Raimondo F, Morosi L, Chinello C, Perego R, Bianchi C, Albo G, Ferrero S, Rocco F, Magni F, Pitto M
Department of Experimental Medicine, University of Milano-Bicocca, Via Cadore 48, 20052 Monza, Italy.
Mol Biosyst. 2012 Apr;8(4):1007-16. doi: 10.1039/c2mb05372a. Epub 2011 Dec 13.
Renal cell carcinoma (RCC) is representing about 3% of all adult cancers. A promising strategy for cancer biomarker discovery is subcellular comparative proteomics, allowing enriching specific cell compartments and assessing differences in protein expression patterns. We investigated the proteomic profile of a peculiar RCC subcellular compartment, plasma membrane microdomains (MD), involved in cell signalling, transport, proliferation and in many human diseases, such as cancer. Subcellular fractions were prepared by differential centrifugation from surgical samples of RCC and adjacent normal kidney (ANK). MD were isolated from plasma-membrane-enriched fractions after Triton X-100 treatment and sucrose density gradient ultracentrifugation. MD derived from RCC and ANK tissues were analyzed after SDS-PAGE separation by LC-ESI-MS/MS. We identified 93 proteins from MD isolated from RCC tissue, and 98 proteins from ANK MD. About 70% of the identified proteins are membrane-associated and about half of these are known as microdomain-associated. GRAVY scores assignment shows that most identified proteins (about 70%) are in the hydrophobic range. We chose a panel of proteins to validate their differential expression by WB. In conclusion, our work shows that RCC microdomain proteome is reproducibly different from ANK, and suggests that mining into such differences may support new biomarker discovery.
肾细胞癌（RCC）约占所有成人癌症的3%。癌症生物标志物发现的一个有前景的策略是亚细胞比较蛋白质组学，它可以富集特定的细胞区室并评估蛋白质表达模式的差异。我们研究了一种特殊的RCC亚细胞区室——质膜微结构域（MD）的蛋白质组概况，该结构域参与细胞信号传导、运输、增殖以及许多人类疾病，如癌症。通过差速离心从RCC手术样本和相邻正常肾组织（ANK）中制备亚细胞组分。经Triton X-100处理和蔗糖密度梯度超速离心后，从富含质膜的组分中分离出MD。通过LC-ESI-MS/MS对SDS-PAGE分离后的RCC和ANK组织来源的MD进行分析。我们从RCC组织分离的MD中鉴定出93种蛋白质，从ANK的MD中鉴定出98种蛋白质。约70%的已鉴定蛋白质与膜相关，其中约一半被认为与微结构域相关。GRAVY评分显示，大多数已鉴定蛋白质（约70%）处于疏水范围内。我们选择了一组蛋白质通过WB验证其差异表达。总之，我们的工作表明RCC微结构域蛋白质组与ANK有可重复的差异，并表明挖掘这些差异可能有助于发现新的生物标志物。
