The reversed-sequence pentapeptides GRGDS and SDGRG are widely used ion mobility spectrometry (IMS) standards as they exhibit distinct gas-phase mobilities despite their identical mass and composition. Here, we investigate the molecular origins of their differing charge-state-dependent conformations using ion mobility mass spectrometry, cryogenic gas-phase infrared spectroscopy, NMR spectroscopy, molecular dynamics simulations, and density functional theory calculations. In aqueous solution, both peptides populate highly flexible conformational ensembles with only minor differences in compactness. Upon desolvation, the peptides collapse into distinct gas-phase structures stabilized by intramolecular hydrogen bonding. For the doubly protonated species, GRGDS adopts a compact cyclic “C-like” fold, whereas SDGRG forms a more extended “S-like” conformation driven by coulombic repulsion between the protonated N-terminus and the arginine side chain. The singly charged ions show a more compact conformation for SDGRG compared to GRGDS, and a combination of different protomers and conformers appears to be present. Crown ether complexation and terminal glycine extensions of the doubly protonated species reduce the differences in IMS, in agreement with contributions from charge–charge interactions in defining the gas-phase folds. These findings provide molecular-level insight into a widely used IMS standard system and contribute to our fundamental understanding of structural rearrangements in peptides upon transfer to the gas phase.