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Core Facilities

Cancer Proteomics

Services

Services

The Cancer Proteomics Core offers a comprehensive array of services pertaining to design, performance and analysis of proteomic and metabolomic studies with special emphasis on clinical samples and expertise in cancer biology and clinical cancer research. The core combines personalized project consultation and study design with comprehensive individualized bioinformatics support for data analysis and new software development.

Consultation is a major service of the facility. Initial consultation occurs at the project planning stage. Staff provides advice on study design, sample size, sample collection and handling and considers questions of clinical and biological relevance to cancer investigation. Close interactions continue throughout the project execution and analysis/interpretation phases. Consultation on all aspects of the project is particular critical for proteomics as workflows and sample processing for each project differ. To implement the optimal proteomics strategy it is crucial to understand the project and the anticipated results in detail. As a consequence, many hours of consultation are provided on project design, project adjustment based on results coming in and, most importantly, analyzing and performing systems biology on the data. The Facility Director or Co-Director participate in most consultations and are kept up-to-date on all ongoing projects by staff.

The core is well equipped with a broad array of state-of-the- art analytical instrumentation for proteomic studies, which include an AB/Sciex 4800 MALDI-TOF/TOF mass spectrometer, a Thermo LTQ Orbitrap XL-ETD mass spectrometer, an AB/Sciex 5500 QTRAP mass spectrometer, various HPLC and NanoLC systems, a Beckman Biomek FX Robotic system, a Perkin Elmer ScanArray Express HT Laser Scanner, as well as state-of-the- art computational platforms that host our website (www.bidmcgenomics.org), all the databases and bioinformatics and statistical analysis tools. The instrumentation is operated by the highly trained technical staff of the core laboratory. The Core has optimized all services and continues to improve on the established methods by carefully dissecting all parameters of each experimental process.

The Bioinformatics Core is the central portal of the Proteomics Core that provides DF/HCC investigators with access to phenotypically annotated proteomics and metabolomics data as well as a large set of analytic procedures (i.e. unsupervised and supervised learning techniques, statistical analysis tools, automated clustering and classification procedures, pathway analysis and generation tools, protein identification database tools, data-visualization tools, annotation tools) with which to explore the data sets for further hypothesis generation.

Finally, a consultative service provided by key members of the core is available at all times for DF/HCC investigators to assess the rationale and feasibility for undertaking specific projects, designing proteomic studies, process and protocol development, proteomic data analysis, and the interpretation of results.

Proteomics Services

•    Protein sample preparation and digestion

•    Protein and peptide profiling ofof bodily fluids and tissue or cell extracts

◦    Profiling by µLC/MS
◦    Multidimensional protein and peptide fractionation by µLC

•    Protein identification by µLC/MS/MS.

◦    coomassie& silver stained gel bands

•    Identification of protein and peptide post-translational modifications.

◦    phosphorylation sites
◦    protein modifications such as acetylation, methylation, and ubiqitination.
◦    By µLC/MS/MS
◦    coomassie stain only, purified proteins

•    Protein-protein interaction (PPI) determination using antibody and tagged based IP-MS

•    Relative quantitation of proteins and peptides, including modified peptides, by µLC/MS/MS

◦    SILAC - stable isotope labeling of amino acids in cell culture which is a biosynthetic approach
◦    GIST - global internal standard technology, a post digestion peptide level labeling technique
◦    ICAT - isotope coded affinity tag -based protein profiling
◦    ITRAQ - isobaric peptide tagging system that enables you to label all primary amines, regardless of peptide class
◦    Targeted proteomics
◦    Metabolomic profiling of cancer cells and tissues
◦    Analytic procedures to explore the data sets for further hypothesis generation

New Services Offered

Quantitative proteomics: Relative and absolute protein quantitation applying isotopic, isobaric, and label-free methods such as iTRAQ, ICAT, SILAC, TMT, GIST, TIC ratio, and spectral counting.
•    Targeted proteomics: With the acquisition of the 5500 QTRAP instrument, the facility now offers a service for targeted quantitative proteomics involving multiple reaction monitoring (MRM) (see Figure).
•    Metabolomics: A new service for targeted metabolomics using selected reaction monitoring (SRM) triple quadrupole MS technology has become very popular for investigating more than 250 polar metabolites in cancer using cell cultures, bodily fluids and tumor tissues. The service for quantification of cellular and non-cellular metabolites makes the CPC the first to provide these analyses at Harvard.
•    Sources for biomarker discovery: While originally focused on serum, the Core has now optimized workflows for quantitative proteomics analysis of urine, saliva, tumor tissue, proximal tumor fluids such as cyst fluid, CSF, and isolated subfractions of cells, such as mitochondria.
•    Protein Depletion: There are new services for depletion of abundant proteins from plasma, urine and other bodily fluids using antibody columns from Sigma (ProteoPrep20) that remove the top 20 proteins from plasma. This service has enabled us to get deeper into the proteome of bodily fluids.
•    Data analysis: New services for bioinformatics and systems biology analysis of proteomic data including pathway analysis tools (Ingenuity, GeneGo) and quantitative proteomics analysis tools (Protein Pilot 3.0).

Bioinformatics Analyses Offered

  • Personalized consulting
  • Customized data analysis and interpretation
  • Web-based bioinformatics tools
  • Comprehensive and innovative Bioinformatics and visualization tools
  • Comprehensive annotation tools
  • Identification of Global Relationships
  • Incorporation of Biological Knowledge
  • Pathway Analysis
  • Publication ready figures
  • The Bioinformatics Core is also responsible for software support and development.