Millea Kevin M, Krull Ira S, Cohen Steven A, Gebler John C, Berger Scott J
Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115, USA.
J Proteome Res. 2006 Jan;5(1):135-46. doi: 10.1021/pr050278w.
In this paper, we present a combined top-down/bottom-up proteomic analysis workflow for the characterization of proteomic samples. This workflow combines protein fractionation (multidimensional chromatographic separation) with parallel online ESI-TOF-MS intact protein analysis, and fraction collection. Collected fractions were digested and protein identifications were produced using MALDI Q-TOF-MS analysis. These identifications were then linked with corresponding ESI-TOF-MS intact protein mass data to permit full protein characterization. This methodology was applied to an E. coli cytosolic protein fraction, and enabled the identification and characterization of proteins exhibiting co-translational processing, post-translational modification, and proteolytic processing events. The approach also provided the ability to distinguish between closely related protein isoforms. The summary of results from this study indicated that roughly one-third of all detected components generated corresponding data from both top-down and bottom-up analyses, and that significant and novel information can be derived from this application of the hybrid analytical methodology.
在本文中，我们展示了一种用于蛋白质组学样本表征的自上而下/自下而上相结合的蛋白质组学分析工作流程。该工作流程将蛋白质分级分离（多维色谱分离）与在线平行电喷雾飞行时间质谱（ESI-TOF-MS）完整蛋白质分析及馏分收集相结合。收集的馏分经酶解后，使用基质辅助激光解吸电离飞行时间质谱（MALDI Q-TOF-MS）分析进行蛋白质鉴定。然后将这些鉴定结果与相应的ESI-TOF-MS完整蛋白质质量数据相关联，以实现对完整蛋白质的表征。该方法应用于大肠杆菌胞质蛋白馏分，能够鉴定和表征表现出共翻译加工、翻译后修饰和蛋白水解加工事件的蛋白质。该方法还能够区分密切相关的蛋白质异构体。本研究结果总结表明，所有检测到的组分中约有三分之一同时产生了自上而下和自下而上分析的相应数据，并且这种混合分析方法的应用能够获得重要的新信息。
