Top-down approaches for measuring expression ratios of intact yeast proteins using Fourier transform mass spectrometry
Top-down approaches for measuring expression ratios of intact yeast proteins using Fourier transform mass spectrometry
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DOI：
10.1021/ac050993p
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发表时间：
2006-02-01
影响因子：
7.4
通讯作者：
Kelleher, NL
中科院分区：
文献类型：
--
作者：
Du, Y;Parks, BA;Kelleher, NL
将小肽的定量方法扩展到 5 kDa 以上的离子，并最终扩展到完整蛋白质的全局定量蛋白质组学，将需要对当前的分析方法进行广泛的改进。在这里，我们使用自上而下的质谱 (MS) 评估生长后 Cys 标记和 N-14/N-15 代谢标记策略，以确定相对蛋白质表达水平及其翻译后修饰。我们发现，用丙烯酰胺 (+71 Da) 与碘乙酰胺 (+57 Da) 进行差异烷基化的完整蛋白质在反相液相色谱分离过程中具有显着的色谱位移（特别是在峰尾），表明自上而下 MS 的烷基化标签中需要稳定同位素。在 N-14/N-15 代谢标记策略中，我们在有氧条件下生长 10 代的酵母中实现了 98% N-15 掺入，并使用傅里叶变换离子回旋共振 MS 确定了 50 个表达比率，将这些细胞与厌氧生长的对照 (N-14) 细胞进行比较。我们设计了用于自上而下分析的定量方法，包括基于信噪比的准确蛋白质比率测定的校正因子。使用先前通过自上而下 MS 鉴定的 200 种酵母蛋白质形式的数据库，我们验证了无需串联 MS 即可进行蛋白质鉴定的完整质量标签概念。总体而言，我们发现自上而下的 MS 有望提供能够使用稳定同位素标记进行大规模蛋白质组分析的工作流程，并确定每个光谱 > 5 个蛋白质比率。
The extension of quantitation methods for small peptides to ions above 5 kDa, and eventually to global quantitative proteomics of intact proteins, will require extensive refinement of current analytical approaches. Here we evaluate postgrowth Cys-labeling and N-14/N-15 metabolic labeling strategies for determination of relative protein expression levels and their posttranslational modifications using top-down mass spectrometry (MS). We show that intact proteins that are differentially alkylated with acrylamide (+71 Da) versus iodoacetamide (+57 Da) have substantial chromatographic shifts during reversed-phase liquid chromatography separation (particularly in peak tails), indicating a requirement for stable isotopes in alkylation tags for top-down MS. In the N-14/N-15 metabolic labeling strategy, we achieve 98% N-15 incorporation in yeast grown 10 generations under aerobic conditions and determine 50 expression ratios using Fourier transform ion cyclotron resonance MS in comparing these cells to anaerobically grown control (N-14) cells. We devise quantitative methods for top-down analyses, including a correction factor for accurate protein ratio determination based upon the signal-to-noise ratio. Using a database of 200 yeast protein forms identified previously by top-down MS, we verify the intact mass tag concept for protein identification without tandem MS. Overall, we find that top-down MS promises work flows capable of large-scale proteome profiling using stable isotope labeling and the determination of > 5 protein ratios per spectrum.