Identification of Recombinant Adeno-Associated Virus Serotypes by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

Recombinant adeno-associated viruses (rAAVs) are widely used as vectors for gene therapy because they can target specific tissues, exhibit low immunogenicity, and have the capacity for long-term transgene expression. The rAAV serotype determines its tropism, therapeutic efficacy, and suitability for specific clinical applications. Moreover, new mutant serotypes have emerged through capsid engineering approaches. Accurate serotype identification is therefore essential for optimizing vector design and ensuring successful therapeutic outcomes. Conventional methods for identifying rAAV serotypes, such as the enzyme-linked immunosorbent assay, often involve complex procedures and rely on specialized reagents, including antibodies. Meanwhile, liquid chromatography-tandem mass spectrometry-based peptide mapping faces challenges in terms of quality control. These limitations highlight the need for more efficient and practical approaches to streamline the identification of rAAV serotypes. This report proposes a methodology for rAAV serotype identification using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) with new scoring algorithm. The developed method can distinguish multiple rAAV serotypes, including mutant serotypes, by analyzing the specific peptide mass fingerprints of each serotype. This approach eliminates the need for antibodies or extensive sample preparation, thereby offering a reagent-free and cost-effective alternative to conventional methods. Moreover, MALDI-MS requires small sample quantities, making it suitable for high-throughput screening, which can facilitate the development of tailored gene therapy vectors. This study highlights the importance of integrating advanced analytical technologies into gene therapy to enhance precision, reduce costs, and ultimately improve patient outcomes.