Binding of ligands often plays a critical role in modulating a protein's function. We, therefore, developed two additional workflows, shown in Figure , which enable indepth interrogation of protein:ligand complexes. The first workflow, nIM-TDMS of a protein-ligand complex (orange line), is comparable to the single-protein approach, with a single conformer of the intact complex isolated, then probed directly by ECD. Application of this experiment is here demonstrated for 1:1 (protein:ligand) complexes of calmodulin with two ligands, melittin and calmodulin-dependent protein kinase II (CaMKII) fragment 290-309 (Figure 3 lower panel). Whilst it has previously been reported that complete calcium occupancy is not a requirement for the binding of these peptide ligands to calmodulin, we found the n=4 calcium-bound state dominated our spectra for both complexes. As the protein:ligand complexes occupy a lower charge state distribution than the free protein, the species selected for ECD were 8+ rather than 10+, which had a noticeable effect on the fragmentation efficiency, as is expected with electron-based dissociation techniques. It is still possible, however, to identify regions From the native mass spectrum of the calmodulin:melittin (1:1) complex (a), the +8 charge state was quadrupole-isolated and mobility separated, giving a single species ATD (b). The complex was then dissociated into its protein and ligand constituents by increasing the injection energy upon re-injection to the mobility region (c). The ligand (orange) was then mobility selected (d) and subjected to ECD fragmentation, allowing identification of melittin through fragment annotation (e).