SBIR-STTR Award

Among post-translational modifications, protein phosphorylation is particularly relevant to cancer biology and therapy. However, despite advances in proteomics, it is still difficult to pinpoint phosphorylation sites in proteins. The long-term goal of this project is to develop and commercialize a multiplexed method for isolating and identifying phosphorylation sites based on phosphorylation-specific antibodies. This method would contribute to the development of a new generation of drugs tailored to inhibit specific protein kinases with roles in cancer by identifying new phosphorylation sites that could become targets for cancer diagnosis and treatment. During Phase I we will develop an immunoaffinity method for multiplexed phosphopeptide sorting. We will address key issues qualitatively to establish the method and its tools. We will show that the method can isolate phosphopeptides from simple model systems and complex biological samples, evaluating results with MALDI-TOF mass spectrometry. We will show that isolated peptides can be further analyzed to identify sequences and phosphorylation sites using capillary LC-MS/MS, MS3, chemical modification, and enzymatic treatment as needed to generate informative spectra. Novel, ambiguous, or especially important phosphorylation sites will be confirmed by comparing the spectra of peptides synthesized to have the identified sequences and phosphorylation sites to the spectra of the isolated, natural phosphopeptides.

Thesaurus Terms:
immunoaffinity chromatography, molecular site, phosphopeptide, phosphorylation, protein purification, protein sequence antibody, posttranslational modification liquid chromatography mass spectrometry, matrix assisted laser desorption ionization, peptide chemical synthesis

Among post-translational modifications, protein phosphorylation is particularly relevant to cancer biology and therapy. However, despite advances in proteomics, it is still difficult to pinpoint phosphorylation sites in proteins. The long-term goal of this project is to develop and commercialize a multiplexed method for isolating, identifying, and quantifying phosphorylation sites using phosphorylation-specific antibodies. This method would contribute to the development of a new generation of drugs tailored to inhibit specific protein kinases with roles in cancer by identifying new phosphorylation sites that could become targets for cancer diagnosis and treatment. During our IMAT-funded Phase I, we established the feasibility of using an immunoaffinity method to isolate phosphopeptides from complex mixtures, which were then identified by tandem mass spectrometry. In this Phase II application, we will optimize the immunoaffinity method so it can be used to analyze low-level samples, using cell numbers that are comparable to what would be available from patient samples. Once we have optimized the method and established the repertory of antibodies that can be used productively in the method, we will demonstrate the method's utility by applying it to a variety of cancer cell lines, to show the method can probe the major signal transduction networks involved in cancer and can identify oncogenic lesions. We will bring a quantitative dimension to the method by merging it with mass spectrometry methods such as SILAC and Aqua, which are needed for later biomarker discovery and biomarker assay efforts.

Thesaurus Terms:
immunoaffinity chromatography, method development, molecular site, phosphopeptide, phosphorylation antibody, biomarker, diagnosis design /evaluation, immunologic substance development /preparation, leukemia, neoplasm /cancer diagnosis, phosphoserine, protein kinase, protein purification, protein sequence cell line, western blotting