The Children's Hospital of Philadelphia Research Institute (CHOP) Proteomics Core Facility (PCF) 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 bacteria. HPLC and FPLC equipment, columns and resins are available for a variety of purification needs. Endogenous fluorescence, circular dichroism, and analytical ultracentrifugation can be used to characterize folding status of proteins and their interactions with small molecules and other proteins or nucleic acids.
A wide range of state-of-the-art 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 measurements on our high resolution discovery platforms (Orbitrap-Elite and Q-Exactive HF mass spectrometers) for deep proteome coverage or multiplexed targeted quantification on our triple quadrupole mass spectrometer (Xevo-TQS). Unique among regional cores is our ability to quantitatively analyze proteomes, phosphoproteomes, ubiquitylomes, and lysine acetylomes at a deep level through a process of serial enrichment. Under development are refinements of statistical and bioinformatic analyses of proteomic results. It is also possible to tailor sensitive and specific methods for multiplexed protein quantification according to the investigator’s needs. Workflows for all the aforementioned protein and proteome analyses are unique to each project and can involve a range of multi-dimensional separation techniques coupled to the appropriate mass spectrometer. For all but the most routine services and/or first-time submissions, a strong interaction with PCF staff and consultation with Dr. Seeholzer is encouraged throughout all phases of a project: planning, execution, and data reduction/interpretation.
"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."