Proteomic Core

概况.蛋白质组学核心为中心，ISB和当地和世界各地的许多合作者提供尖端的蛋白质组学能力。最近的成就包括：开发分析和计算工具，实现蛋白质组，亚蛋白质组和翻译后修饰的全面和系统的分析;开发软件套件，用于蛋白质组数据集的评估和验证;以及设计和实施靶向定量质谱（MS）实验，以分析蛋白质组和亚细胞蛋白质组。该核心配备了最先进的MS技术（请参阅参考资料），并将继续传播和推广用于高质量定量数据采集和快速实施新技术的工具。核心提供MS操作和实验设计的培训和帮助，并协助蛋白质组学信息学课程（见教育和培训）。由于核心是中心研究的组成部分，我们在下面提供了正在进行的技术开发的描述。 定量蛋白质组学。核心是一个世界领先的开发和应用的标签和无标签的定量蛋白质组学的表达谱和分析的大分子组装。例如，核心，与艾奇逊集团合作，开发了一种新的自动化方法来量化SILAC实验（QTIPs）中的肽，沿着分离大分子复合物的新方法。 这些方法显著改善了体内相关相互作用的鉴定 并导致了对过氧化物酶体诱导中涉及的信号网络的广泛定义。 翻译后修饰的分析。该核心通过开发用于肽分离和鉴定修饰位点的技术，继续为翻译后修饰（PTM）（例如磷酸化和-糖基化）的挑战性分析做出重大贡献。 为了解决与磷酸肽的低丰度相关的问题，核心已经实施了用于样品复杂性降低的分级分离方法和靶富集策略（IMAC或TiO 2），这使得鉴定的磷酸肽的数量从几百个显著增加到数千个。我们已经开发了一种基于冷冻裂解的破坏，尿素增溶方法，以最大限度地减少激酶或磷酸酶活性，并保持蛋白质组的条件特异性磷酸化状态。通过将代谢标记（SILAC）与生物化学、分析和计算方法相结合，我们常规地鉴定出在非常低的拷贝数下对细胞扰动有显著响应的关键磷蛋白。为了进一步增强磷酸肽中磷酸化位点的明确鉴定，核心实现了电子转移解离（ETD）能力以产生保留PTM（例如，不稳定的磷酸-丝氨酸/苏氨酸键）。我们还开发了软件来解释ETD光谱，并进行统计验证，以纳入跨蛋白质组学管道（TPP）。

Overview. The proteomics core provides cutting-edge proteomics capabilities to the Center, ISB and many collaborators locally and worldwide. Recent accomplishments include: development of analytical and computational tools enabling comprehensive and systematic analysis of proteomes, subproteomes, and post translational modifications; development of software suites for evaluation and validation of proteomic datasets; and the design and implementation of targeted quantitative mass spectrometry (MS) experiments to analyze proteomes and subcellular proteomes. The core equipped with state-of-the-art MS technologies (see Resources) and will continue to disseminate and promote tools for high quality quantitative data acquisition and rapid implementation of new technologies. The core provides training and assistance on MS operation and experimental design, and assists with the Proteomics Informatics course (See Education and Training). Because the core is so integral to Center research, we provide below a description of ongoing technology development. Quantitative Proteomics. The core is a world leader in the development and application of both label and label-free quantitative proteomics for expression profiling and analysis of macromolecular assemblages. For example, the core, in collaboration with the Aitchison group, developed a novel automated approach to quantify peptides in SILAC experiments (QTIPs) along with new approaches for isolation of macromolecular complexes. These approaches significantly improved the identification of in vivo relevant interactions and led to extensive definition of signaling networks involved in peroxisome induction"¿¿. Analysis of post-translational modifications. The core continues to make significant contributions to the challenging analysis of post-translational modifications (PTMs) (e.g. phosphorylation and -glycosylation) through technologies developed for peptide isolation and identification of the modified sites. To address issues associated with the low abundance of phosphopeptides, the core has implemented fractionation methods for sample complexity reduction, and target enrichment strategies, (IMAC or Ti02), which have significantly increased the number of identified phosphopeptides from a few hundred to thousands. We have developed a cryolysis-based disruption, urea-solubilization methodology to minimize kinase or phosphatase activity and maintain the condition-specific phosphorylation status of the proteome. By combining metabolic labeling (SILAC) with biochemical, analytical, and computational approaches, we routinely identify key phosphoproteins that are significantly responsive to cellular perturbations at very low copy number. To further enhance the unequivocal identification of the phosphorylated site in phosphopeptides, the core implemented electron transfer dissociation (ETD) capabilities to produce fragmentation spectra that retain PTMs (e.g., labile phospho-serine/threonine bond). We have also developed software to interpret ETD spectra with statistical validation for incorporation into the Trans-Proteomic Pipeline (TPP).