Glasgow Polyomics, College of Medical, Veterinary & Life Sciences, Wolfson Wohl Cancer Research Centre , University of Glasgow , Garscube Estate, Glasgow , United Kingdom G61 1QH.
Institute of Molecular, Cell and Systems Biology , University of Glasgow , Davidson Building, Glasgow , United Kingdom G12 8QQ.
Anal Chem. 2018 Dec 18;90(24):14173-14180. doi: 10.1021/acs.analchem.8b02517. Epub 2018 Dec 3.
Cysteine redox state has been identified as one of the key biological influences behind protein structure and/or function. Altered protein redox state has been shown to cause significant physiological changes and can leave proteins with changed sensitivity to oxidative stress. Protein redox-state changes are recognized as an important mediator of disease, cellular abnormalities, and environmental changes, and therefore their characterization is of interest. Isotopic or isobaric labeling followed by sample multiplexing and analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) allows relative comparison of protein expression levels or of protein redox states between several samples. Combining analysis of protein expression level and redox state into one analysis would add an extra dimension and permit the normalization of protein redox changes with protein abundance. To achieve this, we have developed a quantitation workflow that uses commercially available cysteine-reactive tandem mass tags (iodoTMT) to differentially label cysteine residues, and we have applied it to two Leishmania mexicana cell lines that have previously shown different responses to oxidative stress. The individually labeled samples have been pooled in different combinations to create multiple sixplex samples in order to study the relationship between cysteine oxidation and overall protein expression, as well as providing information about protein oxidation levels in each cell line. The results highlight 11 proteins that are differentially expressed between the two cell lines and/or have significant redox changes. This advanced multiplexing method effectively demonstrates the flexibility of tandem mass tags and how they can be used to maximize the amount of information that can be acquired.
半胱氨酸氧化还原状态被确定为影响蛋白质结构和/或功能的关键生物学因素之一。已表明，蛋白质氧化还原状态的改变会导致显著的生理变化，并使蛋白质对氧化应激的敏感性发生改变。蛋白质氧化还原状态的变化被认为是疾病、细胞异常和环境变化的重要介导因素，因此对其进行特征描述具有重要意义。同位素或等重标记后，通过液相色谱串联质谱（LC-MS/MS）进行样品多重分析，允许在几个样品之间相对比较蛋白质表达水平或蛋白质氧化还原状态。将蛋白质表达水平和氧化还原状态的分析结合到一个分析中，将增加一个额外的维度，并允许用蛋白质丰度对蛋白质氧化还原变化进行归一化。为了实现这一点，我们开发了一种定量工作流程，该流程使用市售的半胱氨酸反应性串联质量标签（碘代 TMT）对半胱氨酸残基进行差异标记，并将其应用于两种先前显示出对氧化应激不同反应的墨西哥利什曼原虫细胞系。将单独标记的样品以不同组合混合，以创建多个六重样品，以研究半胱氨酸氧化与整体蛋白质表达之间的关系，并提供每个细胞系中蛋白质氧化水平的信息。结果突出了 11 种在两种细胞系之间差异表达和/或具有显著氧化还原变化的蛋白质。这种先进的多重化方法有效地展示了串联质量标签的灵活性，以及如何最大限度地利用它们可以获取的信息量。
