Genome-wide activity-based profiling of Arabidopsis proteomes

蛋白质活性的信息对于解释和理解蛋白质的功能是必不可少的，因为蛋白质的活性而不是它的丰度最终决定了它在活着的有机体中的作用。我们的目标是通过一种名为基于活动的蛋白质组图谱(ABPP)的新的全基因组技术来揭示拟南芥的蛋白质组活动层。ABPP可应用于活组织或蛋白质提取物，其基础是标记的小分子(探针)，这些小分子(探针)以活性依赖的方式与广泛的蛋白质的活性部位残基反应。标记的蛋白质可以在蛋白质凝胶上分离和可视化，并通过质谱仪(MS)进行鉴定。该项目旨在通过有机化学家、生物化学家和MS专家之间的密切跨学科合作，从不同的拟南芥蛋白质组中产生来自不同机械类别的100-1000个不同蛋白质的活性信息。将通过异源表达来表征和确认活性。尚未注释的蛋白质的标记位置将被识别和验证，以揭示其催化机制。最后，生成抑制信号，将一个大的冗余蛋白质家族细分为功能单元。数据将通过开放访问的可搜索数据库共享，植物科学家可以在其中检索他们最喜欢的蛋白质(S)的活性信息，并为进一步研究请求探针和方案。

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.