Comprehensive two-dimensional high-performance liquid chromatography for the isolation of overexpressed proteins and proteome mapping
Comprehensive two-dimensional high-performance liquid chromatography for the isolation of overexpressed proteins and proteome mapping
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DOI：
10.1006/abio.1998.2588
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发表时间：
1998-05-01
影响因子：
2.9
通讯作者：
Moseley, MA
中科院分区：
文献类型：
--
作者：
Opiteck, GJ;Ramirez, SM;Moseley, MA
这里描述了一种二维液相色谱系统，该系统使用尺寸排阻液相色谱 (SEC)，然后使用反相液相色谱 (RPLC) 来分离大肠杆菌细胞裂解产生的蛋白质混合物，并分离它们产生的蛋白质。尺寸排阻色谱可以在变性或非变性条件下进行。从第一维洗脱的峰自动进行反相色谱分析，根据不同的疏水性分离大小相似的蛋白质。 RPLC 还可以对分析物进行脱盐，以便无论使用何种 SEC 流动相，都可以在 215 nm 的深紫外区域检测到它们。以这种方式产生的二维 (2D) 色谱图与染色的 2D 凝胶的格式非常相似，因为斑点显示在 X-Y 轴上，强度代表分析物的数量。色谱分离后，分析物通过馏分收集器沉积到六个 96 孔（总共 576 个）聚丙烯微量滴定板中。根据样品浓度，通过基质辅助激光解吸电离飞行时间质谱 (MALDI-TOF/MS) 或电喷雾质谱 (ESI/MS) 对感兴趣的组分进行分析，这两种方法均可产生完整蛋白质的准确（2 至 0.02%）分子量信息，无需任何额外的样品制备、电印迹、脱色等。剩余 97% 的组分可用于其他分析，例如 Edman 测序、氨基酸分析，或通过串联质谱进行蛋白水解消化和测序。该 2D HPLC 蛋白质纯化和鉴定系统用于分离插入大肠杆菌中的非受体酪氨酸激酶 pp60(c-src) 和 β-内酰胺酶的 src 同源 (SH2) 结构域，以及构成大肠杆菌蛋白质组一小部分的许多天然蛋白质。 (C) 1998 年学术出版社。
A two-dimensional liquid chromatographic system is described here which uses size-exclusion liquid chromatography (SEC) followed by reversed-phase liquid chromatography (RPLC) to separate the mixture of proteins resulting from the lysis of Escherichia coli cells and to isolate the proteins that they produce. The size-exclusion chromatography can be conducted under either denaturing or nondenaturing conditions. Peaks eluting from the first dimension are automatically subjected to reversed-phase chromatography to separate similarly sized proteins on the basis of their various hydrophobicities. The RPLC also serves to desalt the analytes so that they can be detected in the deep ultraviolet region at 215 nm regardless of the SEC mobile phase used. The two-dimensional (2D) chromatograms produced in this manner then strongly resemble the format of stained 2D gels, in that spots are displayed on a X-Y axis and intensity represents quantity of analyte. Following chromatographic separation, the analytes are deposited into six 96-well (576 total) polypropylene microtiter plates via a fraction collector. Interesting fractions are analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) or electrospray mass spectrometry (ESI/MS) depending on sample concentration, which both yield accurate (2 to 0.02%) molecular weight information on intact proteins without any additional sample preparation, electroblotting, destaining, etc. The remaining 97% of a fraction can then be used for other analyses, such Edman sequencing, amino acid analysis, or proteolytic digestion and sequencing by tandem mass spectrometry. This 2D HPLC protein purification and identification system was used to isolate the src homology (SH2) domain of the nonreceptor tyrosine kinase pp60(c-src) and beta-lactamase, both inserted into E. coli, as well as a number of native proteins comprising a small portion of the E. coli proteome. (C) 1998 Academic Press.