Comparison of multidimensional shotgun technologies targeting tissue proteomics.
Comparison of multidimensional shotgun technologies targeting tissue proteomics.
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DOI：
10.1002/elps.200900367
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发表时间：
2009-12
期刊：
影响因子：
2.9
通讯作者：
Lee, Cheng S.
中科院分区：
文献类型：
--
作者：
Fang, Xueping;Balgley, Brian M.;Wang, Weijie;Park, Deric M.;Lee, Cheng S.
迫切需要开发促进和加速发现具有治疗和诊断潜力的新型蛋白质生物标志物的技术。比较鸟枪蛋白质组技术，包括毛细管等速电泳（CITP）为基础的多维分离和多维液相色谱系统，因此，在这项研究中，他们的能力，以解决蛋白质的复杂性和相对丰富的多形性胶质母细胞瘤衍生的癌症干细胞固有的挑战。使用单一处理的蛋白质消化物进行比较，其中具有相等的样品加载、相同的第二维分离（反相液相色谱）和质谱条件以及一致的搜索参数和由目标诱饵确定的错误发现率建立的截止值。除了在总肽、不同肽和不同蛋白质鉴定中实现上级总体蛋白质组性能外，CITP蛋白质组平台与光谱计数方法结合的分析再现性通过Pearson R2值0.98和所有定量蛋白质的变异系数15%来确定。相比之下，在强阳离子交换中广泛的级分重叠极大地限制了多维液相色谱分离更深入地挖掘组织蛋白质组的能力，如在各种蛋白质功能类别和关键蛋白质途径中的差覆盖所证明的。CITP蛋白质组技术具有选择性分析物富集和超高分辨率，预计将作为通过组织样本的鸟枪蛋白质组分析发现生物标志物所需的整体工具集的关键组成部分。
A compelling need exists for the development of technologies that facilitate and accelerate the discovery of novel protein biomarkers with therapeutic and diagnostic potential. Comparisons among shotgun proteome technologies, including capillary isotachophoresis (CITP)-based multidimensional separations and multidimensional liquid chromatography system, are therefore performed in this study regarding their abilities to address the challenges of protein complexity and relative abundance inherent in glioblastoma multiforme derived cancer stem cells. Comparisons are conducted using a single processed protein digest with equal sample loading, identical second dimension separation (reversed phase liquid chromatography) and mass spectrometry conditions, and consistent search parameters and cutoff established by the target-decoy determined false discovery rate. Besides achieving superior overall proteome performance in total peptide, distinct peptide, and distinct protein identifications, analytical reproducibility of the CITP proteome platform coupled with the spectral counting approach is determined by a Pearson R2 value of 0.98 and a coefficient of variation of 15% across all proteins quantified. In contrast, extensive fraction overlapping in strong cation exchange greatly limits the ability of multidimensional liquid chromatography separations for mining deeper into the tissue proteome as evidenced by the poor coverage in various protein functional categories and key protein pathways. The CITP proteomic technology, equipped with selective analyte enrichment and ultrahigh resolving power, is expected to serve as a critical component in the overall toolset required for biomarker discovery via shotgun proteomic analysis of tissue specimens.
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