VerBerkmoes Nathan C, Bundy Jonathan L, Hauser Loren, Asano Keiji G, Razumovskaya Jane, Larimer Frank, Hettich Robert L, Stephenson James L
Organic and Biological Mass Spectrometry Group, Chemical Sciences Division, Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, Tennessee 37831-6365, USA.
J Proteome Res. 2002 May-Jun;1(3):239-52. doi: 10.1021/pr025508a.
Here we present a comprehensive method for proteome analysis that integrates both intact protein measurement ("top-down") and proteolytic fragment characterization ("bottom-up") mass spectrometric approaches, capitalizing on the unique capabilities of each method. This integrated approach was applied in a preliminary proteomic analysis of Shewanella oneidensis, a metal-reducing microbe of potential importance to the field of bioremediation. Cellular lysates were examined directly by the "bottom-up" approach as well as fractionated via anion-exchange liquid chromatography for integrated studies. A portion of each fraction was proteolytically digested, with the resulting peptides characterized by on-line liquid chromatography/tandem mass spectrometry. The remaining portion of each fraction containing the intact proteins was examined by high-resolution Fourier transform mass spectrometry. This "top-down" technique provided direct measurement of the molecular masses for the intact proteins and thereby enabled confirmation of post-translational modifications, signal peptides, and gene start sites of proteins detected in the "bottom-up" experiments. A total of 868 proteins from virtually every functional class, including hypotheticals, were identified from this organism.
在此，我们提出一种蛋白质组分析的综合方法，该方法整合了完整蛋白质测量（“自上而下”）和蛋白水解片段表征（“自下而上”）质谱方法，利用了每种方法的独特功能。这种综合方法应用于对希瓦氏菌的初步蛋白质组分析，希瓦氏菌是一种对生物修复领域具有潜在重要性的金属还原微生物。细胞裂解物通过“自下而上”方法直接检测，并通过阴离子交换液相色谱进行分级分离以进行综合研究。每个级分的一部分进行蛋白水解消化，所得肽通过在线液相色谱/串联质谱进行表征。每个级分中包含完整蛋白质的其余部分通过高分辨率傅里叶变换质谱进行检测。这种“自上而下”技术提供了完整蛋白质分子量的直接测量，从而能够确认“自下而上”实验中检测到的蛋白质的翻译后修饰、信号肽和基因起始位点。从这种生物体中总共鉴定出了868种几乎来自每个功能类别的蛋白质，包括假设蛋白。
