Electrostatic Linear Ion Trap Optimization Strategy for High Resolution Charge Detection Mass Spectrometry

Single ion mass measurements allow mass distributions to be recorded for heterogeneous samples that cannot be analyzed by conventional mass spectrometry. In charge detection mass spectrometry (CD-MS), ions are detected using a conducting cylinder coupled to a charge sensitive amplifier. For optimum performance, the detection cylinder is embedded in an electrostatic linear ion trap (ELIT) where trapped ions oscillate between end-caps that act as opposing ion mirrors. The oscillating ions generate a periodic signal that is analyzed by fast Fourier transforms. The frequency yields the m/z, and the magnitude provides the charge. With a charge precision of 0.2 elementary charges, ions can be assigned to their correct charge states with a low error rate, and the m/z resolving power determines the mass resolving power. Previously, the best mass resolving power achieved with CD-MS was 300. We have recently increased the mass resolving power to 700, through the better optimization of the end-cap potentials. To make a more dramatic improvement in the m/z resolving power, it is necessary to find an ELIT geometry and end-cap potentials that can simultaneously make the ion oscillation frequency independent of both the ion energy and ion trajectory (angular divergence and radial offset) of the entering ion. We describe an optimization strategy that allows these conditions to be met while also adjusting the signal duty cycle to 50% to maximize the signal-to-noise ratio for the charge measurement. The optimized ELIT provides an m/z resolving power of over 300 000 in simulations. Coupled with the high precision charge determination available with CD-MS, this will yield a mass resolving power of 300 000. Such a high mass resolving power will be transformative for the analysis of heterogeneous samples

A novel class of self-complementary AAV vectors with multiple advantages based on cceAAV lacking mutant ITR

Self-complementary AAV vectors (scAAV) use a mutant inverted terminal repeat (mITR) for efficient packaging of complementary stranded DNA, enabling rapid transgene expression. However, inefficient resolution at the mITR leads to the packaging of monomeric or subgenomic AAV genomes. These noncanonical particles reduce transgene expression and may affect the safety of gene transfer. To address these issues, we have developed a novel class of scAAV vectors called covalently closed-end double-stranded AAV (cceAAV) that eliminate the mITR resolution step during production. Instead of using a mutant ITR, we used a 56-bp recognition sequence of protelomerase (TelN) to covalently join the top and bottom strands, allowing the vector to be generated with just a single ITR. To produce cceAAV vectors, the vector plasmid is initially digested with TelN, purified, and then subjected to a standard triple-plasmid transfection protocol followed by traditional AAV vector purification procedures. Such cceAAV vectors demonstrate yields comparable to scAAV vectors. Notably, we observed enhanced transgene expression as compared to traditional scAAV vectors. The treatment of mice with hemophilia B with cceAAV-FIX resulted in significantly enhanced long-term FIX expression. The cceAAV vectors hold several advantages over scAAV vectors, potentially leading to the development of improved human gene therapy drugs