Comparison of two-dimensional fractionation techniques for shotgun proteomics.
Comparison of two-dimensional fractionation techniques for shotgun proteomics.
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shot弹枪蛋白质组学的二维分级技术的比较。
DOI：
10.1021/ac8007994
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发表时间：
2008-09-01
影响因子：
7.4
通讯作者：
Li, Lingjun
中科院分区：
文献类型：
--
作者：
Dowell, James A.;Frost, Dustin C.;Zhang, Jiang;Li, Lingjun
二维(2D)分离是一种常用的工具，用于增加自下而上的鸟枪式蛋白质组学的动态范围和蛋白质组覆盖率。然而，很少有报告比较使用低微克样品量的2D方法的相对分离效率。为了系统地评价二维分离技术，我们用七种不同的方法对微克量的大肠杆菌蛋白提取物进行了分级。第一维分离采用反相高效液相色谱(RP-HPLC)、凝胶电泳法(SDS-PAGE)或强阳离子交换(SCX-HPLC)。第二维包括标准反相毛细管高效液相色谱与电喷雾电离四极飞行时间质谱仪(ESI-QTOF MS)联用进行串联质谱分析。通过比较每种方法鉴定的蛋白质总数来评估每种技术的总体性能和相对分级效率。蛋白质水平的反相高效液相色谱分离效果最好，分别鉴定出281种蛋白质和266种蛋白质。在线pH变化SCX和SDS-PAGE分别鉴定出178和139个蛋白质，表现平平。离线的SCX表现最差，鉴定出81个蛋白质。我们还考察了影响分离效率的各种色谱因素，包括分辨率、正交性和样品损失。
Two-dimensional (2D) fractionation is a commonly used tool to increase dynamic range and proteome coverage for bottom-up, shotgun proteomics. However, there are few reports comparing the relative separation efficiencies of 2D methodologies using low microgram sample quantities. In order to systematically evaluate 2D separation techniques, we fractionated microgram quantities of E. coli protein extract by seven different methods. The first dimension of separation was performed with either reverse phase high pressure liquid chromatography (RP-HPLC), gel electrophoresis (SDS-PAGE), or strong cation exchange (SCX-HPLC). The second dimension consisted of a standard reverse phase capillary HPLC coupled to an electrospray ionization quadrupole time-of-flight mass spectrometer (ESI-QTOF MS) for tandem mass spectrometric analysis. The overall performance and relative fractionation efficiencies of each technique were assessed by comparing the total number of proteins identified by each method. The protein-level RP-HPLC and the high pH RP-HPLC peptide-level separations performed the best, identifying 281 and 266 proteins, respectively. The on-line pH variance SCX and the SDS-PAGE returned modest performances with 178 and 139 proteins identified, respectively. The off-line SCX had the worst performance with 81 proteins identified. We also examined various chromatographic factors which contribute to separation efficiency, including resolving power, orthogonality, and sample loss.
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影响因子：
7.4
作者：
Eschelbach, JW;Jorgenson, JW
通讯作者：
Jorgenson, JW
影响因子：
4.4
作者：
Delmotte, Nathanael;Lasaosa, Maria;Huber, Christian G.
通讯作者：
Huber, Christian G.
影响因子：
4.4
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Martosella, J;Zolotarjova, N;Boyes, BE
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Boyes, BE
影响因子：
7.4
作者：
Chen, JZ;Balgley, BM;Lee, CS
通讯作者：
Lee, CS
影响因子：
4.4
作者：
Marshall, J;Jankowski, A;Jackowski, G
通讯作者：
Jackowski, G