Cyanobacteria Membrane Biology Grand Challenge: Systems Analysis of the Dynamics of Membrane Architecture, Composition, and Function- Proteomic, Metabolomic, and Metallomic Characterization
EMSL Project ID
16720
Abstract
The strain Cyanothece sp. ATCC 51142, isolated from benthic waters off the coast near Galveston, Texas, is a unicellular strain approximately 4-7 μm in diameter along the major axis. This organism, which fixes nitrogen at very high rates, has developed a type of temporal regulation in which N2 fixation and photosynthesis occur at different times throughout a diurnal cycle, including very high levels of CO2 fixation and storage during light cycles for use in dark cycles. These aspects of carbon and nitrogen storage make Cyanothece almost unique among well-studied cyanobacteria and make it a key candidate for broad characterization. The analysis of gene products and metabolism in Cyanothece can provide tremendous information about how the photosynthetic unicell compensates for this complex multi-tasking effort.This project is directed towards developing approaches and methods for the rapid detection of large numbers of peptides and metabolites in an extremely broad manner that is highly sensitive (e.g., able to detect low level species), quantitative, and is facilitated by high-throughput measurements. In addition, we will develop procedures and techniques for global metallomic characterization of Synechocystis 6803 and Cyanothece 51142 organisms. In this project, we focus on developing methods based upon the use of high-resolution capillary liquid chromatography combined with either Fourier transform ion cyclotron resonance (FTICR) or high mass accuracy time-of flight (TOF) mass spectrometry (MS) to increase the comprehensiveness, quantitation and throughput of the proteomics and metabolomics analyses, and leverage the significant separations and mass spectrometry capabilities originally developed at PNNL in the Biological Systems Analysis and Mass Spectrometry group.
We will apply various MS/MS methods in combination with accurate mass and LC elution time information to initially identify peptides and define “tags” so that subsequent measurements can be made without the need for routine MS/MS analyses. A similar approach will be taken for cataloging and tracking, initially structurally-unidentified, metabolites. Using mass and time (MT) tags, broad proteome and metabolome measurements can be made with much higher throughput. Under the Specific Aims of the project we will: (1) develop approaches using pulse chase stable isotope labeling to quantitatively measure protein turnover rates and dynamics in time-course studies in proteomic methods using both Synechocystis 6803 and Cyanothece 51142 as model systems, (2) Develop procedures and techniques for tracking large numbers of metabolomic features from Synechocystis 6803 and Cyanothece 51142 in time-course studies encompassing the light and dark cycles. This includes improvements to existing software designed to deisotope and display mass spectral data, and development of novel software to address the specific challenges presented by global metabolomics datasets. In addition, we will begin to structurally characterize any features displaying interesting behavior (i.e. dramatic increase or decrease in concentration) through targeted MS/MS studies and other means, if necessary (eg. NMR, chemical derivatization).and (3) develop procedures and techniques for global metallomic characterization of Synechocystis 6803 and Cyanothece 51142 organisms.
Project Details
Project type
Grand Challenge
Start Date
2005-12-16
End Date
2011-12-18
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
Dynamic proteomic profiling of a unicellular cyanobacterium CyanotheceATCC51142 across light-dark diurnal cycles
Aryal UK, J Stockel, RK Krovvidi, MA Gritsenko, ME Monroe, RJ Moore, DW Koppenaal, RD Smith, HB Pakrasi, and JM Jacobs. 2011. "Dynamic proteomic profiling of a unicellular cyanobacterium Cyanothece ATCC51142 across light-dark diurnal cycles." BMC Systems Biology 5:Article No. 194. doi:10.1186/1752-0509-5-194