One-Dimensional Capillary Liquid Chromatographic Separation Coupled with Tandem Mass Spectrometry Unveils the Escherichia coli Proteome on a Microarray Scale
One-Dimensional Capillary Liquid Chromatographic Separation Coupled with Tandem Mass Spectrometry Unveils the Escherichia coli Proteome on a Microarray Scale
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DOI：
10.1021/ac100343q
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发表时间：
2010-04-01
影响因子：
7.4
通讯作者：
Ishihama, Yasushi
中科院分区：
文献类型：
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作者：
Iwasaki, Mio;Miwa, Shohei;Ishihama, Yasushi
我们成功地鉴定了大肠杆菌（Escherichia coli，E.大肠杆菌）细胞的微阵列规模使用一维毛细管液相色谱-串联质谱（LC-MS/MS）与350厘米长，100 μ m i. d.将所述第一和第二组数据进行比较，硅胶-C-18毛细管整体柱。E.将大肠杆菌胰蛋白酶消化物（4 μ g）注射到柱上，并在小于20 MPa下以500 nL/min的流速施加41小时梯度。共从2602个蛋白质中鉴定出22196个非冗余胰蛋白酶肽，其中包括830个膜蛋白。大肠杆菌细胞（一式三份分析），其中通过转录组分析检测到相等数量的基因。与用15 cm长、3 μ m直径的C-18二氧化硅颗粒填充柱进行常规毛细管LC-MS/MS分析相比，在该系统中获得了平均约5倍的峰响应。较高的响应表明，电离抑制的影响大大减少了高效分离的长整体硅胶柱耦合浅梯度。由于这种高分辨率系统在LC-MS/MS之前不需要任何额外的分离，因此这种“一次性”蛋白质组学方法可以简化鸟枪法蛋白质组学的工作流程，并最大限度地减少样品量，以及减少总分析时间，尽管使用了延长的浅梯度洗脱。
We successfully identified the proteome expressed in Escherichia coli (E. coli) cells on a microarray scale using one-dimensional capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a 350 cm long, 100 mu m i. d., monolithic silica-C-18 capillary column. E. coli tryptic digest (4 mu g) was injected onto the column, and a 41 h gradient was applied with a flow rate of 500 nL/min at less than 20 MPa. In total, 22 196 nonredundant tryptic peptides from 2602 proteins, including 830 membrane proteins, were identified from the E. coli cells (triplicate analysis), in which an equivalent number of genes was detected by transcriptome analysis. Approximately a 5-fold larger peak response on average was obtained in this system, compared with that obtained by conventional capillary LC-MS/MS analysis with a 15 cm long, 3 mu m diameter C-18 silica particle-packed column. The higher response suggests that the influence of ionization suppression was drastically reduced by the high-efficiency separation on the long monolithic silica column coupled with the shallow gradient. Because this high-resolution system does not require any additional separation prior to LC-MS/MS, this "one-shot" proteomics approach can simplify the workflow of shotgun proteomics and minimize the sample amount, as well as reduce the total analysis time, despite the use of prolonged shallow gradient elution.