title: Low Charge and Reduced Mobility of Membrane Protein Complexes Has Implications for Calibration of Collision Cross Section Measurements.
abstract: Ion mobility mass spectrometry of integral membrane proteins provides valuable insights into their architecture and stability. Here we show that, due to their lower charge, the average mobility of native-like membrane protein ions is approximately 30% lower than that of soluble proteins of similar mass. This has implications for drift time measurements, made on traveling wave ion mobility mass spectrometers, which have to be calibrated to extract collision cross sections (Ω). Common calibration strategies employ unfolded or native-like soluble protein standards with masses and mobilities comparable to the protein of interest. We compare Ω values for membrane proteins, derived from standard calibration protocols using soluble proteins, to values measured using an RF-confined drift tube. Our results demonstrate that, while common calibration methods underestimate Ω for native-like or unfolded membrane protein complexes, higher mass soluble calibration standards consistently yield more accurate Ω values. These findings enable us to obtain directly structural information for highly charge-reduced complexes by traveling wave ion mobility mass spectrometry.
authors.affiliation: Department of Chemistry, Physical & Theoretical Chemistry Laboratory, University of Oxford, South Parks Road, Oxford, OX1 3QZ, United Kingdom.
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Allison
authors.affiliation: Department of Chemistry, Physical & Theoretical Chemistry Laboratory, University of Oxford, South Parks Road, Oxford, OX1 3QZ, United Kingdom.
authors.forename: Michael
authors.initials: M
authors.lastname: Landreh
authors.affiliation: Department of Chemistry, Physical & Theoretical Chemistry Laboratory, University of Oxford, South Parks Road, Oxford, OX1 3QZ, United Kingdom.
authors.forename: Justin L P
authors.initials: JLP
authors.lastname: Benesch
authors.affiliation: Department of Chemistry, Physical & Theoretical Chemistry Laboratory, University of Oxford, South Parks Road, Oxford, OX1 3QZ, United Kingdom.
authors.forename: Carol V
authors.initials: CV
authors.lastname: Robinson
mesh_terms: D002138:Calibration; D007477:Ions / Q000032:analysis; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000032:analysis*; D010084:Oxidation-Reduction
chemical_list: D007477:Ions; D008565:Membrane Proteins