PROTEOMICS (Under Construction)



Proteomics and molecular characterization - Andrew A. Quong, PhD, KCC, TJU.
The mission of the Proteomics and Molecular Characterization Shared Resource is to provide a wide range of state-of-the-art protein analysis services. These services are organized into 3 primary areas: Proteomics and Mass Spectrometry, which provides experimental data and analysis of samples with the goal to identify and characterize proteins on a global scale (eg, expression levels, protein complexes, post-translational modifications). Protein identification is accomplished with mass spectrometry. Currently, 2-Dimensional PAGE analysis is the predominant platform used to define differences in protein expression between experimental samples, but we have made significant efforts to expand the services to include multidimensional chromatography-based approaches. In our estimation, this will be the preferred workflow for many investigators especially those involved with clinical studies because the HPCL analysis requires significantly less starting material and because of less variability compared with the gel based separations. Commercial kits for isotopic labeling (eg, SILAC, ICAT and ITRAQ) as well as 18O labeling of tryptic peptides are being developed for KCC members. Peptide synthesis services are available for KCC investigators at very reasonable costs. In addition to routine peptides, the resource has successfully synthesized many specialized peptides. Molecular Interactions provides access to instrumentation for characterizing protein/ligand and nucleic acid/ligand interactions. These instruments include a Biacore 3000 SPR biosensor, a Kinexa 3000 flow fluorimeter, an XL-I analytical ultracentrifuge, a Jasco J-810 UV/Vis spectropolarimeter and a MicroCal VP isothermal titration calorimetry system. In addition to the services contained within these Divisions, the shared resource provides users access to several larger pieces of shared equipment, including the Typhoon 9400 and 2 mass spectrometers.



Proteomics - Leila Choe, PhD, DBI, UD.
The proteomics and mass spectrometry core facility at DBI benefits from significant experience in the performance of 2DE and shotgun proteomics experiments. The group was 1 of the first laboratories to deploy TOF/TOF methods, was the first academic group to use 4plex iTRAQ methods, and the first laboratory to use 8plex iTRAQ methods. Services offered relate to protein identification from gel and solution based samples, shotgun analysis using iTRAQ labeling, and post-translational modification analysis after consultation. Other approaches, including 2DE analysis, are available after consultation. Instrumentation includes: 2 AB4800 MALDI TOF/TOFs; 1 AB4000QTrap with nano MDLC; One AB2000QTrap. with capillary LC; 1 Thermo LCQDeca with capillary LC; Robotic platforms for digest and sample cleanup; and LC MALDI capability. Services include protein identification from gel-purified samples; shotgun proteomics analysis using iTRAQ technology and post-translational modification analysis (after consultation).



Protein production - Yu-Sung Wu, PhD, DBI, UD.
The Protein Production Core is funded by a COBRE (Center of Biomedical Research Excellence) grant from NIH. Dr. Yu-Sung Wu, core director, works closely with multidisciplinary research groups to make optimum use of the protein production facilities system for specific research projects. This facility is designed for large scale protein expression and purification, and is equipped with: Fermentor - New Brunswick Scientific BioFlo 4500 (22L capacity for bacteria and yeast cell growth); Bioreactor - New Brunswick Celligen Plus (7.5L capacity for insect and mammalian cell growth); High Pressure Cell Homogenizer - Avestin EmulsiFlex C5. This instrument is designed for processing large volume cell lysate; High Capacity Centrifuge Rotor - Beckman Coulter JLA 8.1000 for cell culture harvest; High Performance Liquid Chromatography - GE AKTA Explorer 100 and Purifier 10. The unique features of the AKTA system include (1) a single platform for large- and small-scale protein/peptide purification; (2) a wide variety of commercial chromatographic media and/or columns available; and (3) user-friendly software to customize method development. Analytical Ultracentrifuge - The facility also features a Beckman Coulter XL-I analytical ultracentrifuge, which can characterize a variety of biophysical properties of macromolecules such as molecular weight, sedimentation coefficients, diffusion coefficients, equilibrium constants, and stoichiometry.



Cell science - Robert Mason, PhD, Nemours/AIDHC.
The Cell Science Core is a cell culture and protein analysis facility. The core is under the direction of Dr. Robert Mason and supported by Lisa Glazewski, BS. The in-house cell biological services include EBV-cell transformation, establishment of embryonic stem cells, and preparation of primary cell cultures. The in-house protein analysis services include protein separation technologies, western blotting, 2D gel electrophoresis, and differential protein expression analysis. Individual proteins can be identified by extraction, protease digestion and arraying for mass analysis by MS/MS. In association with UD and the University of New Mexico, the cell science core provides a seamless access to advanced imaging and proteomics facilities.