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。这些相互作用由特定细胞中蛋白质伙伴的亲和力和特异性决定。翻译后修饰(PTM)可能产生或排除蛋白质之间的相互作用。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递中特别重要，因为它们代表了蛋白质结构和功能的稳定变化，但这些修饰可以通过酶的相反作用来快速控制，以调节新的信号复合体的形成，或者通过招募和/或激活一系列下游信号分子来诱导现有复合体的变化。*申请人Bisson、Elowe和Lambert是职业生涯早期和中期的研究人员，他们在细胞信号传递方面拥有丰富的专业知识，特别是在蛋白质-蛋白质相互作用和PTM方面。这三家公司都开发了NSERC资助的研究项目，这些项目严重依赖蛋白质组学、蛋白质生物化学和细胞生物学。随着这个工具包中增加高通量的肽筛选，这些研究计划将会有巨大的飞跃。所要求的设备是Intavis ResPep SLI多肽合成器，结合自动斑点合成模块，可以进行定制的多肽阵列合成。*ResPep SLI是一款致力于固相多肽合成的全自动台式仪器。在所要求的构型中，它允许在1-10微克分子范围内并行合成多达96个10-30个残基的多肽。我们还要求一个自动化的斑点合成模块，它可以通过反复将激活的氨基酸作为斑点沉积在纤维素膜上来生成定制的多肽阵列。在一天内，可以在自由定义的网格上平行合成两个膜，每个膜包含多达600个多肽点。这些多肽阵列允许在一次实验中快速筛选数百个多肽，以便执行表位绘制、激酶底物分析、受体结合研究和多肽-核酸阵列。缺乏可获得的多肽阵列合成器是阻碍申请人研究计划进展的主要瓶颈，因为这些高通量方法对于Bisson、Elowe和Lambert研究计划的发展至关重要。*尖端的多肽合成器和SPOT模块，与我们研究所现有的基础设施(如质谱学平台)相结合，将使HQP接触到过多的技术，使他们能够在高通量筛选和蛋白质组学等急需合格人员的领域开发出高度需要的技术专业知识。拟议的工具将加快研究培训，并将为受训者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.