Development of high-throughput mass-spec based sequencing and its application to global analysis of protein phosphorylation

本研究的目的是：1)建立质谱图方法，利用从2D凝胶斑点产生的多肽获得的信息来鉴定基因组数据库中的蛋白质。2)建立一种修饰特异性的方法来检测磷酸化或乙酰化的多肽。3)将所建立的方法应用于信号复合体的全局分析，以阐明未知蛋白的功能。对于这些江豚，我们建立了一个分析系统，其中自动进行凝胶斑点和切除、质量测量和数据库搜索。为了实现高通量分析，该基金购买了样品前处理机器人，并对其进行了改装，使样品板上的自动脱盐和靶向成为可能。我们还建立了一套分析系统，利用该系统，可以在三四元质谱仪上通过亲本扫描对磷酸肽进行特定的检测。通过LC/MS分析，一个样品中30多种蛋白质的高通量鉴定成为可能。这样，我们就可以实现高通量的大批量测序。

The aims of the research are :1) To establish, mass mapping method to identify proteins in the genome database with the information obtained by mass spectrometry of peptides generated from 2D gel spots.2) TO establish a modification-specific method to detect phosphorylated or acetylated peptides.3) To apply thus established methods to the global analysis of signaling complexes to elucidate the functions of "unknown" proteins.For these porpoises, we have established an analysis system, in which gel-spotting and excision, mass measurements and database search are performed automatically. To achieve a high-throughput analysis, the sample-pretreatment robot was purchased by the fund, and modified to make automatic desalting and targeting on the sample plate possible. We have also established an analysis system with which specific detection of phosphopeptides are possible by parent scan in the triple-quad mass spectrometer. The high-throughput identification of more than 30 proteins in a single sample became possible by LC/MS analysis. In this way, we could achieve the high-throughput mass sequencing.