Comparative membrane proteomics: a technical advancement in the search of renal cell carcinoma biomarkers
Comparative membrane proteomics: a technical advancement in the search of renal cell carcinoma biomarkers
复制标题
DOI：
10.1039/c5mb00020c
复制
发表时间：
2015-01-01
影响因子：
--
通讯作者：
Pitto, Marina
中科院分区：
文献类型：
--
作者：
Raimondo, Francesca;Corbetta, Samuele;Pitto, Marina
肾细胞癌(RCC)是最常见的肾癌，占成人恶性肿瘤的3%，具有高转移潜能和放化疗耐药。为了研究参与细胞信号传递、转运和肿瘤转化的细胞膜微区(MD)的蛋白质谱，我们建立了一种自下而上的蛋白质组学方法，作为识别潜在的肾细胞癌生物标志物的起点。我们从肾癌和邻近的正常肾(ANK)组织中纯化MD，通过对非离子洗涤剂的抗性，然后在蔗糖密度梯度中超速离心法。从5例肾癌/ANK组织中提取MD并进行LC-ESI-MS/MS分析。为了确定最多的蛋白质数量，增加膜和疏水性蛋白质的数量，我们首先优化了基于滤膜辅助样品制备(FASP)和MD脱脂的酶消化方法。MS分析鉴定出742个ANK MD和721个RCC MD蛋白，其中分别有53.1%和52.6%是膜结合蛋白。此外，我们通过无标记定量的方法对肾癌MD差异蛋白质组进行了评估，发现在RCC MD中分别有170和126个蛋白质上调和下调。一些差异蛋白，即CA2、CD13和ANXA2，通过免疫修饰进行了验证。这些结果表明，根据样品的不同分子特征，建立不同的膜蛋白蛋白质组学分析方法的重要性。此外，亚细胞蛋白质组学方法提供了一份差异表达蛋白的清单，其中可以寻找肾细胞癌的生物标记物。
Renal Cell Carcinoma (RCC) is the most common kidney cancer, accounting for 3% of adult malignancies, with high metastatic potential and radio-/chemo-resistance. To investigate the protein profile of membrane microdomains (MD), plasma membrane supramolecular structures involved in cell signaling, transport, and neoplastic transformation, we set up a proteomic bottom-up approach as a starting point for the identification of potential RCC biomarkers. We purified MD from RCC and adjacent normal kidney (ANK) tissues, through their resistance to non-ionic detergents followed by ultracentrifugation in sucrose density gradient. MD from 5 RCC/ANK tissues were then pooled and analysed by LC-ESI-MS/MS. In order to identify the highest number of proteins and increase the amount of membrane and hydrophobic ones, we first optimized an enzymatic digestion protocol based on Filter Aided Sample Preparation (FASP), coupled to MD delipidation. The MS analysis led to the identification of 742 ANK MD and 721 RCC MD proteins, of which, respectively, 53.1% and 52.6% were membrane-bound. Additionally, we evaluated RCC MD differential proteome by label-free quantification; 170 and 126 proteins were found to be, respectively, up-regulated and down-regulated in RCC MD. Some differential proteins, namely CA2, CD13, and ANXA2, were subjected to validation by immunodecoration. These results show the importance of setting up different protocols for the proteomic analysis of membrane proteins, specific to the different molecular features of the samples. Furthermore, the subcellular proteomic approach provided a list of differentially expressed proteins among which RCC biomarkers may be looked for.