Development of new and innovative strategies in biological mass spectrometry and proteomics

我的研究计划旨在开发蛋白质组学研究的创新技术和新方法，以发现各种生物系统中的新生化机制。蛋白质组学的最终目标是完全表征细胞中的所有蛋白质。从分析的角度来看，蛋白质组学是一个非常具有挑战性的问题，由于蛋白质组的复杂性，特别是在高等生物中。这是由于蛋白质的多种形式和它们在细胞中存在的宽动态范围。研究蛋白质组最有力的工具是质谱及其相关技术。质谱仪设计的最新进展为开发新方法以应对蛋白质组学挑战提供了新的和令人兴奋的机会。与过去一样，我们的研究计划将高度合作，并涉及生物学家，生物化学家和计算机科学家。迄今为止，蛋白质组学的主要策略是所谓的"自下而上"的方法，其中蛋白质被蛋白酶酶解以产生0.5至3KDa范围内的片段，这些片段适合于通过MS分析进行表征。从鉴定的肽片段推断蛋白质的身份。自下而上的蛋白质组学方法存在的问题之一是序列覆盖率低和翻译后修饰缺失。克服该问题的一种方法是使用不同的蛋白酶进行消化，但这导致仪器时间显著增加，并且难以以自动化和可靠的方式组装所有这些片段。我们将通过设计多蛋白酶消化的新方案来解决这两个限制。与我们的计算机科学家合作，我们将开发新的软件，用于组装所有肽片段，以获得完整的序列。这对于抗体及其药物缀合物的全面表征特别相关。然而，我们的目标是将这种方法扩展到复杂的蛋白质混合物。 在这些研究的同时，我们将使用新的MS技术进行“中间向下”测序，即创建和分析3 - 10KDa范围内的蛋白质片段。这些蛋白质片段将通过在非标准条件下的有限蛋白水解或可替代地通过靶向蛋白质中低频氨基酸的特异性化学切割来产生。这些中等大小的蛋白质片段将在注射到MS中之前通过新方法分离。序列（包括翻译后修饰）将通过最近可用的技术（如EthcD和UVPD）片段化获得。这也将刺激新的和强大的软件的开发，用于分析这些数据。 总之，我们建议开发新的概念和工具，以促进蛋白质组学的进步，这将影响生物学研究的几个领域。我们的研究计划的多学科性质将为研究培训提供一个刺激和肥沃的环境。.

My research program aims at the development of innovative technologies and new approaches in proteomics research to discover novel biochemical mechanisms in a wide range of biological systems. The ultimate goal of proteomics is to fully characterize all proteins in cells. From an analytical perspective proteomics is a very challenging problem due to the complexity of the proteome, especially in higher organisms. This is due to the many forms of proteins and the wide dynamic range in which they are exist in cells. The most powerful tool to study the proteome is mass spectrometry (MS) and its associated technologies. Recent advances in the design of mass spectrometers offer new and exciting opportunities to develop new approaches to tackle the challenges in proteomics. As in the past our research program will be highly collaborative and involve biologists, biochemists and computer scientists. To date the main strategy in proteomics is the so called "bottom-up" approach where proteins are enzymatically digested by a protease to yield fragments in the 0.5to 3KDa range that are amenable for characterization by MS analysis. The identity of the proteins is inferred from the identified peptide fragments. One the remaining problems in bottom-up proteomics is the poor sequence coverage and the missed post-translational modifications. One approach to overcome this problem is to use different proteases for the digestion but this results in significant increase in instrument time and difficulty in assembling all these fragments in an automated and reliable manner. We will address both these limitations by designing new protocols for the multi-protease digestion. With our computer scientist collaborators we will develop new software for the assembly of all the peptide fragments in order to get complete sequences. This is particularly relevant for the full characterization of antibodies and their drug conjugates. However our goal is to extend this approach to complex protein mixtures. In parallel to these studies we will use new MS techniques to perform "middle-down" sequencing, that is creating and analysing protein fragments in the range of 3-10KDa. These protein fragments will be generated by limited proteolysis in non-standard conditions or alternatively by specific chemical cleavage targeting amino acids of low frequency in proteins. These mid size protein fragments will be separated by new methods prior to injection in the MS. The sequences, including the post-translational modifications, will be obtained by fragmentation with recently available techniques like EthcD and UVPD. This will also spur the development of new and robust software for the analysis of these data. In summary we propose to develop new concepts and tools to promote advances in proteomics that will impact several areas of biological research. The multidisciplinary nature of our research program will provide a stimulating and fertile environment for research training. .