Photocleavable ICAT Reagents for Quantitative Proteomics

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DESCRIPTION (provided by applicant): Protein expression profiling is becoming one of the most attractive tools for monitoring the course of neoplastic disease as well as identifying diagnostic biomarkers associated with the disease. While several proteomic tools have been extensively investigated for this purpose, including two-dimensional gel electrophoresis and mass spectrometry, quantitative proteomics is still difficult to achieve due to the limited accuracy and inability of these methods to survey the entire proteome. The goal of this project is to overcome these limitations by developing a new approach to quantitative proteomics based on the use of PhotoCleavable Isotope Coded Affinity Tags (PC-ICAT) along with mass spectrometry. PC-ICAT reagents are composed of four distinct elements: a biotin affinity tag, a photodeavable linker which exhibits rapid and efficient photocleavage, a variable mass linker and a chemically reactive group. The light and heavy versions of the PC-ICAT reagents are used to tag two different samples, for example extracts from normal and disease related tissues. After digestion, capture and photorelease, the relative abundance of the tagged peptides in each sample is measured using LC-MS. During Phase I of this project, a sulfhydryl reactive PC-ICAT maleimide reagent has been synthesized and extensively evaluated using both model peptides and proteins. Significantly, the performance of PC-ICAT exhibits superior performance compared with the conventional (CAT reagent. During Phase II, we will extend the usefulness of PC-ICAT in proteomic research and clinical applications by: 1) synthesizing and evaluating an amine reactive version of the PC-ICAT reagent, 2) evaluating a solid supported PC-ICAT reagent and 3) exploring the use of PC-ICAT reagents for protein expression profiling and biomarker discovery. For this purpose, we will apply PC-ICAT reagents and LC-MS to study cell lysates derived from normal and malignant human prostate cell lines. Dr. Cathy Costello, an expert in the area of mass spectrometry and proteomics will serve as a consultant during Phase II of this project.