Project Grant R44GM142351

MULTI-RADICAL PROTEIN FOOTPRINTING (MRPF) PLATFORM - THE GENNEXT PHASE II SBIR SUBMSSION ENTITLED "MULTI-RADICAL PROTEIN FOOTPRINTING (MRPF) PLATFORM," IS RESPONSIVE TO THE ACKOWLEDGED NEED FOR NEW AND IMPROVED TOOLS FOR HIGHER ORDER STRUCTURAL ANALYSIS (HOS) OF BIOPHARMACEUTICALS AND MEMBRANE PROTEIN TARGET STUDIES. AN EMERGING HOS ANALYSIS TECHNIQUE IS RADICAL FOOT- PRINTING (RPF). RPF INVOLVES THE IRREVERSIBLE LABELING OF A PROTEIN'S EXTERIOR BY REACTION WITH RADICALS WITH SUBSEQUENT MS ANALYSIS TO IDENTIFY THE OUTER PORTIONS OF THE PROTEIN. WE HAVE DEVELOPED COMMERCIAL SOLUTIONS TO PERFORM HYDROXYL RADICAL PROTEIN FOOTPRINTING (HRPF) EMPLOYING OH RADICALS KNOWN AS THE FOX PROTEIN FOOTPRINTING SYSTEM. A NEW RPF TECHNIQUE THAT RELIES UPON TRIFLUOROMETHYL (TFM) RADICALS CREATED BY OH ATTACK OF AQUEOUS SODIUM TRIFLINATE HAS BEEN DEVELOPED. TFM PROTEIN FOOTPRINTING (TFMPF) IS HIGHLY COMPLEMENTARY TO HRPF. WHEN USED TOGETHER, HRPF AND TFMPF PROVIDE IMPROVED SPATIAL COVERAGE THAN EITHER ALONE. IN OUR PHASE I PROGRAM, WE CREATED A FACILE MEANS TO ASSESS EFFECTIVE RADICAL LOAD DURING CF3 LABELING EXPERIMENTS BY CREATING AN IN-LINE FLUORESCENCE DETECTOR THAT MEASURED THE PHOTOMETRIC RESPONSE OF COUMARIN-BASED INTERNAL STANDARD RADICAL DOSIMETERS TO OH AND CF3 ATTACK. IN OUR PHASE II PROGRAM, WE BUILD UPON OUR PHASE I SUCCESS TO CREATE A PRE-COMMERCIAL SYSTEM THAT WILL ENABLE SELECTABLE OH OR SIMULTANEOUS OH AND CF3 LABELING, WITH IN-LINE RADICAL DOSIMETRY, AND OPTIMIZED DATA PROCESSING SOFTWARE. IN DOING SO, WE WILL EXTEND OUR PRODUCTS TO ALSO SUPPORT TFMPF, CREATING A POWERFUL AND IMPACTFUL IMPROVEMENT TO BIOPHARMACEUTICAL HOS. GENNEXT TECHNOLOGIES IS THE WORLD'S ONLY COMPANY COMMERCIALIZING INSTRUMENTS AND SOLUTIONS FOR RPF HOS ANALYSIS. OUR OVERARCHING GOAL IS TO CONVERT THE RPF PROCESS FROM AN ACADEMIC RESEARCH EXPERIMENT INTO A BROADLY ADOPTED AND VALUABLE HOS ANALYTICAL TOOL. WE FIRMLY BELIEVE THAT OUR PROPOSAL WILL SIGNIFICANTLY IMPACT HOS ANALYSIS WITH TRANSFORMATIVE BENEFITS TO BIOPHARMACEUTICAL RESEARCH.