Biomedical Proteomics Group, Department of Structural Biology and Bioinformatics, University of Geneva, Geneva, Switzerland.
J Proteome Res. 2011 Jan 7;10(1):249-58. doi: 10.1021/pr100535f. Epub 2010 Nov 18.
Highly complex and dynamic protein mixtures are hardly comprehensively resolved by direct shotgun proteomic analysis. As many proteins of biological interest are of low abundance, numerous analytical methodologies have been developed to reduce sample complexity and go deeper into proteomes. The present work describes an analytical strategy to perform cysteinyl-peptide subset enrichment and relative quantification through successive cysteine and amine-isobaric tagging. A cysteine-reactive covalent capture tag (C³T) allowed derivatization of cysteines and specific isolation on a covalent capture (CC) resin. The 6-plex amine-reactive tandem mass tags (TMT) served for relative quantification of the targeted peptides. The strategy was first evaluated on a model protein mixture with increasing concentrations to assess the specificity of the enrichment and the quantitative performances of the workflow. It was then applied to human cerebrospinal fluid (CSF) from post-mortem and ante-mortem samples. These studies confirmed the specificity of the C³T and the CC technique to cysteine-containing peptides. The model protein mixture analysis showed high precision and accuracy of the quantification with coefficients of variation and mean absolute errors of less than 10% on average. The CSF experiments demonstrated the potential of the strategy to study complex biological samples and identify differential brain-related proteins. In addition, the quantification data were highly correlated with a classical TMT experiment (i.e., without C³T cysteine-tagging and enrichment steps). Altogether, these results legitimate the use of this quantitative C³T strategy to enrich and relatively quantify cysteine-containing peptides in complex mixtures.
高度复杂和动态的蛋白质混合物很难通过直接的鸟枪法蛋白质组学分析进行全面解析。由于许多生物感兴趣的蛋白质丰度较低，因此已经开发了许多分析方法来降低样品复杂性并深入研究蛋白质组。本工作描述了一种通过连续半胱氨酸和胺等摩尔质量标签（isobaric tagging）进行半胱氨酸肽亚组富集和相对定量的分析策略。一种半胱氨酸反应性共价捕获标签（C³T）允许对半胱氨酸进行衍生化，并在共价捕获（CC）树脂上进行特异性分离。6 重胺反应性串联质量标签（TMT）用于靶向肽的相对定量。该策略首先在模型蛋白质混合物中进行评估，随着浓度的增加来评估富集的特异性和工作流程的定量性能。然后将其应用于死后和生前的人脑脊液（CSF）。这些研究证实了 C³T 和 CC 技术对半胱氨酸肽的特异性。模型蛋白质混合物分析显示，定量具有很高的精度和准确性，平均变异系数和平均绝对误差小于 10%。CSF 实验证明了该策略在研究复杂生物样品和鉴定与大脑相关的差异蛋白方面的潜力。此外，定量数据与经典 TMT 实验（即，没有 C³T 半胱氨酸标记和富集步骤）高度相关。总之，这些结果证明了使用这种定量 C³T 策略对半胱氨酸肽在复杂混合物中进行富集和相对定量的合理性。
