Dept. Biochemistry and Immunology
Medical School of Ribeirão Preto
University of Sao Paulo
Ribeirao Preto – Sao Paulo – Brazil
Cancer arises from progressive accumulation of cellular changes promoted by mutations in cell genome that alter the complex pathways regulating cell cycle. During these changes, altered mechanisms increase expression of specific proteins in tumor, which culminate in advanced stages with elevated levels of such proteins in tissue, proximal fluids and blood. These protein biomarkers are therefore important targets to reflect tumor burden and eventually could reach patient bedside to diagnose disease. In the last few years, proteomics technology advanced significantly in terms of sensitivity and mass accuracy allowing routine in-depth discovery studies in human tissues and body fluids. Such development contributed with extensive lists of new protein biomarker candidates that now urge to be validated in extended sample sets. Since the same highthroughput proteomic technology applied for discovery of protein candidates is not suitable to accurately quantitate specific proteins in multiple patient samples, a new scenario appears, where sets of proteins are defined from prior biological knowledge and then quantified by targeted proteomics. Our group is applying this targeted strategy to evaluate cancer biomarker signatures during progression of several types of cancers of epithelial origin. Using in vitro models and different types of patient samples, we are evaluating the influence of several signaling mechanisms and pathways involved in the development and modulation of biomarker candidates in cancer. For that, proteotypic peptides reflecting tryptic fragments of 14 proteins of interest were chosen and synthesized using standard Fmoc solid phase chemistry. LC-MS/MS methods of Multiple Reaction Monitoring were developed to specifically quantitate such peptides based on their best fragment ions. This 14-plex molecular signature of cancer biomarker candidates, composed of proteins CDH1, CDH2, HE4, FN1, MMP2, MMP9, MSLN, OLFM4, PSA, SNAIL, SOD2, TIMP1, TWIST and VIME has been used to quantitate tryptic digest of protein extracts, obtained from cell culture models, tumors and tumor fluids. In addition, new proteins have been planned to be added to our panel so a more comprehensive set of alterations could be covered. Our data already demonstrates certain promising protein alterations that will be extensively analyzed in the near future in large sets of patient samples. With such approach, we expect to uncover and validate novel protein targets with potential application in diagnostics of epithelial cancer.