Lucitt Margaret B, Price Thomas S, Pizarro Angel, Wu Weichen, Yocum Anastasia K, Seiler Christoph, Pack Michael A, Blair Ian A, Fitzgerald Garret A, Grosser Tilo
Institute for Translational Medicine and Therapeutics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Mol Cell Proteomics. 2008 May;7(5):981-94. doi: 10.1074/mcp.M700382-MCP200. Epub 2008 Jan 22.
The model organism zebrafish (Danio rerio) is particularly amenable to studies deciphering regulatory genetic networks in vertebrate development, biology, and pharmacology. Unraveling the functional dynamics of such networks requires precise quantitation of protein expression during organismal growth, which is incrementally challenging with progressive complexity of the systems. In an approach toward such quantitative studies of dynamic network behavior, we applied mass spectrometric methodology and rigorous statistical analysis to create comprehensive, high quality profiles of proteins expressed at two stages of zebrafish development. Proteins of embryos 72 and 120 h postfertilization (hpf) were isolated and analyzed both by two-dimensional (2D) LC followed by ESI-MS/MS and by 2D PAGE followed by MALDI-TOF/TOF protein identification. We detected 1384 proteins from 327,906 peptide sequence identifications at 72 and 120 hpf with false identification rates of less than 1% using 2D LC-ESI-MS/MS. These included only approximately 30% of proteins that were identified by 2D PAGE-MALDI-TOF/TOF. Roughly 10% of all detected proteins were derived from hypothetical or predicted gene models or were entirely unannotated. Comparison of proteins expression by 2D DIGE revealed that proteins involved in energy production and transcription/translation were relatively more abundant at 72 hpf consistent with faster synthesis of cellular proteins during organismal growth at this time compared with 120 hpf. The data are accessible in a database that links protein identifications to existing resources including the Zebrafish Information Network database. This new resource should facilitate the selection of candidate proteins for targeted quantitation and refine systematic genetic network analysis in vertebrate development and biology.
模式生物斑马鱼(Danio rerio)特别适合用于解析脊椎动物发育、生物学和药理学中调控基因网络的研究。要揭示此类网络的功能动态,需要在生物体生长过程中对蛋白质表达进行精确量化,而随着系统复杂性的增加,这一挑战也日益增大。在针对动态网络行为进行此类定量研究的方法中,我们应用了质谱方法和严格的统计分析,以创建斑马鱼发育两个阶段表达的蛋白质的全面、高质量图谱。分别对受精后72小时(hpf)和120小时的胚胎蛋白质进行分离,并通过二维(2D)液相色谱(LC)结合电喷雾串联质谱(ESI-MS/MS)以及二维聚丙烯酰胺凝胶电泳(2D PAGE)结合基质辅助激光解吸电离飞行时间串联质谱(MALDI-TOF/TOF)蛋白质鉴定两种方法进行分析。使用二维液相色谱 - 电喷雾串联质谱(2D LC-ESI-MS/MS),我们在72 hpf和120 hpf时从327,906个肽序列鉴定中检测到1384种蛋白质,错误鉴定率低于1%。这些蛋白质中只有约30%是通过二维聚丙烯酰胺凝胶电泳 - 基质辅助激光解吸电离飞行时间串联质谱(2D PAGE-MALDI-TOF/TOF)鉴定出来的。所有检测到的蛋白质中约10%来自假设或预测的基因模型,或者完全没有注释。二维差异凝胶电泳(2D DIGE)对蛋白质表达的比较显示,与能量产生以及转录/翻译相关的蛋白质在72 hpf时相对更为丰富,这与此时生物体生长过程中细胞蛋白质合成速度快于120 hpf时一致。这些数据可通过一个数据库获取,该数据库将蛋白质鉴定与包括斑马鱼信息网络数据库在内的现有资源相链接。这一新资源应有助于选择用于靶向定量的候选蛋白质,并完善脊椎动物发育和生物学中的系统遗传网络分析。