Quantitative Proteomics of Xenopus Embryos I, Sample Preparation
Quantitative Proteomics of Xenopus Embryos I, Sample Preparation
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DOI：
10.1007/978-1-4939-8784-9_13
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发表时间：
2018-01-01
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影响因子：
--
通讯作者：
Wuhr, Martin
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文献类型：
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作者：
Gupta, Meera;Sonnett, Matthew;Wuhr, Martin
非洲爪蟾卵母细胞和胚胎是最适合于定量蛋白质组学的模型系统。这是由于大量蛋白质材料的可用性和易于物理操作。此外，简单的体外受精提供了细胞周期和发育阶段的完美同步胚胎。在这里，我们详细介绍了在过去几年中开发的多路复用蛋白质组学的样品制备与TMT标签，然后使用MultiNotch MS3方法进行定量质谱分析的协议。在这种方法中，每种条件在肽水平上用同量异序标签进行条形码化。条形码化后，将样品合并，并在质谱仪上定量类似于100，000种肽的相对丰度。在肽被标记和组合之前，样品制备过程的高再现性对于获得无偏数据是最重要的。否则，样品处理的差异可能会无意中表现为生物学变化。我们详细介绍和应用我们的样本工作流程上的非洲爪蟾胚胎的10个发育阶段的胚胎时间序列，从鸡蛋到第35阶段（孵化前）。我们的随附论文（第14章）详细介绍了分析给定样品制备质量的生物信息学管道和转换X光谱的策略。laevis肽转化为生物学上可解释的数据。
Xenopus oocytes and embryos are model systems optimally suited for quantitative proteomics. This is due to the availability of large amount of protein material and the ease of physical manipulation. Furthermore, facile in vitro fertilization provides superbly synchronized embryos for cell cycle and developmental stages. Here, we detail protocols developed over the last few years for sample preparation of multiplexed proteomics with TMT-tags followed by quantitative mass spectrometry analysis using the MultiNotch MS3 approach. In this approach, each condition is barcoded with an isobaric tag at the peptide level. After barcoding, samples are combined and the relative abundance of similar to 100,000 peptides is quantified on a mass spectrometer. High reproducibility of the sample preparation process prior to peptides being tagged and combined is of upmost importance for obtaining unbiased data. Otherwise, differences in sample handling can inadvertently appear as biological changes. We detail and exemplify the application of our sample workflow on an embryonic time-series of ten developmental stages of Xenopus laevis embryos ranging from the egg to stage 35 (just before hatching). Our accompanying paper (Chapter 14) details a bioinformatics pipeline to analyze the quality of the given sample preparation and strategies to convert spectra of X. laevis peptides into biologically interpretable data.