Proteomics in muscle and motility systems

蛋白质分离、质谱和生物信息学的最新进展使同时分析细胞和组织中的大量蛋白质成为可能。这些蛋白质的相互作用，特别是那些对特定挑战作出反应的蛋白质，可以通过巧妙的筛选技术（如噬菌体表达或酵母两种杂交系统）快速识别。这些研究被统称为“蛋白质组学”，极大地丰富了我们对正常和病变组织的细胞生理学和病理学的认识。系统地识别自然细胞环境中相互作用的蛋白质之间的分子界面是对复杂和动态细胞事件的分子理解的下一个主要挑战。通过靶向关键分子界面，这种分子界面的概况也将作为药物开发的宝贵工具。我们正在启动这样一个蛋白质组学项目，并专注于化学交联方法的应用，以系统和全局地识别复杂系统中的蛋白质-蛋白质界面，如肌肉细胞的收缩机制、细胞骨架细丝和非肌肉细胞的细胞器。首先，高分辨率凝胶电泳和分馏技术用于鉴定和索引骨骼肌和心肌组织中的表达蛋白。其次，邻接关系是通过与各种试剂的化学交联来定义的。第三，通过分离交联复合物、切割蛋白质和分离交联肽来鉴定邻近蛋白质的分子界面，用常规化学方法或质谱法进行蛋白质测序。第四，合成界面上的肽，并通过毛细管电泳和生物传感器技术进一步表征它们之间的相互作用。将建立一个已知和未知身份的蛋白质分子界面的蛋白质序列数据库，以补充和增强基因组和蛋白质组数据库在基础和临床研究中的效用。我们正在评估这个多方面项目的每个步骤的可行性和整合潜力。在过去的一年里，我们成功地应用了一种荧光交联剂来促进交联肽的鉴定和纯化。用这种交联剂绘制了星云片段与钙调蛋白之间的界面，并应用质谱法鉴定了交联在一起的肽序列。分子内交联模式也揭示了新的折叠模式。我们正在将这项研究扩展到其他交联剂和运动系统中更复杂的蛋白质组装。

Recent advances in protein separation, mass spectrometry and bioinformatics have enabled the simultaneous analysis of a large number of proteins in cells and tissues. The interaction of these proteins, especially those that respond to specific challenges, are rapidly identified by ingenious screening techniques such as the phage expression or yeast two hybrid systems. These studies, collectively termed "proteomics", have greatly enriched our knowledge of cellular physiology and pathology in normal and diseased tissues. A systematic identification of molecular interfaces between interacting proteins in natural cellular environment represents the next major challenge toward a molecular understanding of the complex and dynamic cellular events. Such a profile of molecular interfaces would also be invaluable as a tool in drug development by targeting crucial molecular interfaces. We are initiating such a proteomics project and focusing on the application of chemical cross-linking approach toward a systematic and global identification of protein-protein interfaces in complex systems such as the contractile machinery of muscle cells, the cytoskeletal filaments and organelles of nonmuscle cells. First, high-resolution gel electrophoresis and fractionation techniques are used to identify and index expressed proteins in skeletal and heart muscle tissues. Second, the neighboring relationship is defined by chemical crosslinking with a wide range of reagents. Third, the molecular interfaces of neighboring proteins are identified by isolating the crosslinked complexes, cleaving the proteins and isolating crosslinked peptides for protein sequencing with conventional chemical methods or by mass spectrometry. Fourth, the peptides at the interfaces will be synthesized and their interaction characterized further by capillary electrophoresis and biosensor techniques. A protein sequence database of molecular interfaces of proteins with known and unknown identities will be established to complement and enhance the utility of genome and proteome databases for basic and clinical research. We are evaluating the feasibility and integration potential of each steps of this multifaceted project. In the past year, we have succeeded in applying a fluorogenic crosslinker to facilitate the identification and purification of crosslinked peptides. The interface between a nebulin fragment and calmodulin has been mapped by such a crosslinker and the application of mass spectrometry to identify the peptide sequences that are crosslinked together. Novel folding patterns were also revealed by the intramolecualr crosslinking patterns. We are extending this study to other crosslinkers and more complex protein assemblies in motile systems.