Martens Lennart, Van Damme Petra, Van Damme Jozef, Staes An, Timmerman Evy, Ghesquière Bart, Thomas Grégoire R, Vandekerckhove Joël, Gevaert Kris
Department of Medical Protein Research, Flanders Interuniversity Institute for Biotechnology, Department of Biochemistry, Ghent University, A. Baertsoenkaai 3, B-9000 Ghent, Belgium.
Proteomics. 2005 Aug;5(12):3193-204. doi: 10.1002/pmic.200401142.
Several studies have been published in which holistic approaches were used to characterise the proteome and transcriptome of human platelets. The key intent being that a deeper understanding of the normal and aberrant physiological functions of platelets can only be achieved if most biomolecular building blocks are mapped. Here we present the application of recently developed novel technologies that overcome some of the shortcomings of gel-based proteomics. Central in our approach is the so-called combined fractional diagonal chromatography (COFRADIC)-technology in which sets of representative peptides are sorted in a diagonal RP chromatographic system through a specific modification of their side chain. In this study we combined three different COFRADIC sorting techniques to analyse the proteome of human platelets. Methionyl, cysteinyl and amino terminal peptides were isolated and analysed by MS/MS. Merging the peptide identifications obtained after database searching resulted in a core set of 641 platelet proteins, which comprises the largest set identified today. In comparison to previously published platelet proteomes, we identified 404 novel platelet proteins containing a high number of hydrophobic membrane proteins and hypothetical proteins. Furthermore we discuss the observed characteristics and potential benefits of each of the different COFRADIC technologies for proteome analysis and highlight important issues that need to be considered when searching sequence databases using data obtained in peptide-centric, non-gel proteomics studies.
已有多项研究发表，这些研究采用整体方法来表征人类血小板的蛋白质组和转录组。关键目的在于，只有绘制出大多数生物分子构建模块，才能更深入地了解血小板的正常和异常生理功能。在此，我们展示了最近开发的新技术的应用，这些技术克服了基于凝胶的蛋白质组学的一些缺点。我们方法的核心是所谓的组合分数对角线色谱法（COFRADIC）技术，在该技术中，代表性肽组通过其侧链的特定修饰在对角线反相色谱系统中进行分类。在本研究中，我们结合了三种不同的COFRADIC分类技术来分析人类血小板的蛋白质组。甲硫氨酰、半胱氨酰和氨基末端肽通过串联质谱进行分离和分析。合并数据库搜索后获得的肽鉴定结果，得到了一个由641种血小板蛋白组成的核心集，这是目前鉴定出的最大的蛋白集。与之前发表的血小板蛋白质组相比，我们鉴定出了404种新的血小板蛋白，其中包含大量疏水膜蛋白和假设蛋白。此外，我们讨论了每种不同的COFRADIC技术在蛋白质组分析中观察到的特征和潜在优势，并强调了在使用以肽为中心的非凝胶蛋白质组学研究获得的数据搜索序列数据库时需要考虑的重要问题。
