Project Grant R41GM148104

A MICROFLUIDIC/NANOFLUIDIC SYSTEM FOR RAPID SINGLE CELL PROTEOMICS - PROTEOMICS TYPICALLY INVOLVES THE ANALYSIS OF THE PROTEIN COMPONENTS OF LARGE POPULATIONS OF CELLS. NEARLY ALL THE OF A CELL'S MACHINERY ARE PROTEINS, SO MANY DISEASES AND RESPONSES TO STIMULI AND STRESSORS ARE MANIFESTED AT THE PROTEIN LEVEL. PROTEOMES ARE CHALLENGING TO CHARACTERIZE FULLY FOR MANY REASONS. SOME OF THE KEY ATTRIBUTES OF A PROTEOME THAT MAKE ITS CHARACTERIZATION SO CHALLENGING ARE: THE LARGE NUMBER OF PROTEINS EXPRESSED IN A SINGLE CELL, THE WIDE DYNAMIC RANGE OF PROTEIN RELATIVE ABUNDANCE, AND THE NUMEROUS MODIFICATIONS THAT CAN SWITCH PROTEINS BETWEEN ACTIVE AN INACTIVE STATES. TO FURTHER INCREASE COMPLEXITY, SIGNIFICANT HETEROGENEITY EXISTS AT THE CELLULAR LEVEL THAT IS AVERAGED OUT BY BULK ANALYSES. TO BETTER UNDERSTAND BIOLOGICAL AND HEALTH IMPLICATIONS OF CELLULAR HETEROGENEITY, SINGLE CELL PROTEOMICS HAS EMERGED. HOWEVER, IMPROVEMENTS IN THE ENABLING ANALYTICAL TECHNOLOGIES ARE NEEDED AS SINGLE-CELL PROTEOMICS ADDS THE ADDITIONAL CHALLENGE OF EXTREMELY SMALL SAMPLE SIZE. THIS APPLICATION FOCUSES ON THE DEVELOPMENT OF A NOVEL ELECTROKINETIC MICROFLUIDIC/NANOFLUIDIC SYSTEM FOR IMPROVED SINGLE-CELL PROTEOME ANALYSES THAT IS A SIGNIFICANT DEVIATION FROM THE CURRENT ULTRA-PERFORMANCE LIQUID CHROMATOGRAPHY (UPLC)-MASS SPECTROMETRY (MS) BASED APPROACHES. THE PROPOSED ELECTROKINETIC MICROFLUIDIC/NANOFLUIDIC SYSTEM IS EXPECTED TO OFFER IMPROVED PERFORMANCE FROM: REDUCED SAMPLE LOSS, INCREASED SAMPLE CONCENTRATION ELUTED INTO THE MASS SPECTROMETER, REDUCED PEPTIDE CONTAMINATION, HIGH EFFICIENCY SEPARATIONS, AND RAPID ANALYSES. THE AIMS OF THIS APPLICATION FOCUS ON: 1) FABRICATION AND OPTIMIZATION OF THE KEY FUNCTIONAL MODULES, AND 2) INTEGRATION OF THESE FUNCTIONAL MODULES INTO A COMPLETE SYSTEM AND MEASUREMENT OF THE PERFORMANCE OF THE COMPLETE SYSTEM. THE PRIMARY METRICS FOR SYSTEM PERFORMANCE WILL BE THE SPEED OF THE ANALYSIS AND THE NUMBER OF PROTEIN GROUPS IDENTIFIED. IF SUCCESSFUL, THIS APPLICATION WILL PROVIDE A NOVEL SYSTEM FOR SINGLE-CELL PROTEOMICS THAT WILL IMPROVE THE ANALYSIS SPEED WITHOUT SACRIFICING DEPTH OF PROTEOME COVERAGE.