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Florida Faculty and Professional Staff
Jennifer Ann Busby
Assistant Professor
Department of Molecular Therapeutics
TSRI - 2004
Joint Appointments Associate Scientific Director II , Translational Research Institute
Education
Research Focus
Proteomics core facility at Scripps Florida
The Proteomics Facility at Scripps Florida has general HPLC and mass spectrometry capabilities for protein and peptide identification using nanoflow chromatography followed by MS and MS/MS analysis. Current instrumentation includes a Thermo Electron Finnigan LTQ ion trap mass spectrometer which is used predominantly for protein and peptide identification and a Thermo Electron Finnigan TSQ Quantum triple quadrupole mass spectrometer which is used for relative quantitation experiments. Each mass spectrometer is interfaced to nano-flow ESI sources and capillary HPLC columns. Expertise exists for the identification and mapping of post-translational modifications on proteins and peptides. As a core facility the Proteomics group is responsible for providing collaborative mass spectrometry services to other Scripps Florida faculty. These collaborative projects will necessarily drive technology development in the lab. Current efforts in technology development include on-line sample cleanup and digestion, relative quantitation of serum proteins and creation of software for differential analysis of mass spectrometry data.
During the lifetime of a protein it can have several locations and duties within the cell. Location, current status and three dimensional structures of proteins are all influenced by static and dynamic chemical modifications that occur to proteins after translation. These modifications are varied from small methyl groups, which have been shown to be a part of the histone codes, to large lipid and glycosylation modifications which act as cellular markers and signaling molecules. Mass spectrometry has the unique advantage of being able to detect both the small and large changes in mass that occur based on these modifications.
Phosphorylation is one post-translational modification that receives attention due to its important role in signaling pathways. Unfortunately phosphorylation is still relatively rare and enrichment techniques are needed to identify sites of phosphorylation. In the core facility here at Scripps Florida we use Immobilized Metal Affinity Chromatography (IMAC) to enrich complex samples for phosphorylated peptides. This mapping of phosphorylation sites has been used to identify kinase and phosphatase activities as well as determine differential protein binding affinities based on modification site.
Selected References
Roig,J., Groen,A., Caldwell,J., Avruch,J.(2005) Active Nercc1 protein kinase concentrates at centrosomes early in mitosis, and is necessary for proper spindle assembly. MolBiol Cell Aug 3, Epub (in press), 2005.
Hogan,K.T., Sutton,J.N., Chu,K.U., Busby,J.A.C., Shabanowitz,J., Hunt,D.F., Slingluff,C.L. Jr. Use of Selected Reaction Monitoring Mass Spectrometry for the Detection of Specific MHC class I Peptide Antigens on A3 Supertype Family Mambers. Cancer Immunol Immunother 54:359, 2005.
Stover,D.R, Caldwell,J., Marto, J.M., Root,K.R., Mestan,J., Stumm,M., Ornatsky,O., Orsi,C., Radosevic,N., Liao,L., Fabbro,D., Moran,M. Differential Phosphoprofiles of EGF and GFR Kinase Inhibitor-Treated Human Tumor Cells and Mouse Xenografts. Clin Prot J. 1: 69, 2004.
Thompson,L.W., Hogan,K.T., Caldwell,J.A., Pierce,R.A., Hendrickson,R.C., Deacon,D.H., Settlage,R.E., Brinkerhoff,L.H., Ehgelhard,V.H., Shabanowitz,J., Hunt,D.F., Slingfluff,C.L. Jr. Preventing the spontaneous modification of an HLA-A2 restricted peptide at an N-terminal glutamine or an internal cysteine residue enhances peptide antigenicity. J.Immunother. 27(3):177, 2004.
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