High throughput analysis of protein interactions and modifications using peptide arrays

后生动物中的信号转导是通过蛋白质相互作用网络的动态组织来实现的。这些相互作用由给定细胞中蛋白质伴侣的亲和力和特异性决定。翻译后修饰（PTM）可以产生或阻止蛋白质-蛋白质相互作用。特别是，磷酸化和乙酰化是真核生物中最常见的两种PTM。这些修饰在细胞信号传导中特别重要，因为它们代表了蛋白质结构和功能的稳定改变，但这些改变可以通过酶的相反作用来快速控制，以调节新信号传导复合物的形成，或通过募集和/或激活下游信号传导分子的集合来诱导现有复合物的变化。申请人Bisson，Elowe和Lambert是职业生涯早期和中期的研究人员，他们在细胞信号传导方面具有很强的专业知识，特别是蛋白质-蛋白质相互作用和PTM。这三个国家都开发了NSERC资助的研究项目，这些项目在很大程度上依赖于蛋白质组学、蛋白质生物化学和细胞生物学。这些研究计划将极大地飞跃与高通量肽筛选在这个工具包中添加。要求的设备是Intavis ResPep SLi肽合成仪，与自动化SPOT合成模块相结合，以允许定制肽阵列合成。* ResPep SLi是一款全自动台式仪器，专用于固相肽合成。在所要求的配置中，它允许以1-10 μmol规模平行合成多达96个10-30个残基的肽。我们还要求一个自动化的SPOT合成模块，它可以通过在纤维素膜上重复沉积活化的氨基酸作为斑点来生成定制的肽阵列。在一天内，可以在自由定义的网格上平行合成两个膜，每个膜含有多达600个肽点。这些肽阵列允许在单个实验中快速筛选数百种肽，以进行表位作图、激酶底物分析、受体结合研究和肽-核酸阵列。缺乏可访问的肽阵列合成器是延迟申请人研究项目进展的主要瓶颈，因为这些高通量方法对于Bisson，Elowe和Lambert研究项目的发展至关重要。尖端的肽合成仪和SPOT模块，结合我们研究所现有的基础设施（例如质谱平台），将使HQP接触到大量的技术，使他们能够在急需合格人员的领域开发高度寻求的技术专长，如高通量筛选和蛋白质组学。拟议的工具将加速研究培训，并将为受训人员提供进行创新研究的坚实基础。

Signal transduction in metazoans is effected via the dynamic organization of protein interaction networks. These interactions are determined by the affinity and the specificity of the protein partners in a given cell. Post-translational modifications (PTMs) may create or preclude protein-protein interactions. In particular, phosphorylation and acetylation are two of the most frequent PTMs in eukaryotes. These modifications are of particular importance in cell signalling, as they represent stable alterations of protein structure and function, yet ones that may be rapidly controlled through the opposing action of enzymes to regulate the formation of new signalling complexes, or induce changes in existing complexes, via the recruitment and/or activation of a collection of downstream signalling molecules.******The applicants Bisson, Elowe and Lambert are early- and mid-career investigators who have a strong expertise in cell signalling, in particular with protein-protein interactions and PTMs. All three have developed NSERC-funded research programs that rely heavily on proteomics, protein biochemistry and cell biology. These research programs would tremendously leap forward with the addition of high-throughput peptide screening in this toolkit. The requested equipment is an Intavis ResPep SLi peptide synthesizer, combined with an automated SPOT synthesis module, to allow custom peptide array synthesis. ******The ResPep SLi is a fully automated benchtop instrument dedicated to solid-phase peptide synthesis. In the requested configuration, it allows for the parallel synthesis of up to 96 peptides of 10-30 residues in a 1-10 µmol scale. We also request an automated SPOT synthesis module, which may generate custom peptide arrays via repeated deposition of activated amino acids as spots on a cellulose membrane. Two membranes each containing up to 600 peptide spots may be synthesized in parallel on freely defined grids, in a single day. These peptide arrays allow for the rapid screening of hundreds of peptides in a single experiment, in order to perform epitope mapping, analysis of kinase substrates, receptor binding studies and peptide-nucleic acid arrays. The lack of an accessible peptide array synthesizer is a major bottleneck that delays the progress of the applicant's research programs, as these high-throughput approaches are essential for growth of the Bisson, Elowe and Lambert research programs.******The cutting-edge peptide synthesizer and SPOT module, combined with infrastructure available within our Institute (e.g. mass spectrometry platforms), will expose HQP to a plethora of techniques that will allow them to develop a highly sought technical expertise in areas that are in dire need of qualified personnel such as high-throughput screening and proteomics. The proposed instrument will accelerate research training and will provide trainees with a solid base to perform innovative research.