Sample preparation of AAV2 for peptide mapping was performed using a SMART Digest magnetic bead bulk pepsin kit and following protocol previously described by Guapo et al. , with sample digestion performed in triplicate. Briefly for each digestion, 4 µg (35 µL) of AAV sample were denatured and reduced by bringing the AAV sample to a total volume of 50 µL with LC-MS grade water, before adding 148 µL of SMART Digest buffer and 2 µL of TCEP (0.5 M), producing a 200 µL solution with a final TCEP concentration of 5 mM. Pepsin beads were prepared by mixing 15 µL of pepsin beads with 100 µL SMART Digest buffer and a wash solution for the digest was prepared by mixing 50 µL of SMART Digest buffer with 150 µL of LC-MS grade water. 200 µL of denatured AAV sample, 100 µL of pepsin bead solution, and 200 µL of wash solution were then added to different wells of a 96-deepwell plate (Thermo Fisher Scientific, Vantaa, Finland). Sample digestion on the pepsin beads was performed using a Thermo Scientific™ KingFisher™ Duo Prime purification system under the control of Thermo Scientific™ BindIt software, version 4.0. The sample was incubated on the pepsin beads for 40 min at 70°C, set to medium mixing speed to prevent sedimentation of the beads. After incubation, the beads were removed, with the remaining sample volume transferred to 1.5 mL Eppendorf protein LoBind tubes (Eppendorf, Dublin). Samples were then acidified with the addition of trifluoroacetic acid (TFA) at a final concentration of 0.1% (v.v -1 ) for 5 min followed by centrifugation at 14000 ´ g for 5 min to pellet any pepsin beads that might not have been removed. Subsequently, samples were transferred to clean 1.5 mL Eppendorf protein LoBind tubes and evaporated to dryness using vacuum centrifugation.