Banfi Cristina, Guarino Anna, Brioschi Maura, Ghilardi Stefania, Mastrullo Valeria, Tremoli Elena, Polvani Gianluca
Centro Cardiologico Monzino IRCCS;
Cardiovascular Tissue Bank of Milan, Centro Cardiologico Monzino IRCCS.
J Vis Exp. 2017 Jun 14(124):55762. doi: 10.3791/55762.
Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit the large-scale identification and quantification of the proteins present in complex biological systems.The knowledge gained from a proteomic approach can potentially lead to a better understanding of the pathogenic mechanisms underlying diseases, allowing for the identification of novel diagnostic and prognostic disease markers, and, hopefully, of therapeutic targets. However, the cardiac mitral valve represents a very challenging sample for proteomic analysis because of the low cellularity in proteoglycan and collagen-enriched extracellular matrix. This makes it challenging to extract proteins for a global proteomic analysis. This work describes a protocol that is compatible with subsequent protein analysis, such as quantitative proteomics and immunoblotting. This can allow for the correlation of data concerning protein expression with data on quantitative mRNA expression and non-quantitative immunohistochemical analysis. Indeed, these approaches, when performed together, will lead to a more comprehensive understanding of the molecular mechanisms underlying diseases, from mRNA to post-translational protein modification. Thus, this method can be relevant to researchers interested in the study of cardiac valve physiopathology.
由于能够对复杂生物系统中存在的蛋白质进行大规模鉴定和定量的技术的发展，细胞蛋白质组分析有助于阐明疾病背后的分子机制。从蛋白质组学方法中获得的知识有可能使人们更好地理解疾病的致病机制，从而能够识别新的疾病诊断和预后标志物，并且有望识别出治疗靶点。然而，由于富含蛋白聚糖和胶原蛋白的细胞外基质中细胞数量较少，心脏二尖瓣是蛋白质组分析极具挑战性的样本。这使得提取用于全局蛋白质组分析的蛋白质具有挑战性。这项工作描述了一种与后续蛋白质分析（如定量蛋白质组学和免疫印迹）兼容的方案。这可以使有关蛋白质表达的数据与定量mRNA表达数据和非定量免疫组织化学分析数据相关联。实际上，这些方法一起实施时，将有助于从mRNA到翻译后蛋白质修饰更全面地理解疾病背后的分子机制。因此，该方法对于对心脏瓣膜生理病理学研究感兴趣的研究人员可能具有重要意义。
