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

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

3 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.

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

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

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

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

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