To ensure a safe and efficient biotherapeutic protein, candidates are being characterized by a variety of analytical tools. LC-MS/MS is a key tool for assessing product quality attributes (PQAs). To understand PQAs and critical quality attributes (CQAs) fully, identification, localization, and relative quantification are key. Bottom-up approaches are considered the golden standard to assess these. However, fragmentation techniques such as CID and even ExD show certain limitations when it comes to differentiation of amino acid isomers and characterization of dissociation-labile modifications, or to efficiency for low charged analytes and relative quantitative power. Here, the novel fragmentation technique electron activated dissociation (EAD) was employed for peptide mapping of multispecific monoclonal antibody (mAb) and standard mAb samples. The samples were denatured, reduced and enzymatically digested prior to injection onto a novel QTOF instrument (ZenoTOF 7600 system, SCIEX) with data-dependent acquisition in either CID or EAD mode. A specific focus was put on assessing challenging PTMs, such as glycosylations, amino acid isomers and the localization of a sulfation on a singly charged peptide. Results for glycosylated peptides show that fragments providing positioning information could be detected with EAD. Amino acid isomers could be differentiated by EAD data based on signature fragments. For a peptide from the CDR containing three possible aspartic acid isomerization sites, it was possible for the first time to determine the site of isomerization. Sulfation is a rather atypical modification for a biotherapeutic candidate. However, it was found to be critical for antigen binding. Using EAD available on the ZenoTOF 7600 system, the localization of the sulfation on the singly charged peptide with two potential sites could be revealed. The fragmentation pattern obtained from CID did not allow for any of these findings. Furthermore, the EAD method proved to be suitable for reliable, relative quantification of percentages of modification.