Programmed cell death plays an important role during animal development, and defects in this process result in a variety of human disorders including cancer and autoimmunity. A family of cysteine proteases, called Caspases, are conserved throughout animals and function to dismantle cells during programmed cell death by proteolysis. The goal of this project is to develop, optimize, and apply new multidimensional microfluidics technology for the rapid profiling of protein modifications based on changes in isoelectric point (pi) and molecular weight (MW) during programmed cell death, and identification of modified proteins via mass spectrometry. By using the fruit fly Drosophila melanogaster as a model system, these studies will explore pathways and identify biomarkers associated with Caspase activation during cell death in developing animals which will provide important insight into human cell death pathways. This challenge will be addressed through the development and application of a microfluidic platform capable of ultra-high-throughput multidimensional protein separation, followed by extremely sensitive protein quantification and identification, enabling effective screening of protein modifications. By offering significant reductions in sample requirements, the platforms will also serve to greatly improve the efficiency of Drosophila proteomic studies, and provide important benefits for downstream clinical applications of the technology. The proposed research will couple our team's expertise in programmed cell death studies and bioinformatics with experience in the development of capillary electrophoresis, microfluidic, and mass spectrometry proteomic instrumentation. Dr. DeVoe (Univ. of Maryland) will lead the project as PI, and take responsibility for overall coordination between the personnel and organizations involved in the research. He will be the leader for all activities involving microfabrication, micro and nanofluidics, and system engineering. Dr. Lee (Univ. of Maryland) will direct the activities in protein separation development and mass spectrometry analysis. Dr. Baehrecke (Univ. of Maryland Biotechnology Institute) will lead the investigation of the programmed cell death studies, and analysis of the resulting protein profiling data. Dr. Rudnick (Calibrant Biosystems) will work in concert with Drs. Baehrecke and Lee to develop and apply bioinformatics tools relevant to the programmed cell death studies. Dr. English (Univ. of Maryland) will collaborate with Drs. DeVoe and Lee on the development and implementation of ultrasensitive confocal microscopy systems for nanofluidic separation platforms. Dr. Ivory (Washington State Univ.) will work with Dr. DeVoe to develop electrokinetic simulations to be employed in optimizing the microfluidic separation systems in order to meet the stated performance goals for ultra-high-throughput protein profiling.