Hoehenwarter Wolfgang, van Dongen Joost T, Wienkoop Stefanie, Steinfath Matthias, Hummel Jan, Erban Alexander, Sulpice Ronan, Regierer Babette, Kopka Joachim, Geigenberger Peter, Weckwerth Wolfram
Max Planck Institute of Molecular Plant Physiology, Potsdam, Germany.
Proteomics. 2008 Oct;8(20):4214-25. doi: 10.1002/pmic.200701047.
The dynamics of a proteome can only be addressed with large-scale, high-throughput methods. To cope with the inherent complexity, techniques based on targeted quantification using proteotypic peptides are arising. This is an essential systems biology approach; however, for the exploratory discovery of unexpected markers, nontargeted detection of proteins, and protein modifications is indispensable. We present a rapid label-free shotgun proteomics approach that extracts relevant phenotype-specific peptide product ion spectra in an automated workflow without prior identification. These product ion spectra are subsequently sequenced with database search and de novo prediction algorithms. We analyzed six potato tuber cultivars grown on three plots of two geographically separated fields in Germany. For data mining about 1.5 million spectra from 107 analyses were aligned and statistically examined in approximately 1 day. Several cultivar-specific protein markers were detected. Based on de novo-sequencing a dominant protein polymorphism not detectable in the available EST-databases was assigned exclusively to a specific potato cultivar. The approach is applicable to organisms with unsequenced or incomplete genomes and to the automated extraction of relevant mass spectra that potentially cannot be identified by genome/EST-based search algorithms.
蛋白质组的动态变化只能通过大规模、高通量的方法来研究。为了应对其固有的复杂性，基于使用蛋白型肽进行靶向定量的技术应运而生。这是一种重要的系统生物学方法；然而，对于探索性地发现意外的标志物、非靶向蛋白质检测以及蛋白质修饰而言，非靶向蛋白质检测是必不可少的。我们提出了一种快速的无标记鸟枪法蛋白质组学方法，该方法可在无需预先鉴定的自动化流程中提取相关的表型特异性肽产物离子谱。随后，使用数据库搜索和从头预测算法对这些产物离子谱进行测序。我们分析了在德国两个地理上分开的田地的三块土地上种植的六个马铃薯品种。为了进行数据挖掘，在大约1天的时间里，对来自107次分析的约150万个谱进行了比对和统计检验。检测到了几个品种特异性的蛋白质标志物。基于从头测序，在可用的EST数据库中未检测到的一种主要蛋白质多态性被专门指定给一个特定的马铃薯品种。该方法适用于基因组未测序或不完整的生物体，以及适用于自动提取可能无法通过基于基因组/EST的搜索算法鉴定的相关质谱。
