一种用于鸟枪法蛋白质组学的自动化多维蛋白质鉴定技术。

An automated multidimensional protein identification technology for shotgun proteomics.

作者信息

Wolters D A, Washburn M P, Yates J R

机构信息

Torrey Mesa Research Institute, San Diego, California 92121, USA.

出版信息

Anal Chem. 2001 Dec 1;73(23):5683-90. doi: 10.1021/ac010617e.

DOI:10.1021/ac010617e

PMID:11774908

Abstract

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 10000 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.

摘要

我们描述了一种用于鸟枪法蛋白质组学的自动化方法，即多维蛋白质鉴定技术（MudPIT），它将多维液相色谱与电喷雾电离串联质谱相结合。多维液相色谱方法在双相柱中整合了强阳离子交换（SCX）树脂和反相树脂。我们详细介绍了相对于Link等人（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）所描述系统的改进，该系统可分离并获取数千种肽的串联质谱。肽通过增加pI从SCX相洗脱，并且从SCX材料上的洗脱在一次分析中均匀分布。此外，我们描述了MudPIT的色谱基准。两次分析之间，MudPIT的重现性在0.5%以内。此外，在复杂肽混合物中，最丰富和最不丰富的蛋白质/肽之间的动态范围已被证明为10000比1。通过改进样品制备以及分离过程，该方法通过鉴定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。