Lemeer Simone, Pinkse Martijn W H, Mohammed Shabaz, van Breukelen Bas, den Hertog Jeroen, Slijper Monique, Heck Albert J R
Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands.
J Proteome Res. 2008 Apr;7(4):1555-64. doi: 10.1021/pr700667w. Epub 2008 Feb 29.
In the developing embryo, as in many other biological processes, complex signaling pathways are under tight control of reversible phosphorylation, guiding cell proliferation, differentiation, and growth. Therefore the large-scale identification of signaling proteins and their post-translational modifications is crucial to understand the proteome biology of the developing zebrafish embryo. Here, we used an automated, robust, and sensitive online TiO 2-based LC-MS/MS setup to enrich for phosphorylated peptides from 1 day old zebrafish embryos. We identified, with high confidence, 1067 endogenous phosphorylation sites in a sample taken from 60 embryos (approximately 180 microg), 321 from 10 embryos, and 47 phosphorylation sites from a single embryo, illustrating the sensitivity of the method. This data set, representing by far the largest for zebrafish, was further exploited by searching for serine/threonine or tyrosine kinase motifs using Scansite. For one-third of the identified phosphopeptides a potential kinase motif could be predicted, where it appeared that Cdk5 kinase, p38MAPK, PKA, and Casein Kinase 2 substrates were the most predominant motifs present, underpinning the importance of these kinases in signaling pathways in embryonic development. The phosphopeptide data set was further interrogated using alignments with phosphopeptides identified in recent large-scale phosphoproteomics screens in human and mouse samples. These alignments revealed conservation of phosphorylation sites in several proteins suggesting preserved function in embryonic development.
在发育中的胚胎中，如同在许多其他生物过程中一样，复杂的信号通路受到可逆磷酸化的严格控制，引导细胞增殖、分化和生长。因此，大规模鉴定信号蛋白及其翻译后修饰对于理解斑马鱼胚胎发育的蛋白质组生物学至关重要。在这里，我们使用了一种自动化、稳健且灵敏的基于TiO₂ 的在线液相色谱 - 串联质谱装置，从1日龄斑马鱼胚胎中富集磷酸化肽段。我们从60个胚胎（约180微克）的样本中高置信度地鉴定出1067个内源性磷酸化位点，从10个胚胎中鉴定出321个，从单个胚胎中鉴定出47个磷酸化位点，这说明了该方法的灵敏度。这个数据集是迄今为止斑马鱼最大的数据集，我们进一步利用Scansite搜索丝氨酸/苏氨酸或酪氨酸激酶基序对其进行了分析。对于三分之一已鉴定的磷酸化肽段，可以预测到潜在的激酶基序，其中Cdk5激酶、p38丝裂原活化蛋白激酶、蛋白激酶A和酪蛋白激酶2的底物似乎是最主要的基序，这强调了这些激酶在胚胎发育信号通路中的重要性。我们还将该磷酸化肽段数据集与最近在人类和小鼠样本的大规模磷酸蛋白质组学筛选中鉴定出的磷酸化肽段进行比对，进一步分析该数据集。这些比对揭示了几种蛋白质中磷酸化位点的保守性，表明其在胚胎发育中具有保守功能。
