Grant: Discovery Grants Program - Individual

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. .