Genomes to Life Contractor-Grantee Workshop III
February 6-9, 2005, Washington, D.C.
Genomics:GTL Program Projects
Oak Ridge National Laboratory and Pacific Northwest National Laboratory
9
High-Throughput Analysis of Protein Complexes in the Center for Molecular and Cellular Systems
Vladimir Kery*2 (vladimir.kery@pnl.gov), Dale A. Pelletier1, Joshua N. Adkins2, Deanna L. Auberry2, Frank R. Collart3, Linda J. Foote1, Brian S. Hooker2, Peter Hoyt1, Gregory B. Hurst1, Stephen J. Kennel1, Trish K. Lankford1, Chiann-Tso Lin2, Eric A. Livesay2, Tse-Yuan S. Lu1, Cathy K. McKeown1, Priscilla A. Moore2, Ronald J. Moore2, and Kristin D. Victry2
1Oak Ridge National Laboratory, Oak Ridge, TN; 2Pacific Northwest National Laboratory, Richland, WA; and 3Argonne National Laboratory, Argonne, IL
The Genomics:GTL Center for Molecular and Cellular Systems has implemented an integrated high-throughput “pipeline” for identifying the components of protein complexes from two bacterial species of interest to the DOE: Shewanella oneidensis, and Rhodopseudomonas palustris. This integrated pipeline uses two complementary approaches to isolate and identify protein complexes using affinity-tagged proteins—an endogenous approach, and an exogenous approach. In the exogenous approach, the targets of interest are cloned in a high-throughput procedure. Proteins are then expressed in E. coli and purified on Ni2+ agarose. Dialyzed purified tagged proteins are reattached to fresh Ni2+ agarose and exposed to lysate from the host cell of interest, thus forming protein complexes with host target proteins in vitro. In the endogenous approach, plasmids expressing the tagged protein of interest are transformed into the native host, and complexes are purified by tandem affinity purification using resins selective for the hexahistidine tag and the V5 epitope. In both approaches, the complexes are eluted from the beads under denaturing conditions, digested with trypsin and identified using automated liquid chromatography/electrospray tandem mass spectrometry in combination with SEQUEST™ analysis of the data. All main liquid handling procedures in protein and protein complex purification as well as MS sample preparation and MS measurement are automated. We are completing automation of data processing and bioinformatics. A laboratory information management system (LIMS) has been implemented for integrating all aspects of sample tracking, analysis and data flow. Between ORNL and PNNL, we have to date attempted expression of nearly 400 different genes as affinity-tagged fusion proteins, completed over 5000 “pulldown” experiments on these genes (including replicates) and identified several hundred different proteins in the pulldown samples. Distinguishing authentic interactors from non-specific interactors in the identified proteins has been an important aspect of this work. Our process was validated on a number of well known bacterial protein complexes; RNA polymerase, RNA degradosome, F1F0-ATP synthase, GroESL, and others. Some interesting findings on composition of other newly identified protein complexes are being further validated and investigated (e. g. peptidoglycan biosynthesis complexes involving genes Mur A, Mur C and Mur E of S. oneidensis etc.). While we have initially focused our efforts on R. palustris and S. oneidensis, the processes that we have developed are universally applicable to any organism of interest. Our aim is to scale up this process to provide a fully automated capability for high-throughput analysis of protein complexes with the goal of increasing throughput that would allow characterization of greater than 5,000 complexes per year.
* Presenting author
