Fully automated online multi-dimensional protein profiling system for complex mixtures
Fully automated online multi-dimensional protein profiling system for complex mixtures
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DOI：
10.1016/j.chroma.2004.09.078
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发表时间：
2004-11-19
影响因子：
4.1
通讯作者：
Nishimura, T
中科院分区：
文献类型：
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作者：
Fujii, K;Nakano, T;Nishimura, T
为了对高度复杂的蛋白质混合物进行高通量蛋白质组分析，我们构建了一个全自动的多维蛋白质谱在线分析系统，该系统利用二维液相色谱和串联质谱（2D-LC-MS-MS）的组合，基于我们先前描述的成熟的离线系统[K.藤井中野、T. Kawamura，F. Alzhei，Y.班多河Wang，T. Nishimura，J. Proteome Res.3（2004）712]。双阀切换系统。在可编程自动进样器上进行在线二维色谱分析，具有强阳离子交换（SCX）和反相（RP）分离功能。在RP色谱平衡期间进行SCX分离，基于6次SCX分级，在4小时内在全自动条件下进行整个分析序列，二维RP色谱运行时间为40分钟。为了评价其在蛋白质检测和鉴定中的性能，对6种标准蛋白和酵母20 S蛋白酶体的质谱进行了分析，并与一维反相色谱系统（1D-LC-MS-MS）的结果进行了比较。2D-LC-MS-MS系统表明，检测到的肽片段的数量和蛋白质覆盖率均增加了一倍以上。此外，该多维蛋白质谱分析系统还应用于人类26 S蛋白酶体，这是一种高度复杂的蛋白质混合物。因此，723个肽片段被鉴定为31个蛋白酶体组分，以及样品中的其他共存蛋白质。鉴定可以以平均63%的序列覆盖率全面地进行，并且另外，在N-末端具有修饰。这些结果表明，在这里描述的在线2D-LC-MS-MS系统能够以高通量的方式分析高度复杂的蛋白质混合物，并且它将适用于动态蛋白质组学。(C)2004 Elsevier B. V.保留所有权利。
For high throughput proteome analysis of highly complex protein mixtures, we have constructed a fully automated online system for multi-dimensional protein profiling, which utilizes a combination of two-dimensional liquid chromatography and tandem mass spectrometry (2D-LC-MS-MS), based on our well-established offline system described previously [K. Fujii. Nakano, T. Kawamura, F. Usui, Y. Bando, R. Wang, T. Nishimura, J. Proteome Res. 3 (2004) 712]. A two-valve switching system. on a programmable auto sample injector is utilized for online two-dimensional chromatography with strong cation-exchange (SCX) and reversed-phase (RP) separations. The SCX separation is carried out during the equilibration of RP chromatography and the entire sequence of analysis was performed under fully automated conditions within 4 h, based on six SCX fractionations, and 40 min running time for the two-dimensional RP chromatography. In order to evaluate its performance in the detection and identification of proteins, digests of six standard proteins and yeast 20S proteasome have been analyzed and their results were compared to those obtained by the one-dimensional reversed-phase chromatography system (1D-LC-MS-MS). The 2D-LC-MS-MS system demonstrated that both the number of peptide fragments detected and the protein coverage had more than doubled. Furthermore, this multi-dimensional protein profiling system was also applied to the human 26S proteasome, which is one of the highly complex protein mixtures. Consequently, 723 peptide fragments were identified as 31 proteasome components, together with other coexisting proteins in the sample. The identification could be comprehensively performed with a 63% sequence coverage on an average, and additionally, with modifications at the N-terminus. These results indicated that the online 2D-LC-MS-MS system being described here is capable of analyzing highly complex protein mixtures in a high throughput manner, and that it would be applicable to dynamic proteomics. (C) 2004 Elsevier B.V. All rights reserved.