An automated multidimensional protein identification technology for shotgun proteomics
An automated multidimensional protein identification technology for shotgun proteomics
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DOI：
10.1021/ac010617e
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发表时间：
2001-12-01
影响因子：
7.4
通讯作者：
Yates, JR
中科院分区：
文献类型：
--
作者：
Wolters, DA;Washburn, MP;Yates, JR
我们描述了一种多维蛋白质识别技术(MudPIT)的自动化鸟枪式蛋白质组学方法，该技术结合了多维液相色谱和电喷雾电离串联质谱仪。该多维液相色谱方法将强阳离子交换树脂(SCX)和反相树脂集成在一个两相色谱柱中。我们详细介绍了对Link等人描述的系统的改进。(LINK，A.J.；Eng，J.；Schieltz，D.M.；Carmack，E.；Mize，G.J.；Morris，D.R.；Garvik，B.M.；Yates，J.R.，III.NAT。生物技术。1999，17,676-682)，分离并获取数千个肽的串联质谱图。通过增加等电点，多肽从SCX相洗脱下来，从SCX材料上洗脱出来的东西在分析过程中均匀分布。此外，我们还描述了MudPIT的色谱基准。在两次分析之间，泥坑的重复性在0.5%以内。此外，还证明了复杂多肽混合物中最丰富的和最不丰富的蛋白质/肽之间的动态范围为10000：1。通过改进样品制备和分离，该方法通过识别所有功能和物理类别的蛋白质，改进了蛋白质组的整体分析。
We describe an automated method for shotgun proteomics named multidimensional protein identification technology (MudPIT, which combines multidimensional liquid chromatography with electrospray ionization tandem mass spectrometry. The multidimensional liquid chromatography method integrates a strong cation-exchange (SCX) resin and reversed-phase resin in a biphasic column. We detail the improvements over a system described by Link et al. (Link, A. J.; Eng, J.; Schieltz, D. M.; Carmack, E.; Mize, G. J.; Morris, D. R.; Garvik, B. M.; Yates, J. R., III. Nat. Biotechnol. 1999, 17, 676-682) that separates and acquires tandem mass spectra for thousands of peptides. Peptides elute off the SCX phase by increasing pI, and elution off the SCX material is evenly distributed across an analysis. In addition, we describe the chromatographic benchmarks of MudPIT. MudPIT was reproducible within 0.5% between two analyses. Furthermore, a dynamic range of 10 000 to 1 between the most abundant and least abundant proteins/peptides in a complex peptide mixture has been demonstrated. By improving sample preparation along with separations, the method improves the overall analysis of proteomes by identifying proteins of all functional and physical classes.