Robust Analysis of the Yeast Proteome under 50 kDa by Molecular-Mass-Based Fractionation and Top-Down Mass Spectrometry
Robust Analysis of the Yeast Proteome under 50 kDa by Molecular-Mass-Based Fractionation and Top-Down Mass Spectrometry
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DOI：
10.1021/ac202384v
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发表时间：
2012-01-03
影响因子：
7.4
通讯作者：
Kelleher, Neil L.
中科院分区：
文献类型：
--
作者：
Kellie, John F.;Catherman, Adam D.;Kelleher, Neil L.
随着自上而下的质谱分析过程的不断成熟，我们对改进方法的下一部分进行了基准测试，该方法采用了管式凝胶电泳（TGE）装置，通过分子质量分离完整的蛋白质。自上而下的蛋白质组学是以一种稳健的方式完成的，可以鉴定数百种独特的蛋白质，其中许多蛋白质对应于多种蛋白质形式。TGE平台将从酵母蛋白质组中提取的0-50 kDa蛋白质分离成12个级分，然后进行自动纳米毛细管LC-MS/MS，一式三份。该过程可以在不到72小时内完成。从这项研究中，530个独特的蛋白质和1103个不同的蛋白质物种被确定和表征，从而代表了最高的覆盖率，迄今为止的酿酒酵母蛋白质组使用自上而下的蛋白质组学。这项工作标志着基于直接测量和完整蛋白质片段化的蛋白质组学成熟的重要一步。
As the process of top-down mass spectrometry continues to mature, we benchmark the next installment of an improving methodology that incorporates a tube-gel electrophoresis (TGE) device to separate intact proteins by molecular mass. Top-down proteomics is accomplished in a robust fashion to yield the identification of hundreds of unique proteins, many of which correspond to multiple protein forms. The TGE platform separates 0-50 kDa proteins extracted from the yeast proteome into 12 fractions prior to automated nanocapillary LC-MS/MS in technical triplicate. The process may be completed in less than 72 h. From this study, 530 unique proteins and 1103 distinct protein species were identified and characterized, thus representing the highest coverage to date of the Saccharomyces cerevisiae proteome using top-down proteomics. The work signifies a significant step in the maturation of proteomics based on direct measurement and fragmentation of intact proteins.