A Multiplexed Quantitative Strategy for Membrane Proteomics OPPORTUNITIES FOR MINING THERAPEUTIC TARGETS FOR AUTOSOMAL DOMINANT POLYCYSTIC KIDNEY DISEASE
A Multiplexed Quantitative Strategy for Membrane Proteomics OPPORTUNITIES FOR MINING THERAPEUTIC TARGETS FOR AUTOSOMAL DOMINANT POLYCYSTIC KIDNEY DISEASE
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DOI：
10.1074/mcp.m800068-mcp200
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发表时间：
2008-10-01
影响因子：
7
通讯作者：
Chen, Yu-Ju
中科院分区：
文献类型：
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作者：
Han, Chia-Li;Chien, Chih-Wei;Chen, Yu-Ju
为了实现膜蛋白质组的多重、全面和稳健的定量，我们报告了一种结合凝胶辅助消化、iTRAQ（用于相对和绝对定量的同量异位素标签）标记和LC-MS/MS的策略。D.），证明了该定量平台的高度一致性和再现性。在严格的鉴定标准（错误发现率=0%）下，该策略有效地定量了膜蛋白;多达520种蛋白质（91%）是膜蛋白，每种蛋白质的定量基于平均每个完整膜蛋白14.1个肽。除了对大多数定量蛋白质的信号强度的显著改善之外，最显著的是，拓扑分析显示，在从具有多达19个跨膜螺旋的完整膜蛋白检测跨膜肽方面实现了最大的改善。据我们所知，这种覆盖水平超过了以前使用MS所达到的覆盖水平，并且与其他方法相比，提供了上级定量准确度。我们将这种方法应用于常染色体显性多囊肾病（ADPKD）小鼠模型中表型表达的第一个蛋白质组学描绘。通过表征来自野生型与PKD 1敲除小鼠的肾细胞质膜，对791种蛋白质进行定量，并且67种和37种蛋白质分别显示> 2倍的上调和下调。这些差异表达的膜蛋白中的一些涉及ADPKD中主要异常的潜在机制，包括上皮细胞增殖和凋亡、细胞-细胞和细胞-基质相互作用、离子和液体分泌以及膜蛋白极性。在这些蛋白质中，靶向治疗某些转运蛋白/受体，如表皮生长因子受体，已被证明是有效的ADPKD的临床前研究;其他已知的药物靶点在各种疾病。我们的方法证明了比较膜蛋白质组学如何能够深入了解ADPKD的分子机制，并确定潜在的药物靶点，这可能会导致新的治疗机会，以预防或延缓疾病。Molecular & Cellular Proteomics 7：1983-1997，2008.
Toward multiplexed, comprehensive, and robust quantitation of the membrane proteome, we report a strategy combining gel-assisted digestion, iTRAQ ( isobaric tags for relative and absolute quantitation) labeling, and LC-MS/MS. Quantitation of four independently purified membrane fractions from HeLa cells gave high accuracy (< 8% error) and precision (< 12% relative S. D.), demonstrating a high degree of consistency and reproducibility of this quantitation platform. Under stringent identification criteria (false discovery rate =0%), the strategy efficiently quantified membrane proteins; as many as 520 proteins (91%) were membrane proteins, each quantified based on an average of 14.1 peptides per integral membrane protein. In addition to significant improvements in signal intensity for most quantified proteins, most remarkably, topological analysis revealed that the biggest improvement was achieved in detection of transmembrane peptides from integral membrane proteins with up to 19 transmembrane helices. To the best of our knowledge, this level of coverage exceeds that achieved previously using MS and provides superior quantitation accuracy compared with other methods. We applied this approach to the first proteomics delineation of phenotypic expression in a mouse model of autosomal dominant polycystic kidney disease (ADPKD). By characterizing kidney cell plasma membrane from wild-type versus PKD1 knock-out mice, 791 proteins were quantified, and 67 and 37 proteins showed > 2-fold up-regulation and down-regulation, respectively. Some of these differentially expressed membrane proteins are involved in the mechanisms underlying major abnormalities in ADPKD, including epithelial cell proliferation and apoptosis, cell-cell and cell-matrix interactions, ion and fluid secretion, and membrane protein polarity. Among these proteins, targeting therapeutics to certain transporters/receptors, such as epidermal growth factor receptor, has proven effective in preclinical studies of ADPKD; others are known drug targets in various diseases. Our method demonstrates how comparative membrane proteomics can provide insight into the molecular mechanisms underlying ADPKD and the identification of potential drug targets, which may lead to new therapeutic opportunities to prevent or retard the disease. Molecular & Cellular Proteomics 7: 1983-1997, 2008.