Core Center Resources:

Background and Resources for Proteomics

Proteomics and Molecular Characterization – Andrew A. Quong, Ph.D., 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 for KCC investigators. These services are organized into three 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 (e.g. 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 (e.g. SILAC, ICAT and ITRAQ) as well as 18O labeling of tryptic peptides are being developed for KCC members.

Peptide synthesis services 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.

Equipment

• IPGphor isoelectric focusing unit (GE Healthcare) and an Ettan DALTsix gel box (GE Healthcare) for running the 1st and 2nd dimensions of 2D-PAGE gels, respectively.
• Typhoon 9400 Variable Mode Imager (GE Healthcare) for gel imaging.
• Ettan Spothandling Workstation (GE Healthcare) for robotic for gel processing.
• PBS2 SELDI-ToF (Ciphergen Biosystems)
• MALDI-ToF/Pro (GE Healthcare)
• 4800 MALDI-TOF/TOF (Applied Biosystems)
• ProteomeX Workstation (ThermoElectron). This platform is comprised of a Surveyor HPLC coupled to an LCQ Deca XP Plus Electrospray-ion trap Mass Spectrometer.
• HPLC (Agilent 1100) with automatic fraction collection.
• 4000 QTRAP (Applied Biosystems) coupled to a two-dimensional nano-flow HPLC (ABI TEMPO).
• Model 9050 PLUS Peptide Synthesizer (Applied Biosystems).
• Biacore 3000 Surface Plasmon Resonance Biosensor.
• Kinexa 3000 flow fluorimeter.
• XL-I Analytical Ultracentrifuge.
• Jasco J-810 UV/Vis Spectropolarimeter.
• Microcal VP Isothermal Titration Calorimetry System.

Proteomics – Leila Choe, Ph.D., DBI, UD

The proteomics and mass spectrometry core facility at the Delaware Biotechnology Institute benefits from significant experience in the performance of 2DE and shotgun proteomics experiments. The group was one 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. Equipment includes:

Equipment:

• Two AB4800 MALDI TOF/TOFs
• One AB4000QTrap with nano MDLC
• One AB2000QTrap with capillary LC
• One Thermo LCQDeca with capillary LC
• Robotic platforms for digest and sample cleanup
• LC MALDI capability

Services:

• Protein identification from gel-purified samples.
• Shotgun protoemics analysis using iTRAQ technology (after consultation).
• Post-translational modification analysis (after consultation).

Protein Production – Yu-Sung Wu, Ph.D., DBI, UD

The protein production core is funded by a COBRE (Center of Biomedical Research Excellence) grant from NIH (Abraham Lenhoff, Chemical Engineering, P.I.) 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. 

Mass Spectroscopy – John Dykins, Chemistry/Biochemstry, UD

The mass spectroscopy core facility is grouped into three areas; the first laboratory houses the high performance instruments for more detailed and precise analyses, a second lab houses three open-access user-friendly instruments that are available for researchers to use 24/7, and finally, a high-performance MALDI instrument is located in Dr. Murray V. Johnston’s Lab.

• Protein Sequencing : Micromass model Ultima Q Tof (Quadrupole-Time of Flight tandem mass spectrometer). This is an LC-MS-MS instrument primarily used to identify proteins by sequencing the products of a digested protein. Product molecular ions and subsequent collisional activation to generate fragments are automatic. Software generated or de novo peptide sequencing can be compared to protein databases for identification.
• Protein Digester : Bruker Daltonics model Proteineer DP This automated digest and prep station performs protein digests. Digest products can be automatically spotted to a MALDI sample plate or captured in MTP format for LC-MSMS analyses.
• GC-MS (Gas Chromatography-Mass Spectrometry) Agilent models 6850 GC and 5973 MS with autosampler. Used primarily for separation of small organic mixtures, incorporates a NIST library to aid identification.
• LC-MS (Liquid Chromatography-Mass Spectrometry) Thermo-Finnigan model LCQ using Electrospray Ionisation (ESI) with an integrated autosampler. Used for biological samples and polar organics. The instrument can be switched between column mode for mixture separations and loop injections for quick analyses of purer samples.
• MALDI (Matrix Assisted Laser Desorption Ionisation) Bruker Daltonics model Omniflex. Used primarily for biological samples to determine molecular weight and purity.

Cell Science – Robert Mason, Ph.D., Nemours

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 the University of Delaware and the University of New Mexico, the cell science core provides a seamless access to advanced imaging and proteomics facilities.

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