Adeno-associated viral (AAV) vectors have emerged as gene therapy and vaccine delivery systems. Differential scanning fluorimetry or differential scanning calorimetry is commonly used to measure the thermal stability of AAVs, but these global methods are unable to distinguish the stabilities of different AAV subpopulations in the same sample. To address this challenge, we combined charge detection-mass spectrometry (CD-MS) with a variable temperature (VT) electrospray source that controls the temperature of the solution prior to electrospray. Using VT-CD-MS, we measured the thermal stabilities of empty and filled capsids. We found that filled AAVs ejected their cargo first and formed intermediate empty capsids before completely dissociating. Finally, we observed that pH stress caused a major decrease in thermal stability. This new approach better characterizes the thermal dissociation of AAVs, providing the simultaneous measurement of the stabilities and dissociation pathways of different subpopulations.

This work describes the development of phenyl diazenyl piperidine triazene derivatives that can be activated to release aryl diazonium ions for labeling of proteins using light. These probes show marked bench stability at room temperature and can be photoisomerized via low-intensity UVA irradiation at physiological pH. Upon isomerization, the triazenes are rendered more basic and readily protonate to release reactive aryl diazonium ions. It was discovered that the intensity and duration of the UV light was essential to the observed diazonium ion reactivity in competition with the traditionally observed photolytic radical pathways. The combination of their synthetic efficiency coupled with their overall stability makes triazenes an attractive candidate for use in bioconjugation applications. Bioorthogonal handles on the triazenes are used to demonstrate the ease by which proteins can be modified.

Native mass spectrometry (MS) and charge detection-mass spectrometry (CD-MS) have become versatile tools for characterizing a wide range of proteins and macromolecular complexes. Both commonly use nanoelectrospray ionization (nESI) from pulled borosilicate needles, but some analytes are known to nonspecifically adsorb to the glass, which may lower sensitivity and limit the quality of the data. To improve the sensitivity of native MS and CD-MS, we modified the surface of nESI needles with inert surface modifiers, including polyethyleneglycol. We found that the surface modification improved the signal intensity for native MS of proteins and for CD-MS of adenoassociated viral capsids. Based on mechanistic comparisons, we hypothesize that the improvement is more likely due to an increased flow rate with coated ESI needles rather than less nonspecific adsorption. In any case, these surface-modified needles provide a simple and inexpensive method for improving the sensitivity of challenging analytes.

Native mass spectrometry (MS) and charge detection-mass spectrometry (CD-MS) have become versatile tools for characterizing a wide range of proteins and macromolecular complexes. Both commonly use nano-electrospray ionization (nESI) from pulled borosilicate needles, but some analytes are known to nonspecifically adsorb to the glass, which may lower sensitivity and limit the quality of the data. To improve the sensitivity of native MS and CD-MS, we modified the surface of nESI needles with inert surface modifiers, including polyethylene-glycol. We found that the surface modification improved the signal intensity for native MS of proteins and for CD-MS of adeno-associated viral capsids. These surface modified needles provide a simple and inexpensive method for improving the sensitivity of challenging analytes.

Adeno-associated virus (AAV) capsids are among the leading gene delivery platforms used to treat a vast array of human diseases and conditions. AAVs exist in a variety of serotypes due to differences in viral protein sequences, with distinct serotypes targeting specific cells and tissues. As the utility of AAVs in gene therapy increases, ensuring their specific composition is imperative for correct targeting and gene delivery. From a quality control perspective, current analytical tools are limited in their selectivty for viral protein (VP) subunits due to their sequence similiaries, instrumental difficulties in assessing the large molecular weights of intact capsids, and the uncertainity in distinguishing empty and filled capsids. To address these challenges, we combine two distinct analytical workflows that assess the intact capsids and VP subunits separately. First, charge detection-mass spectrometry (CD-MS) was applied for characterization of the intact capsids and then liquid chromatography, ion mobility spectrometry, and mass spectrometry (LC-IMS-MS) separations were used for capsid denaturing measurements. This multi-method combination was applied to 3 AAV serotypes (AAV2, AAV6, and AAV8) to evaluate their intact empty and filled capsid ratios and then examine the distinct VP sequences and modifications present.