We applied nIM-TDMS to study the protein calmodulin, which undergoes conformational changes upon binding to calcium and small peptides. The protein was electrosprayed from native-like conditions, yielding a charge state distribution from 5+ to 12+, corresponding to a species consistent with calmodulin (theoretical Mw 16791.5 Da). The quadrupole was then used to isolate the 10+ charge state (Figure ), which exhibits a mixed calcium-binding occupancy of n = 0-5 (Supplementary Figure ) reflecting the protein's reported state within its native environment. Due to the resolving power of the quadrupole and the close spacing between calcium-bound calmodulin states it was not found to be within the capabilities of the instrument to cleanly isolate a distinct calmodulin-Ca 2+ population, hence the use of a mixed-calcium binding state for these studies. Ion mobility separation of the 10+ charge state revealed two conformational families, as demonstrated by the arrival time distributions (ATD) in Figure . Following calibration, these species equate to conformers with CCS values of 1539 Å 2 and 1966 Å 2 respectively, which are consistent with literature measured and theoretical CCS values for globular (1PRW) and dumbbell (3CLN) calmodulin. Details on the CCS calibration method used can be found in the Supplementary Methods of this manuscript. Using the tandem cIM capabilities, each conformational family was then isolated from the rest of the population within the instrument, Figure , forming a clean ion population. Individual conformers were then subjected to ECD. The two families exhibited different dissociation patterns, with the extended one showing considerably more fragmentation, made up of the c and z ions associated with electron-based methods, alongside y ions at 67% backbone cleavage, compared to 27% for the compact form (Figure upper panel). Fragmentation of the extended population was spread over a greater proportion of the sequence, while the compact structure's dissociation was predominantly at the protein termini. Preferential fragmentation at the termini, combined with non-specific behavior in regards to cleavage between residues, largely correlates with published data regarding the amino acid cleavage preferences by ECD rather than slow-heating methods, providing confidence that the observed variation between conformers is structurally-driven rather than a feature of the dissociation mechanism. It should be noted, however, that some contribution to the variation in fragmentation pattern could be attributed to differences in calcium binding state between the two conformations (Supplementary Figure ), however given the consistency of our observation with literature findings, we believe that this contribution is likely minor and that structural differences drive the observed changes. Further interrogation of this contribution could be achieved by searching for fragment ions where Ca 2+ ions remain bound, however, this remains challenging at present due to a dearth of suitable software to identify these highly mobile modifications to fragments. In comparison, performing a similar experiment without conformer selection (Supplementary Figure ) resulted in a fragmentation pattern corresponding to 63% backbone cleavage, a similar or improved value compared to literature reports for nTDMS of calmodulin, but containing overlapping fragments from both families. This highlights the necessity of using a conformation-specific approach to correctly attribute fragmentation findings to protein structure. By performing top-down fragmentation on isolated conformational families, we were next able to directly link the observed fragmentation patterns to structural elements of the protein. We highlight the bonds that fragmented on the PDB structures most representative of our compact and extended forms of calmodulin in the upper panel of Figure . The central region of the protein, specifically residues 60- 114, had a greater number of fragmentation sites in the extended conformer than the compact form, particularly z and y ions. This additional fragmentation can be directly linked to the increased solvent accessibility of the central helix of calmodulin (residues 65-93) in the extended structure, which has previously been reported in covalent labelling-MS studies and solution state structural methods. This observation is consistent with the general trend observed in the data, that higher levels of bond dissociation are directly linked to higher levels of solvent accessibility. Detailed comparison to established methods for characterizing protein solvent accessibility, such as hydrogen-deuterium exchange (HDX), would be a helpful in confirming this observation, however HDX provides insight only into the bulk solution state. As such, the method is not specific enough to provide information directly on single conformations to enable direct comparison to the nIM-TDMS workflow. Beyond the clear link between solvent accessibility and dissociation propensity, we did not observe a strong correlation between either amount of fragmentation or ion type with the specific secondary-order protein structures, for example there was no observation of the 3-4 residue cleavage pattern sometimes associated with helical structure. Whilst there are clearly fragmentation differences between some structural regions, for example the helix between 119-127 has minimal fragmentation compared to several z and y ions in the dual helix-sheet region between 134-145, we do not feel that these contrasts are significantly consistent across regions to draw definitive conclusions on secondary structure-specific fragmentation patterns. This observation is consistent with the variation in published literature in this area, it seems that there remain insufficient examples in order to form a universally accepted opinion on the relationship between ECD fragmentation patterns and structural protein regions.