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Spatial and Temporal Proteomics for Characterizing Protein Dynamics and Posttranslational Modifications


EMSL Project ID
42901

Abstract

The development of technologies for systems biology is considered a vital component of DOE energy and environmental missions for attaining a systems-level understanding of microbes and plants. While mass spectrometry-based proteomics technologies have played a significant role in whole proteome analyses, proteomic studies to date have primarily focused on global protein identification and changes in protein expression levels. The more important aspects of cellular regulation by spatial (subcellular) and temporal protein dynamics, and posttranslational modifications are still largely unstudied, especially in environmental eukaryotes such as plants, algae and fungi, due to the lack of effective analytical technologies. In response to this specific fundamental challenge, this project aims to develop a suite of quantitative proteomics technologies that enable spatially-resolved measurements of subcellular protein abundance changes and the dynamics of posttranslational modifications (e.g. glycosylation, phosphorylation, ubiquitination, and proteolytic processing) in environmental eukaryotes to gain understanding of the regulation of cellular function. Our vision is that such 'spatial and temporal' proteomics capabilities not only provide core value for current systems biology efforts, but that they also add unique datasets for refining gene models and genome annotation. The resulting unique layers of biological information significantly increase the knowledge base for a biological system under study and provide essential quantitative data for future predictive modeling efforts of that system. Our focus is on characterizing glycosylation, phosphorylation, ubiquitination, and proteolytic processing, which involves developing novel enrichment strategies for identifying glycopeptides, phosphopeptides, ubiquitinated peptides, and protein N-terminal and C-terminal peptides. To achieve spatially resolved proteomic measurements, we will further develop a 'universal' quantitative approach for subcellular proteomics that integrates subcellular fractionation with an isotope-labeled reference. The integration of subcellular fractionation, posttranslational modifications, and quantitative proteomics technologies establishes a general approach for enabling spatial and temporal proteomics. The effectiveness and utility of these technologies for biological applications will be demonstrated using filamentous fungus Aspergillus niger, an organism that plays an important role in biofuel production and global carbon cycling, to attain a better understanding of how its morphology is regulated. We anticipate that the unique suite of technologies will have broad application in diverse studies of microbial and plant organisms and in systems biology studies aimed at better understanding of cellular machineries.

Project Details

Start Date
2011-02-10
End Date
2014-02-23
Status
Closed

Team

Principal Investigator

Weijun Qian
Institution
Pacific Northwest National Laboratory

Related Publications

Guo J, AY Nguyen, Z Dai, D Su, MJ Gaffrey, RJ Moore, JM Jacobs, ME Monroe, RD Smith, DW Koppenaal, HB Pakrasi, and W Qian. 2014. "Proteome-wide Light/Dark Modulation of Protein Thiol Oxidation in Cyanobacteria Revealed by Quantitative Site-Specific Redox Proteomics." Molecular & Cellular Proteomics. MCP Epub ahead of print:, doi:pii: mcp.M114.041160
Guo J, MJ Gaffrey, D Su, T Liu, DG Camp, II, RD Smith, and W Qian. 2014. "Resin-assisted enrichment of thios as a general strategy for proteomic profiling of cysteine-based reversible modifications." Nature Protocols 9(1):64-75. doi:10.1038/nprot.2013.161
Kim JS, Z Dai, UK Aryal, RJ Moore, DG Camp, II, SE Baker, RD Smith, and W Qian. 2013. "Resin-assisted Enrichment of N-terminal Peptides for Characterizing Proteolytic Processing ." Analytical Chemistry 85(14):6826-6832. doi:10.1021 /ac401000q
Zhang H, F Yang, W Qian, RN Brown, Y Wang, ED Merkley, JH Park, ME Monroe, SO Purvine, RJ Moore, L Shi, JK Fredrickson, L Pasa-Tolic, RD Smith, and MS Lipton. 2011. "Identification of c-Type Heme-Containing Peptides Using Non-Activated IMAC Resin Enrichment and Higher-Energy Collisional Dissociation." Analytical Chemistry 83(19):7260-7268. doi:10.1021/ac2000829.