GTL User Facilities Facility II Whole Proteome Analysis

GTL User Facilities Facility II: Whole Proteome Analysis Michelle V. Buchanan

Genomes to Life User Facilities for 21 st Century Biology Facility II Whole Proteome Analysis l Measure proteome and metabolites under well- defined conditions l Gain first insight into function of proteins l what pathways and processes are present under what conditions l regulatory network structure and connectivity for individual processes Facility III Facility IV 2

GTL Facility II Whole Proteome Analysis Key capabilities will include: • • • Growth of organisms under controlled conditions High-throughput approaches for sample preparation prior to analysis. High throughput techniques for the identification and quantification of proteins, metabolites. New computational tools for interpretation and modeling whole proteome data Databases and tools for interpreting, archiving, and disseminating data and models to the greater biological community. 3

Genome-scale, comprehensive determination of microbial proteomes will require high throughput approaches for sample preparation and analysis l Novel cell cultivation l Microsample handling l On-line analysis of metabolites l Single cell analysis l Robotics and automation l Imaging l Chip expression assays l Computation and Informatics MS of 3 modifications of a single protein 2 D Gel of intact proteins 4 2 D representation of LC/MS data from shewanella

Deinococcus radiodurans Proteome l BER pilot project developed MS-based approach for proteome analysis using FTICR and accurate mass tags (AMTs) l Permits proteome to be rapidly identified l Automated data collection and interpretation 83% of ORFs predicted by genome sequence detected by MS Protein Synthesis Hypothetical Amino Acid Biosynthesis Purines, Pyrimidines, Nucleotides, Nucleosides Energy Metabolism % Coverage Transport and Binding Proteins DNA Metabolism Conserved Hypothetical Biosynthesis of Cofactors 5

Protein Analysis of Whole Cells on Microfluidic Devices (-) high voltage emulsifier waste cells lysis + injection separation channel (+) high voltage Note: arrows depict direction of flow. J. M. Ramsey, et al 6

Impact of Facility II l High fidelity data openly accessible to enable scientific studies l Economy of scale l Critical base information for Facilities III and IV • • • Proteome database Models of pathways, processes and regulatory networks Empower the general microbiological and biological communities 7

Facility II Whole Proteome Analysis Gene Sequence Microbe Growth Metabolite Analysis Protein Processing Mass Spectrometry Data Interpretation/ Archival, modeling Validation High validity data resource for biological community Facility IV Cellular Systems Facility I Reagents Facility III Complexes 8