Quantitative membrane proteomics applying narrow range peptide isoelectric focusing for studies of small cell lung cancer resistance mechanisms
Quantitative membrane proteomics applying narrow range peptide isoelectric focusing for studies of small cell lung cancer resistance mechanisms
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DOI：
10.1002/pmic.200800174
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发表时间：
2008-08-01
期刊：
影响因子：
3.4
通讯作者：
Jakobsson, Per-Johan
中科院分区：
文献类型：
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作者：
Eriksson, Hanna;Lengqvist, Johan;Jakobsson, Per-Johan
耐药性通常与膜相关药物流出系统的上调相关，因此全局膜蛋白质组学方法是寻找耐药表型新成分的宝贵工具。在此，我们比较了肺癌细胞系 H69 及其同基因阿霉素耐药亚细胞系 H69AR 的微粒体蛋白质组。所使用的方法包括微粒体制备、iTRAQ 标记、随后在固定 pH 梯度中进行窄范围肽 IEF (IPG-IEF) 和 LC-MS/MS 分析。我们证明微粒体制备和 iTRAQ 标记在蛋白质含量和组成方面是可重复的。使用窄范围肽 IPG-IEF 分离的基本原理通过其以下能力得到证明：(i) 将样品的复杂性降低三分之二，同时保持高蛋白质组覆盖率 (96%)，(ii) 提供高分离效率，以及 (iii) 允许进行肽验证并可能识别转录后修饰。分析五分之一的 IEF 组分（有效 pH 范围为 4.0-4.5）后，总共鉴定出 3704 个蛋白质，其中 527 个预测为膜蛋白。 Serca 2 是发现差异表达的蛋白质之一，它是一种位于内质网膜上的钙泵，可能会导致对阿霉素的细胞凋亡反应发生变化。
Drug resistance is often associated with upregulation of membrane-associated drug-efflux systems, and thus global membrane proteomics methods are valuable tools in the search for novel components of drug resistance phenotypes. Herein we have compared the microsomal proteome from the lung cancer cell line H69 and its isogenic Doxorubicin-resistant subcell line H69AR. The method used includes microsome preparation, iTRAQ labeling followed by narrow range peptide IEF in an immobilized pH-gradient (IPG-IEF) and LC-MS/MS analysis. We demonstrate that the microsomal preparation and iTRAQ labeling is reproducible regarding protein content and composition. The rationale using narrow range peptide IPG-IEF separation is demonstrated by its ability to: (i) lowering the complexity of the sample by two-thirds while keeping high proteome coverage (96%), (ii) providing high separation efficiency, and (iii) allowing for peptide validation and possibly identifications of post-transcriptional modifications. After analyzing one-fifth of the IEF fractions (effective pH range of 4.0-4.5), a total of 3704 proteins were identified, among which 527 were predicted to be membrane proteins. One of the proteins found to be differentially expressed was Serca 2, a calcium pump located in the ER membrane that potentially could result in changes of apoptotic response toward Doxorubicin.