This study demonstrates the ability of travelling wave ion mobility-mass spectrometry to measure collision cross-sections of ions in the negative mode, using a calibration based approach. Here, negative mode ion mobility-mass spectrometry was utilised to understand structural transitions of calmodulin upon Ca2+ binding and complexation with model peptides melittin and the plasma membrane Ca2+ pump C20W peptide. Coexisting calmodulin conformers were distinguished on the basis of their mass and cross-section, and identified as relatively folded and unfolded populations, with good agreement in collision cross-section to known calmodulin geometries. Titration of calcium tartrate to physiologically relevant Ca2+ levels provided evidence for intermediately metalated species during the transition from apo- to holo-calmodulin, with collision cross-section measurements indicating that higher Ca2+ occupancy is correlated with more compact structures. The binding of two representative peptides which exemplify canonical compact (melittin) and extended (C20W) peptide-calmodulin binding models has also been interrogated by ion mobility mass spectrometry. Peptide binding to calmodulin involves intermediates with metalation states from 1–4 Ca2+, which demonstrate relatively collapsed structures, suggesting neither the existence of holo-calmodulin or a pre-folded calmodulin conformation is a prerequisite for binding target peptides or proteins. The biological importance of the different metal unsaturated calmodulin complexes, if any, is yet to be understood.
Collision Cross Sections of Charge-Reduced Proteins and Protein Complexes: A Database for Collision Cross Section Calibration
