Protein Characterization Laboratory (PCL) offers various technologies to CCR investigators to characterize proteins and metabolites. The laboratory develops and applies state-of-the-art analytical technologies, primarily mass spectrometry, liquid chromatography, and Surface Plasmon Resonance (SPR), to advance the understanding of cellular function at the protein, proteomics, metabolite and metabolomic levels.
PCL engages in both short and long-term collaborations based on the need of each project. PCL is operated by Leidos Biomedical Research Inc. on behalf of NCI as part of the Frederick National Laboratory.
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.
Contact:
Thorkell Andresson, Ph.D.
301-846-7190
240-840-1383
andressont@mail.nih.gov