Expertise and Technology

Protein and proteomics analysis:   

The PCL specializes in the identification and characterization of individual proteins, global proteomics analysis from variety of different starting materials, targeted and global analysis of macromolecular post-translational modifications, characterization of entire protein complexes and other macromolecular interactions, protein cross linking mapping and intact protein mass analysis.
• Protein identification and characterization from different sample types using variety of proteases, separation, and mass spectrometry methods.
• Characterization of complex proteomes (e.g., serum, tissue, cell lysates)
• Global comparative proteomic quantitation using TMT, SILAC, dimethyl labeling and/or LFQ
• Identification and characterization of protein modifications – PTM site mapping
• Global comparative Posttranslational Modification (PTM) quantitation using TMT, SILAC, dimethyl labeling, LFQ and affinity enrichment for modifications such as phosphorylation, ubiquitination, and acetylation
• MHC I and II peptide identification and global immunopeptidome analysis.
• Protein complex isolation and characterization 
• Validation of protein-protein interaction
• Identification of protein-nucleic acid (DNA, RNA) interaction
• Protein contact mapping using mass spectrometry cleavable cross linkers
• Intact protein analysis, protein-drug conjugate analysis
• Analytical protein and peptide fractionation using different methods such as size exclusion chromatography (SEC), reverse phase (RP) and ion exchange (SCX).

Available instrumentation includes Orbitrap Eclipse and Orbitrap Fusion Tribrid mass spectrometers and Q Exactive HF, coupled to nano flow liquid chromatography HPLCs.

Metabolite and metabolomics analysis:  

The laboratory specializes in identification and quantification of small molecules and metabolites utilizing targeted MRM assays in complex biological samples. Conducts analysis of key cellular metabolite pathway using targeted MRM panel assays.  And carries out untargeted metabolomics profiling in variety of biological matrixes.
• Relative and absolute quantification of metabolites using MRM based targeted assay incorporating isotopic standards for increased identification and quantification accuracy 
• Develops and establishes custom targeted metabolite assays for small molecules and metabolites.
• Analysis of entire metabolic pathways using targeted MRM panel assays covering key pathways involved in the metabolism of cells and cancer.
• Over 150 available targeted MRM assays spanning many of the key cellular metabolite pathways such as the glycolysis, pentose phosphate, TCA, oxidative phosphorylation, amino acids and numerus derivatives, nucleotides (purine and pyrimidine) and short- medium and long chain fatty acids.    
• Identification and quantitation of steroid hormones (estrogen, progesterone, and androgen) in different biological matrixes
• Identification and quantification of modified nucleotides (DNA and RNA).
• Pharmacokinetics (PK) analysis, develop quantitative MRM assay for known and novel drugs and small molecules.  Analyzing drug metabolites in relevant biofluids and tissues.
• Purity determination and structural analysis of small molecules
• Isotope tracing to monitor metabolite pathway activities
• Global untargeted metabolomics analysis.  Samples are analyzed under two different LC conditions using Reverser Phase chromatography (RP) and Hydrophilic interaction chromatography (HILIC) ran under two different pH conditons (pH3 and pH9), in both negative and positive ionization mode.  Spiked in isotopic standards, water blanks, pool samples and reference samples are used for data filtration, normalization, and QC assessment.

Available instrumentation includes two high resolution orbitrap Q Exactives and four triple quadrupole (3x TSQ Quantiva and 1x TSQ Altis from Thermo) mass spectrometers coupled to high flow HPLC.

Surface Plasmon Resonance (SPR):

The PCL characterizes interactions between variety of molecules such as, proteins – protein, protein – small molecule, protein – nucleic acids and protein – peptide interactions, using Surface Plasmon Resonance.  
• Measuring the binding affinity (Kd) between two molecules.
• Estimation of the association (On) and dissociation (Off) rate in real time.
• Develop and establish experimental conditions as needed to measure the binding affinity and the kinetic constant between two molecules

Available instrumentation Biacore T200.

User Guidelines

PCL services are available to all CCR Investigators.  Excess capacity can be made available to other NIH Investigators on a case-by-case basis. To request services from this CCR dedicated core facility, you must submit your requisition through NAS.

Established Technologies

• Clone construction for protein expression
• Expression in microbial, insect, and mammalian cells
• Small-scale parallel expression and purification testing
• Protein purification from micrograms to grams

User Guidelines

PEL services are available to all CCR investigators. To request services from this CCR-dedicated core facility, you must submit your requisition through NAS. Excess capacity can be made available to other NCI Investigators on a case-by-case basis.

Expertise

• We develop, validate and perform quantitative biomarker studies in clinical trials of investigational oncology agents. We cover areas including pharmacodynamics studies and predictive biomarker analysis
• Our capabilities include designing correlative studies, developing and validating assays for biomarker analysis, implementing them in clinical correlative studies, and analyzing and reporting data.
• We also have preclinical programs to identify effective novel therapies in sarcoma models, and to discover biomarkers predictive of responses.
• We collaborate with researchers clinical studies via the participation of drug trial protocols. We are also involved in preclinical studies in biomarker discovery and validation.
• We are in a very unique position at NCI to develop new and highly quantitative biomarker assays for clinical trials.

Research areas

• Oncology clinical research
• Biomarker analysis in oncology drug trials
• Pharmacodynamics
• Predictive biomarkers
• Preclinical investigations
• Clinical correlative Studies
• Translational oncology research

Quantitative assay coverage for clinical studies

• Cytokines
• Cell signaling pathway markers
• Angiogenic agents
• Growth factors
• Immunoassay development

User Guidelines

The Molecular Targets Core is open to all CCR clinical investigators.

Instrumentation

• Thermo Exploris480 Orbitrap mass spectrometer with Dionex UltiMate 3000 nanoUPLC
• Thermo Orbitrap Fusion mass spectrometer with Dionex UlitMate 3000 nanoUPLC
• SCIEX X500B Q-TOF mass spectrometer with Exion UPLC
• Agilent 6495 Triple Quadrupole mass spectrometer with Agilent UPLC
• Thermo Q Exactive Plus mass spectrometer with Dionex UltiMate 3000 UPLC
• LEAP automated liquid handling system for hydrogen/deuterium exchange mass spectrometry
• Thermo iCAP-Q ICP-MS mass spectrometer

Established Technologies

Mass Spectrometry

• Characterization of protein or RNA interactomes
• Identification of proteins in complexes, organelles, subcellular fractions
• Global protein quantitation (label-free, SILAC, TMT/iTRAQ, reductive dimethylation)
• Characterization of sites of post-translational modification, including phosphorylation, acetylation, methylation, and ubiquitination
• Targeted quantitation of proteins
• Cross-linking mass spectrometry
• Limited proteolysis mass spectrometry
• ICP-MS analysis of inorganic atoms and compounds
• Determination of accurate mass of intact proteins

Developing Technologies

Mass Spectrometry

• Quantitation of RNA nucleosides
• Hydrogen-deuterium exchange mass spectrometry for studying protein flexibility and dynamics of conformational change in different conditions or upon interaction with a ligand

User Guidelines

Projects must be approved by resource advisory committee through an online proposal process.

Established Technologies

Automated capillary immunoassay system – Simple Western technology

• High-resolution MW (size-based) or isoelectric-focusing (IEF, charge-based) separation, followed by target-specific immunoprobing to profile proteins and respective post-translational modification isoforms
• Nanogram level of protein analysis for extremely small and precious samples, e.g. stem cells, primary cells, micro-dissected samples, fine needle aspirates, non- or minimally- invasive collected patient specimens etc.
• Provides precise and accurate measurement of protein expression level and activation status, highly quantitative data, good assay sensitivity, dynamic range and reproducibility
• About three hundred assays covering key signaling pathways have been established and successfully applied for functional proteomic research, will validate assays for other targets as needed
• Assay readiness for bench to bed transfer

In-solution multiplex ELISA assays – Luminex xMAP Technology

• Up to 50-plex simultaneous protein target analysis with small sample consumption
• Analysis of cytokine, metabolite, immune response, serum/plasma biomarkers etc.
• Clinical applicable assay performance

Immunofluorescence imaging platform – CODEX (CO-Detection by indEXing) technology

• Provides full spatial context and is not limited to just regions of interest (ROI)
• Provides single-cell resolution down to 600 nm or 250 nm depending on microscope objective used (20X and 40X respectively)
• Single-step staining and gentle fluorophore removal preserves the sample for ROI analysis
• Simple, benchtop fluidics system that is cost-effective and simple to implement in any research lab

Developing Technologies

Single-cell western system

• Western-based protein analysis on 1000-2000 single-cells in parallel
• Measures proteins hard-to-detect by FACS, such as isoforms, phosphorylated proteins, transcription factors, intracellular proteins etc.
• Single cell signaling profiling and population heterogeneity study

We actively evaluate, develop, acquire and implement innovative protein analysis technologies to accommodate the demands from the CCR/NCI/NIH community.

User Guidelines

• Projects are collaborative and subject to the approval of resource advisory committee.
• Investigators are expected to share the cost of certain consumable reagents, for which CCR investigators are eligible for a 50% subsidy from the CCR Office of Science and Technology Resource (OSTR).
• Staff are available to assist on project design, data interpretation and study milestone evaluation, as well as general consultation on proteomic analysis questions.