（Thakur，S.S.，Geiger，T.，Chatterjee，B.，Bandilla，Bandilla P.，Frohlich，F。，Cox，J。和Mann，M。（2011）LC-MS/MS高度敏感的蛋白质组覆盖率，没有预分量。 ）。在这里，我们将超高压液相色谱系统与新型的基准Orbitrap质谱仪（Q Extrive）融为一体，以对酵母蛋白质组的几乎完整，快速和稳健的分析进行。单次滤清器样品制备（FASP）准备和LYSC消化的酵母细胞裂解物的平均为3923蛋白。对六次运行的组合分析将这些值提高到4000多个已识别的蛋白质/运行，接近标准条件下表达的蛋白质总数，中位序列覆盖率为23％。由于缺乏分馏步骤，仅需要微量的样品。因此，现在可以在测量时间和高灵敏度的几个小时内大部分覆盖酵母模型蛋白质组。京都百科全书中蛋白质的中位基因和基因组途径至少10个成员的途径为88％，预计未涵盖的途径在所使用的条件下不会活跃。为了研究酵母蛋白质组的扰动，我们开发了一种代表不同蛋白质组状态的外部，重型赖氨酸标记的Silac酵母标准。使用此尖峰标准来测量酵母蛋白质组的热休克响应。热休克反应的生物信息学分析表明，与翻译相关的功能显着下调，包括核仁过程。相反，与应力相关的途径被上调。此处描述的蛋白质组学技术是直接，快速和健壮的，有可能在酵母和其他生物学研究界中广泛使用。
Yeast remains an important model for systems biology and for evaluating proteomics strategies. In-depth shotgun proteomics studies have reached nearly comprehensive coverage, and rapid, targeted approaches have been developed for this organism. Recently, we demonstrated that single LC-MS/MS analysis using long columns and gradients coupled to a linear ion trap Orbitrap instrument had an unexpectedly large dynamic range of protein identification (Thakur, S. S., Geiger, T., Chatterjee, B., Bandilla, P., Frohlich, F., Cox, J., and Mann, M. (2011) Deep and highly sensitive proteome coverage by LC-MS/MS without prefractionation. Mol. Cell Proteomics 10, 10.1074/mcp.M110.003699). Here we couple an ultra high pressure liquid chromatography system to a novel bench top Orbitrap mass spectrometer (Q Exactive) with the goal of nearly complete, rapid, and robust analysis of the yeast proteome. Single runs of filter-aided sample preparation (FASP)-prepared and LysC-digested yeast cell lysates identified an average of 3923 proteins. Combined analysis of six single runs improved these values to more than 4000 identified proteins/run, close to the total number of proteins expressed under standard conditions, with median sequence coverage of 23%. Because of the absence of fractionation steps, only minuscule amounts of sample are required. Thus the yeast model proteome can now largely be covered within a few hours of measurement time and at high sensitivity. Median coverage of proteins in Kyoto Encyclopedia of Genes and Genomes pathways with at least 10 members was 88%, and pathways not covered were not expected to be active under the conditions used. To study perturbations of the yeast proteome, we developed an external, heavy lysine-labeled SILAC yeast standard representing different proteome states. This spike-in standard was employed to measure the heat shock response of the yeast proteome. Bioinformatic analysis of the heat shock response revealed that translation-related functions were down-regulated prominently, including nucleolar processes. Conversely, stress-related pathways were up-regulated. The proteomic technology described here is straightforward, rapid, and robust, potentially enabling widespread use in the yeast and other biological research communities.