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Children's Hospital of Philadelphia Research Institute
Children's Hospital of Philadelphia Research Institute
3615 Civic Center Boulevard, ARC/806
Philadelphia, PA 19104
http://stokes.chop.edu/proteinSteven Seeholzer
Last Updated: 03/01/2012
The Hospital of Philadelphia Research Institute (CHOP) Protein and Proteomics Core Facility provides a variety of protein and proteomics services for investigators at CHOP, University of Pennsylvania, and outside institutions. These services include producing and characterizing proteins, investigating protein-protein interactions, and characterizing whole proteomes. Some services are provided on a user-operated, sign-up basis, while others are performed as full-service by the dedicated facility personnel. Protein expression services include recombinant protein production in both bacteria and insect cells. HPLC and FPLC equipment, columns and resins are available for a variety of purification needs. Endogenous fluorescence, circular dichroism, analytical ultracentrifugation, and surface plasmon resonance can be used to characterize the folding status of proteins and their interactions with small molecules and other proteins or nucleic acids. A wide range of proteomics experiments are possible. These include but are not limited to intact mass determination, post-translational modification analysis, protein identification, and targeted (e.g. co-immunoprecipitation) or comprehensive (e.g. protein expression profiling) proteome analysis. Proteome analysis may employ a number of different isotope-labeling strategies enabling quantitative estimates. Unique among regional cores is our ability to quantitatively analyze phosphoproteomes at a deep level. Under development are techniques for characterizing lysine acetylomes and glycoproteomes and use of our triple quadrupole mass spectrometer for quantitative peptide and protein analysis. Also, installation of our Orbitrap-Elite mass spectrometer (Dec 2011) with electron transfer dissociation allows us to consider top-down proteomics projects as well. Workflows for all the aforementioned analyses are unique to each project and can involve a range of multi-dimensional separation techniques coupled to the appropriate instrument.
Services are offerred outside of Children's Hospital of Philadelphia Research Institute
Laboratory:
The CHOP Protein and Proteomics core is located in a 2000 sq. ft. laboratory on the 8th floor of the Abramson Research Center. The lab contains all of the resources of the facility. Physical space comprises benches, 2 chemical hoods, a refrigerated Gem box, tissue culture room and three equipment rooms. The usual range of equipment needed for protein production and biochemical and cell biological experiments is available. The lab is also very well equipped with the more specialized instrumentation and computational capabilities necessary for proteomics experiments.
Major pieces of equipment in the lab Spectrometers Thermo LTQ-Orbitrap-Elite hybrid linear-Orbitrap-D20 with ETD (installed 2011/12); Thermo LTQ-Orbitrap XL hybrid linear-orbitrap (installed 2008/05) ; Waters Xevo TQS triple quadrupole with Trizaic ion source (installed Sources 2ea. New Objective PicoView dual column nano-ESI sources, ADVION NanoMate 96 well ESI source, Thermo micro-ion sources, HESI probe for Orbitrap Elite HERAsafe laminar flow hood, 2x New Brunswick InnOva4230 refrigerated incubator/shakers, New Brunswick C24KC refrigerated 3000 Surface Plasmon Optima XL analytical equipment 2ea. Eksigent nano LC2D Ultra HPLC systems with autosampler and CHIP-LC Nanoflex (dedicated to LTQ mass spectrometers), 2ea. Waters nanoAcquity 2D UPLC systems (one dedicated to triple quad MS, the other reserved for method development), Shimadzu HPLC system (5 to 400 uL/min), 2 ea. Beckman-Coulter Gold HPLC system (0.2 to 2 mL/min, currently dedicated to hydrophyllic interaction chromatography phosphoproteome projects), GE Amersham Biosciences AKTA FPLC system with wide range of chromatographic J-810 spectropolarimeter (with fluorescence, variable temperature, and automatic titration); Agilent 8453 UV-Vis spectrophotometer, Nanodrop BioSciences, Inc. Barocycler NEP2320 (for proteome sample MM400 ball mill grinder (for cryogenic tissue homogenization)
Computing Infrastructure & Software:
In addition to the computers dedicated to instrument control and data acquisition the lab is equipped with an additional 8 Windows XP workstations and servers. Four of these have fully licensed Thermo Xcalibur with Bioworks and SEQUEST database search software installed and two with Proteome Discoverer. Another 64 bit hex-core dual CPU Windows XP server is dedicated to database searching with Matrix Science MASCOT software. APEX Quantitative Proteomics Tool (J. Craig Venter Institute) is implemented for label-free quantification experiments. The SageN Sorcerer2 5 CPU LINUX cluster is dedicated to SEQUEST database searches and calculation of probability of phosphorylation site localization using the ASCORE algorithm. Another two hex-core dual cpu Windows XP workstations are used primarily for MaxQuant processing of SILAC quantification experiments as well as phosphoproteome experiments. The Institute for Systems Biology Trans-Proteomic Pipeline software is fully implemented on most of our workstations.
All laboratory computers are Ethernet connected and we have all the space we require (presently 10 Terabytes) on our institutional Storage Area Network for data archiving and secure redundant backup.
Office:
The director, Dr. Seeholzer, has a 100 sq. ft. office nearby the lab with a secretarial office beside that.
Staff: The protein and Proteomics core is staffed with an additional 2 Ph.D. level, and one each MS and BS level scientists comprising a collective 96 years of post-baccalaureate experience.
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Facility Details
About This Facility
Services and Equipment
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