Genome-wide activity-based profiling of Arabidopsis proteomes

关于蛋白质活性的信息对于蛋白质功能的注释和理解至关重要，因为它是蛋白质的活性，而不是它的丰度，最终决定了它在生物体中的作用。我们的目标是通过一种新的全基因组技术，即基于活性的蛋白质组分析（ABPP），揭示拟南芥的蛋白质组活性层。ABPP可以应用于活组织或蛋白质提取物，并且基于标记的小分子（探针），这些小分子以活性依赖的方式与多种蛋白质的活性位点残基反应。标记的蛋白质可以在蛋白质凝胶上分离和可视化，并通过质谱（MS）进行鉴定。该项目旨在通过有机化学家、生物化学家和质谱专家之间的密切跨学科合作，从拟南芥不同蛋白质组中获得100-1000种不同机制类别的不同蛋白质的活性信息。活性将通过异源表达进行表征和确认。尚未注释的蛋白质的标记位点将被鉴定和验证，以揭示其催化机制。最后，抑制信号将产生亚分类一个大的冗余蛋白家族到功能单位。数据将通过一个开放访问的可搜索数据库共享，植物科学家可以检索他们喜欢的蛋白质的活性信息，并为进一步的研究请求探针和协议。

Information on protein activity is essential to the annotation and understanding of protein functions since it is the activity of a protein, rather than its abundance, that ultimately dictates its role in a living organism. We aim to uncover the proteome activity layer of Arabidopsis through a novel, genome-wide technology called activity-based proteome profiling (ABPP). ABPP can be applied on living tissues or on protein extracts and is based on labeled small molecules (probes) that react with active site residues of a broad range of proteins in an activity-dependent manner. Labeled proteins can be separated and visualized on protein gels and identified by mass spectrometry (MS). This project aims to generate activity information of 100-1000 different proteins from various mechanistic classes from different Arabidopsis proteomes through an intimate interdisciplinary collaboration between an organic chemist, biochemist and MS-specialist. Activities will be characterized and confirmed by heterologous expression. Labeling sites of as yet non-annotated proteins will be identified and validated to reveal their catalytic mechanisms. Finally, inhibitory signatures will be generated to sub-classify a large redundant protein family into functional units. Data will be shared through an open-access searchable database where plant scientists can retrieve activity information for their favorite protein(s) and request probes and protocols for further studies.