Reddy Chinnapapagari Satheesh Kumar, Holzgreve Wolfgang, Hahn Sinuhe
Laboratory of Prenatal Medicine, University Women's Hospital/Department of Research, Basel, Switzerland.
Methods Mol Biol. 2008;444:311-26. doi: 10.1007/978-1-59745-066-9_25.
Proteomics has brought with it the hope of identifying novel biomarkers for the fetal aneuploidies. This hope is built on the ability of proteomic technologies, such as two-dimensional gel electrophoresis (2-DE) with immobilized pH gradients combined with protein identification by mass spectrometry (MS). The large dynamic range of plasma necessitates the effective removal of abundant plasma proteins to allow analysis of the lower concentration analytes. There are many factors that make this research very challenging, beginning with standardization of sample collection, consistent sample preparation, and continuing through the entire analytical process. Therefore, reproducible sample complexity reduction methods such as depletions or fractionations are an essential first step in biomarker discovery experiments. For qualitative and quantitative evaluation of the proteome, the fluorescent dye stains offer several advantages over traditional staining methods. The sensitivity of the fluorescent dye stains such as SYPRO Ruby is comparable with that of silver staining and also has a broad dynamic range, which allows accurate protein quantification being compatible with MS methods for protein identification. Despite its limitations for proteome analysis 2-DE is currently the workhorse for proteomics. Taking into account the factors such as cost, availability and ease of use, 2-DE electrophoresis is one of the most apposite approaches toward the methodical characterization of proteomes.
蛋白质组学带来了识别胎儿非整倍体新型生物标志物的希望。这种希望基于蛋白质组学技术的能力,比如结合了固定化pH梯度的二维凝胶电泳(2-DE)以及通过质谱(MS)进行蛋白质鉴定。血浆的动态范围很大,因此有必要有效去除丰富的血浆蛋白,以便分析低浓度的分析物。从样本采集的标准化、一致的样本制备,一直到整个分析过程,有许多因素使得这项研究极具挑战性。因此,诸如去除或分级分离等可重复的样本复杂度降低方法是生物标志物发现实验必不可少的第一步。对于蛋白质组的定性和定量评估,荧光染料染色相对于传统染色方法具有几个优点。诸如SYPRO Ruby等荧光染料染色的灵敏度与银染相当,并且还具有较宽的动态范围,这使得能够进行准确的蛋白质定量,并且与用于蛋白质鉴定的质谱方法兼容。尽管二维凝胶电泳在蛋白质组分析方面存在局限性,但它目前仍是蛋白质组学的主力技术。考虑到成本、可用性和易用性等因素,二维凝胶电泳是对蛋白质组进行系统表征的最合适方法之一。