CCR Protein Characterization Laboratory (PCL)

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 identification and characterization from different sample types using a 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).
• 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 glycolysis, pentose phosphate, TCA, oxidative phosphorylation, amino acids and numerous 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 matrices
• 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 conditions (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.
• 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

Protein and proteomics analysis:

The PCL specializes in the identification and characterization of individual proteins, global proteomics analysis from a 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.

Metabolite and metabolomics analysis:

The laboratory specializes in the identification and quantification of small molecules and metabolites utilizing targeted MRM assays in complex biological samples. Conducts analysis of key cellular metabolite pathways using targeted MRM panel assays. And carries out untargeted metabolomics profiling in a variety of biological matrices.

Surface Plasmon Resonance (SPR):

The PCL characterizes interactions between a variety of molecules such as proteins – protein, protein – small molecule, protein – nucleic acids, and protein – peptide interactions, using Surface Plasmon Resonance.