Understanding protein structure is essential for elucidating its function. Cross-linking mass spectrometry (XL-MS) has been widely recognized as a powerful tool for analyzing protein complex structures. However, the effect of cross-linker backbone structure on protein dynamic conformation analysis remains less understood. In this study, we investigated the impact of cross-linker backbone structure on resolving the dynamic conformations of Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1), which features a blend of relatively steady intradomain structures and dynamic interdomain regions. Three cross-linkers with varying arm lengths and different oxygen-containing backbones, Disuccinimidyl tartrate (DST), Bis(succinimidyl) di(ethylene glycol) (BS(PEG)