Description:
Summary
Anti-peptide capture reagents can be used to identify and quantify proteins containing a target peptide sequence with a number of applications in biological research and bioassay development. For instance, they are used routinely in techniques such as immunoprecipitation, Western blot, and immunohistological identification and protein localization. A recently-developed application of such reagents is Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA), which utilizes antibodies to enrich peptides from complex matrices for quantitation of proteins by stable isotope dilution mass spectrometry. SISCAPA has the potential for simultaneous quantification of multiple targets from a given sample. There is growing interest in the development of such multiplex protein assays, including large-scale biomarker candidate verification studies and analyses of targeted biological pathways. Concurrent quantification of multiple protein analytes is highly desirable in these applications to minimize sample requirements, handling, and assay costs per analyte, while maximizing throughput.
Project Goals
The goal of this project is to develop new technologies that generate reproducible, well-characterized anti-peptide capture reagents for use in affinity-enriched proteomic studies for the cancer research community. An important characteristic of the desired reagents is the ability to immunoprecipitate their target peptide with high affinity. These reagents should be comparable or superior to ELISA-based antibody technologies in terms of specificity, affinity, and sensitivity and be reproducibly generated in a cost-effective and efficient (e.g. renewable) manner. Currently, mice are often used to generate monoclonal antibodies or alternative capture reagents, but peptides do not always elicit a potent immune response, which can result in low yield of antibodies to effectively immunoprecipitate the target peptides. The desired technology will likely be one that produces a strong immune response to peptide antigens, and may include the use of species other than mice for the generation of antibodies, since other species may have more diverse epitope recognition and improved immune response to small-size epitopes.
The development of these affinity capture reagents will be done in coordination with NCI's Clinical Proteomic Technologies for Cancer (CPTC) (http://proteomics.cancer.gov). A list of proteins or proteotypic peptides derived from cancer biomarker candidates may be requested from CPTC. Furthermore, these capture reagents must be made available as a resource to the scientific community. The suggested choices of performance platforms that the affinity reagents must be compared to include mass spectrometry-based quantitative assays, immunoprecipitation, ELISA-based assays, Western blot, and immunohistochemistry. In addition, other considerations should include sensitivity/specificity/affinity information for the reagents, and method comparison with gold standard practices, precision, and LOD/LOQ.
Phase I Activities and Expected Deliverables
• Develop proof-of-concept strategies and/or technologies that reliably generate anti-peptide capture reagents that can immunoprecipitate the target peptides; this includes, but is not limited to, strategies/technologies that can produce stronger immune responses to peptide antigens than current technologies
• Demonstrate that the capture reagents developed through this technology can repeatedly and reproducibly immunoprecipitate the target peptides
• Work with the Clinical Proteomic Technologies for Cancer (CPTC) community (http://proteomics.cancer.gov), private and public sector to identify appropriate minimum characterization criteria for validation of the assays
• In coordination with CPTC program staff, select and generate affinity reagents to at least ten proteotypic peptides and demonstrate high affinity (Kd of10-9 M or better), specificity and immunoprecipitation performance
• If requested, be prepared to make available to NCI sufficient reagents to perform 10 test runs for each of the ten peptides for independent evaluation • Present findings to an NCI CPTC Evaluation Panel and demonstrate any additional characteristics (e.g. capture of corresponding full-length protein) and how the capture reagents have improved cost effectiveness and throughput capabilities in production and method feasibility of screening of large numbers of hybridomas while conserving time and resources
• Propose quantitative feasibility milestones
Phase II Activities and Expected Deliverables
• Implement the new fully functional anti-peptide capture reagent development strategies/technologies and project plan for development of at least 100 anti-peptide capture reagents capable of immunoprecipitation in coordination with CPTC program staff
o Reagents should be able to capture the target peptides of interest from complex biological mixtures such as blood, plasma, or tissue
• Demonstrate whether the antibodies can immunoprecipitate full-length proteins
• Test performance criteria against affinity, specificity, immunoprecipitation and affinity-enriched SRM-MS (Selected Reaction Monitoring-Mass Spectrometry) platforms or clinical-grade ELISA tests if available
• Work with CPTC to integrate capture reagents into proteomic research platforms
