Linking protein fractionation with multidimensional monolithic reversed-phase peptide chromatography/mass spectrometry enhances protein identification from complex mixtures even in the presence of abundant proteins
Linking protein fractionation with multidimensional monolithic reversed-phase peptide chromatography/mass spectrometry enhances protein identification from complex mixtures even in the presence of abundant proteins
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DOI：
10.1002/rcm.1376
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发表时间：
2004-01-01
影响因子：
2
通讯作者：
Weckwerth, W
中科院分区：
文献类型：
--
作者：
Wienkoop, S;Glinski, M;Weckwerth, W
最近，多维鸟枪蛋白质组学已被证明是一种替代技术，能够从单个样本中识别数百种蛋白质。该技术的两个主要限制是存在高丰度蛋白质（例如植物叶组织中的 RUBISCO）和大量共洗脱肽，这些肽超出了传统颗粒填充柱的装载和分离能力以及由于离子抑制而导致的电喷雾电离能力。在这里，拟南芥叶蛋白提取物的快速高效液相色谱 (FPLC) 预分级与随后使用整体硅胶 C18 毛细管柱提高分辨率的二维液相色谱/质谱联用，可以在单个 4 mg 总蛋白植物叶组织样品中鉴定出 1032 种独特的蛋白质。将肽 ID 重新分配给不同的 FPLC 蛋白质片段增强了鉴定程序，特别是在存在蛋白质亚型的情况下。所提出的策略可用于检测传统多维色谱/质谱方法中未见的蛋白质。版权所有 (C) 2004 John Wiley Sons, Ltd.
Recently, multidimensional shotgun proteomics has proven to be an alternative technology able to identify hundreds of proteins from single samples. Two major limitations of the technology are the presence of high abundance proteins (e.g. RUBISCO in plant leaf tissue) and the enormous number of co-eluting peptides that overstrain the loading and resolving capacity of conventional particle-packed columns as well as the capacity of electrospray ionisation due to ion suppression. Here, the coupling of fast performance liquid chromatography (FPLC) pre-fractionation of an Arabidopsis leaf protein extract and subsequent two-dimensional liquid chromatography/mass spectrometry with improved resolution using a monolithic silica C18 capillary column allowed the identification of 1032 unique proteins in a single 4 mg total protein plant leaf tissue sample. The reassignment of peptide IDs to distinct FPLC protein fractions enhances the identification procedure, especially in the case of present protein isoforms. The proposed strategy is useful to detect proteins otherwise not seen in conventional multidimensional chromatography/mass spectrometry approaches. Copyright (C) 2004 John Wiley Sons, Ltd.