The isolation of specific fragment ions corresponding to each conformation would have also been possible through computational methods such as TWIMExtract. However, by instead isolating these conformers in-instrument our method minimizes the risk for spectral contamination. Furthermore, when applying this post-collection processing approach to our own data (Supplementary Figure ), we found the differences in fragmentation pattern between conformers to be far less pronounced than when using in-instrument isolation. It should also be noted that isolating a single conformer allows for further experimental investigations to gain deeper insights into its properties before undergoing ECD fragmentation. For instance, an isolated conformer could be trapped in the gas phase over extended periods of time prior to ECD to study gas phase annealing phenomena in more detail. ECD fragmentation data is displayed against the calmodulin sequence in the circular plots above, with colored rectangles indicating different secondary structural elements (alpha helicesorange, beta strandsred, EF-handblue, calcium binding sitescyan). The three main ion fragment series observed (c, z, y ions) are shown as concentric circle slices. In the upper plot the outermost circle shows the c fragments for the compact and extended conformers, followed by the z ions for the compact and extended conformers, and the innermost circle shows the y ions for the compact and extended conformers. Each fragment is represented as a colored dot, with the color indicating the normalized fragment ion intensity. In the center of the circle, the PBD structures representing the compact (1PRW) and extended conformers (3CLN) are shown, with fragmentation sites mapped in purple.