Adeno-Associated Virus Capsid Protein Peptide Mapping by Analytical and Micro Flow Reversed Phase Chromatography Coupled to High Resolution Mass Spectrometry

In this study, we employ microflow LC-HRMS to enhance sequence coverage of AAV9 viral proteins and compare the results with conventional analytical flow. Our approach utilizes microflow LC-HRMS with a 0.300 mm column, showcasing a tenfold increase in ion sensitivity, peak area, and S/N ratio compared to the 2.1 mm analytical column. The tryptic digestion method with micro flow outperforms analytical flow, achieving 98% peptide sequence coverage with a minimal 1 µL injection volume compared to the 20 µL required for analytical flow. This not only improves the quality of MS2 data but also boosts ion intensities, providing superior sensitivity and requiring only 5% of the sample needed for analytical scale LC-HRMS. Our findings underscore the significance of microflow LC-HRMS in enhancing the analytical capabilities for AAV9 protein analysis, demonstrating improved sequence coverage, sensitivity, and reduced sample requirements compared to conventional analytical methods.