Molecular dynamics (MD) is an essential tool for correlating collision cross-section data determined by ion mobility spectrometry (IMS) with candidate (calculated) structures. Conventional methods used for ion structure determination rely on comparing the measured cross-sections with the calculated collision cross-section for the lowest energy structure(s) taken from a large pool of candidate structures generated through multiple tiers of simulated annealing. We are developing methods to evaluate candidate structures from an ensemble of many conformations rather than the lowest energy structure. Here, we describe computational simulations and clustering methods to assign backbone conformations for singly-protonated ions of the model peptide (NH 2 -Met-Ile-Phe-Ala-Gly-Ile-Lys-COOH) formed by both MALDI and ESI, and compare the structures of MIFAGIK derivatives to test the 'sensitivity' of the cluster analysis method. Cluster analysis suggests that [MIFAGIK ϩ H] ϩ ions formed by MALDI have a predominantly turn structure even though the low-energy ions prefer partial helical conformers. T he emphasis of mass spectrometry based biological chemistry is shifting from compound identification to structural studies of large biomolecules and biomolecule complexes [1][2][3][4][5][6][7], including membrane proteins [8]. The next phase of 'omics' related research must be aimed at obtaining and predicting additional dimensions of information, such as secondary, tertiary, and quaternary structures and linkage-specific information for glycans. Although sophisticated structural characterization tools, such as NMR and XRD, provide the most information, high throughput analysis of complex biological mixtures obtained by using these techniques is an underdeveloped technology. On the other hand, IMS is much more than a separation device, the structural information derived from 2D conformation space afforded by IM-MS is potentially well-suited to both high throughput applications and complex biological samples.A number of laboratories have focused their research on developing IM-MS for biophysical studies of peptides and proteins [9 -14]. In previous work, we showed that a large proportion of singly charged peptide ions (formed by MALDI) appear on a single trendline in 2D mobility-m/z plots, i.e., plots of arrival-time distribution (ATD) or ion-neutral collision cross-section (⍀) versus m/z [15]; however, a few ion signals deviate (Ͼ3% to ϳ20%) from the expected trendline and nonpeptidic ion signals appear on separate, compound class specific trendlines [14,15]. Ruotolo et al. showed that gas-phase [M ϩ H] ϩ ions of LLGNVLVVVLAR (derived from bovine hemoglobin) prefer extended (helical) structure(s) resulting in a larger collision cross-section than random coil structures having the same or similar m/z values [12,13], while some post-translationally modified (PTM) peptide ions (phosphopeptides) tend to pack more tightly than the unmodified protonated peptide ions owing to intra-molecular charge-solvation and/or formation of salt-...