Wilson Richard, Belluoccio Daniele, Bateman John F
Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Victoria 3052, Australia.
Methods. 2008 May;45(1):22-31. doi: 10.1016/j.ymeth.2008.01.008.
While the analysis of the cartilage proteome is important for our comprehensive understanding of the development and disease of this important tissue, several unique features of cartilage present some technical obstacles. Firstly, cartilage is difficult to obtain in adequate quantities for many protein analyses, especially from mice which are otherwise powerful experimental models. Furthermore, the cartilage extracellular matrix contains an insoluble network of collagen II-containing fibrils that are integrated within an abundant anionic network of aggrecan and hyaluronan aggregates. These interacting networks provide a structural scaffold for the covalent and non-covalent attachment of other proteins and glycoproteins. Consequently, proteomic analysis of cartilage requires extraction of proteins with chaotropic agents to achieve and significant protein solubilization. Finally, isolated chondrocytes are phenotypically unstable, which requires rapid isolation of cells or the use of specific culture conditions. Despite these problems, recent improvements in the sensitivity and reproducibility of two-dimensional electrophoresis (2-DE) and tandem mass spectrometry (MS/MS) techniques, combined with improved tissue preparation and sample pre-fractionation approaches, have made the proteomic characterization of cartilage tissues possible. Here we review the approaches that have been used and describe in detail protocols for the proteomic analysis of cartilage tissues and cells.
虽然对软骨蛋白质组的分析对于我们全面了解这种重要组织的发育和疾病至关重要，但软骨的几个独特特征带来了一些技术障碍。首先，要获得足够数量的软骨用于许多蛋白质分析很困难，尤其是从小鼠身上获取，而小鼠在其他方面是强大的实验模型。此外，软骨细胞外基质包含一个不溶性的含胶原蛋白II的纤维网络，该网络整合在丰富的聚集蛋白聚糖和透明质酸聚集体的阴离子网络中。这些相互作用的网络为其他蛋白质和糖蛋白的共价和非共价附着提供了结构支架。因此，软骨的蛋白质组分析需要用离液剂提取蛋白质以实现显著的蛋白质溶解。最后，分离的软骨细胞在表型上不稳定，这需要快速分离细胞或使用特定的培养条件。尽管存在这些问题，但二维电泳（2-DE）和串联质谱（MS/MS）技术在灵敏度和重现性方面的最新改进，结合改进的组织制备和样品预分级方法，使得软骨组织的蛋白质组表征成为可能。在这里，我们回顾了已使用的方法，并详细描述了软骨组织和细胞蛋白质组分析的方案。
