PTMs on biotherapeutic proteins are monitored as potential CQAs to inform process development, batch consistency, product stability and more, as their presence can influence product quality, efficacy, and potentially patient safety . Comprehensive PTM analysis is crucial for in-depth characterization of full-length AAV VPs, as it aids in the proper identification of different VP proteoforms present and any potential consequences their presence might cause. For PTM determination, peptide mapping and relative PTM quantitation of AAV2 using on-bead pepsin digestion and nano LC separation was performed in parallel to full-length VP analysis, following the procedure described by Guapo et al . One hundred percent sequence coverage was obtained after filtering identified peptides in BPF as described in Section 2.7 for improved peptide mapping confidence (Figure ). Here it should be noted the VP1 sequence searched for peptide mapping was A2-735 as it is well known that for AAV2 the N-terminal methionine (M, Met) residue is cleaved within the cell (Figure ). Eighteen PTMs were identified with relative abundances greater than 1% including 2 acetylations on alanine (A, Ala) residues, 3 deamidations on asparagine (N, Asn) residues, 5 oxidations on methionine residues, 1 oxidation-to-kynurenine on a tryptophan (W, Trp) residue, 3 succinimides formed from aspartic acid (D, Asp) residues (from now on referred to as succinimide D), 1 phosphorylation on a serine (S, Ser) residue, and loss of ammonia (NH3) from 3 glutamine (Q, Gln) residues (Table ). *The peptide sequence shown is that of the most abundant peptide to contain its associated modification.