The Protein Core Facility (PCF) at CHOP addresses the overwhelming need for the technological resources to identify, produce and characterize new proteins. The PCF has four operating modules available: Protein expression, Characterization, Protein-Protein Interactions, and Proteomics. While many of our services are routine, we are also committed to collaborating with the CHOP community to overcome difficult research obstacles. We strongly encourage CHOP researchers to bring their research needs to our attention, so that we can try and provide solutions.
PROTEIN EXPRESSION AND PURIFICATION: With the advent of high-throughput DNA sequencing technology and the identification of an increasing number of genes and ORF's, there is an increasing demand for fast and efficient protein production. While some proteins can be expressed in bacteria, other proteins require other expression systems. These might include proteins that require certain chaperones for folding, are toxic to bacteria, contain multiple disulfides, have glycosylation sites, or require other extensive post-translational modifications for their activity. The PCF provides protein production in insect cells (sf9, sf21) using the Baculovirus expression system. We also offer assistance with expression of bacterial proteins or in vitro translation. For purification of the recombinant proteins we offer affinity, ion exchange and size exclusion chromatography.
PROTEIN CHARACTERIZATION: In addition to offering expression services, the PCF provides analyses such as mass spectrometry, gel electrophoresis, analytical and/or preparative chromatography, spectroscopy (fluorescence, circular dichroism, ultraviolet-visible), ultracentrifugation, and phosphorimaging. We also offer assistance in refolding recombinant proteins.
PROTEIN-PROTEIN INTERACTION: The identification of interacting protein partners is fundamental to the understanding of signaling networks and drug-discovery. The PCF has a number of tools for identifying new protein-protein interactions. Options include the SELDI protein-chip analyzer, analytical ultra-centrifugation, and Surface Plasmon Resonance (Biacore). We are also implementing, in collaboration with Invitrogen, an new protein-interaction array service. Once new partners are detected, we offer protein identification using mass spectrometry. Please consult with Steven Seeholzer for more information regarding these techniques.
PROTEOMICS: The PCF is committed to providinging the latest technologies in mass-spectrometry, multi-dimensional chromatography, protein-arrays etc. Utilizing these tools will enable us to uncover more new protein sequences and protein-protein partners so that we might have a better understanding of proteins within specific networks and at the center of pediatric diseases. This knowledge will be critical if we are to successfully understand and treat medical problems that arise at the biochemical level.
"GE Amersham Biosciences AKTA FPLC (plus a wide range of chromatographic media)"
"Thermo Orbitrap Elite mass spectrometer with ETD with new objective digital picoview nano-esi source and Eksigent nano 2d ultra uplc"
"Thermo Q Exactive HF mass spectrometer with Dionex nano ultimate 3000 UPLC"
"Waters H-class Acquity UPLC standalone"
"Waters NanoAcquity UPLC standalone"
"Waters Xevo TQ-S triple quadrupole mass spectrometer with Waters Nanoacquity UPLC"
"Basic analysis of proteomics data using sequence database search algorithms and visualization software"
"Advanced bioinformatics analyses including data processing and visualization, network and pathway analysis, and identification of statistically significant protein changes"
• Gel-purified protein identification
• Intact protein MW determination
"Protein purification: affinity, anion exchange, and size exclusion chromatography"
"Provide access to: circular dichroism (CD), fluorescence spectroscopy, analytical ultracentrifuge, FPLC protein purification equipment (AKTA), apparatus for cryogenic cell disruption, and UV-visible plate readers"
"Proteomics: whole proteome, sub-proteome, interactome, surfaceome, and membrane proteomics
Post-translational modification analysis: phosphoproteomics, ubiquitylomics, lysine acetylomics, and glycoproteomics
Targeted proteomics: multiplexed Selective Reaction Monitoring (SRM)
Quantitative proteomics: label-free, SILAC, AQUA peptide, SILAM, SRM, and etc."
"Production and distribution of recombinant TAT-Cre recombinase , Taq polymerase, and rMOG"
• Top-down and bottom-up search capabilities
• Modification Fine Control™ for fast and accurate identification, even with 20+ types of modification
• Wildcard Search™ for unknown modifications
• Intact glycopeptide identification
• Scoring models for all types of fragmentation spectra: CID, TOF-TOF, QTOF HCD, ETD/ECD, and EThcD
• Sequence variant search and modification site localization"
"MaxQuant is a quantitative proteomics software package designed for analyzing large-scale mass-spectrometric data sets. It supports all main labeling techniques like SILAC, Di-methyl, TMT and iTRAQ as well as label-free quantification. Also measured spectra of various vendors - Thermo Fisher Scientific, Bruker Daltonics, AB Sciex and Agilent Technologies - can be processed using MaxQuant."
"Perseus is a software package for shotgun proteomics data analysis, which helps researchers to extract biologically meaningful information from their MaxQuant output such as posttranslational modifications. Perseus is not only capable to analyze proteomics data, also systems-wide analyses are possible combining data from all omics sources using various bioinformatics tools. The software contains numerous statistical methods like t-test, clustering, enrichment analysis and principal component analysis. Also normalization, imputation, motif identification and various illustration options like volcano plots, scatter plots, histograms and heatmaps are covered in Perseus."
"Visualize and validate complex MS/MS proteomics experiments
• Compare samples to identify biological relevance.
• Identify regulated isoforms and protein PTMs.
• Drill down into spectrum details and counts.
• Identify proteins intuitively and confidently.
• Create comprehensive lists of target proteins.
• Classify proteins by molecular function or organelle.
• Harness high through-put batch processing."
"SEQUEST correlates uninterpreted tandem mass spectra of peptides with amino acid sequences from protein and nucleotide databases. SEQUEST will determine the amino acid sequence and thus the protein(s) and organism(s) that correspond to the mass spectrum being analyzed."
"Skyline is a freely-available and open source Windows client application for building Selected Reaction Monitoring (SRM) / Multiple Reaction Monitoring (MRM), Parallel Reaction Monitoring (PRM - Targeted MS/MS), Data Independent Acquisition (DIA/SWATH) and targeted DDA with MS1 quantitative methods and analyzing the resulting mass spectrometer data. It aims to employ cutting-edge technologies for creating and iteratively refining targeted methods for large-scale quantitative mass spectrometry studies in life sciences."
"The Trans-Proteomic Pipeline (TPP) includes all of the steps of the ISB MS/MS analysis pipeline, after the database search."
"X! Tandem open source is software that can match tandem mass spectra with peptide sequences, in a process that has come to be known as protein identification."