ABSTRACT

Quantitative proteomic analyses in combination with genetics provide powerful tools in developmental cell signalling research. Drosophila melanogaster is one of the most widely used genetic models for studying development and disease. Here we combined quantitative proteomics with genetic selection to determine changes in the proteome upon depletion of Heartless (Htl) Fibroblast-Growth Factor (FGF) receptor signalling in Drosophila embryos at the gastrula stage. We present a robust, single generation SILAC (stable isotope labelling with amino acids in cell culture) protocol for labelling proteins in early embryos. For the selection of homozygously mutant embryos at the pre-gastrula stage, we developed an independent genetic marker. Our analyses detected quantitative changes in the global proteome of htl mutant embryos during gastrulation. We identified distinct classes of downregulated and upregulated proteins, and network analyses indicate functionally related groups of proteins in each class. In addition, we identified changes in the abundance of phosphopeptides. In summary, our quantitative proteomic analysis reveals global changes in metabolic, nucleoplasmic, cytoskeletal and transport proteins in htl mutant embryos.

摘要

定量蛋白质组学分析与遗传学相结合，为发育细胞信号传导研究提供了强大的工具。黑腹果蝇是研究发育和疾病的最广泛使用的遗传模型之一。在这里，我们将定量蛋白质组学与遗传选择相结合，以确定在果蝇中无心 (Htl) 成纤维细胞生长因子 (FGF) 受体信号耗尽后蛋白质组的变化原肠胚阶段的胚胎。我们提出了一种强大的单代 SILAC（细胞培养中氨基酸的稳定同位素标记）协议，用于标记早期胚胎中的蛋白质。为了在原肠胚前阶段选择纯合突变胚胎，我们开发了一个独立的遗传标记。我们的分析检测到原肠胚形成过程中htl突变胚胎的全局蛋白质组的数量变化。我们确定了不同类别的下调和上调蛋白质，网络分析表明每个类别中功能相关的蛋白质组。此外，我们还确定了磷酸肽丰度的变化。总之，我们的定量蛋白质组学分析揭示了htl中代谢、核质、细胞骨架和转运蛋白的整体变化 突变胚胎。