Conditions for Analysis of Native Protein Structures Using Uniform Field Drift Tube Ion Mobility Mass Spectrometry and Characterization of Stable Calibrants for TWIM-MS

  • Julian A. Harrison
  • Celine Kelso
  • Tara L. Pukala
  • Jennifer L. BeckEmail author
Research Article


Determination of collisional cross sections (CCS) by travelling wave ion mobility mass spectrometry (TWIM-MS) requires calibration against standards for which the CCS has been measured previously by drift tube ion mobility mass spectrometry (DTIM-MS). The different extents of collisional activation in TWIM-MS and DTIM-MS can give rise to discrepancies in the CCS of calibrants across the two platforms. Furthermore, the conditions required to ionize and transmit large, folded proteins and assemblies may variably affect the structure of the calibrants and analytes. Stable hetero-oligomeric phospholipase A2 (PDx) and its subunits were characterized as calibrants for TWIM-MS. Conditions for acquisition of native-like TWIM (Synapt G1 HDMS) and DTIM (Agilent 6560 IM-Q-TOF) mass spectra were optimized to ensure the spectra exhibited similar charge state distributions. CCS measurements (DTIM-MS) for ubiquitin, cytochrome c, holo-myoglobin, serum albumin and glutamate dehydrogenase were in good agreement with other recent results determined using this and other DTIM-MS instruments. PDx and its β and γ subunits were stable across a wide range of cone and trap voltages in TWIM-MS and were stable in the presence of organic solvents. The CCS of PDx and its subunits were determined by DTIM-MS and were used as calibrants in determination of CCS of native-like cytochrome c, holo-myoglobin, carbonic anhydrase, serum albumin and haemoglobin in TWIM-MS. The CCS values were in good agreement with those measured by DTIM-MS where available. These experiments demonstrate conditions for analysis of native-like proteins using a commercially available DTIM-MS instrument, characterize robust calibrants for TWIM-MS, and present CCS values determined by DTIM-MS and TWIM-MS for native proteins to add to the current literature database.

Graphical Abstract


Native mass spectrometry Drift tube ion mobility mass spectrometry Travelling wave ion mobility mass spectrometry 



Drift tube ion mobility mass spectrometry


Electrospray ionization


Electrospray ionization mass spectrometry


Glutamate dehydrogenase


Human serum albumin


Ion mobility mass spectrometry


Phospholipase A2/paradoxin from Oxyuranus microlepidotus


Travelling wave ion mobility mass spectrometry



The Waters Synapt G1 HDMS mass spectrometer (University of Wollongong, UOW) used in this work was funded by the Australian Research Council (LE0882289). The Agilent 6560 IM-Q-TOF mass spectrometer (University of Adelaide) was accessed through a collaborative partnership with Agilent Technologies. The work was also supported by Molecular Horizons (UOW).

Supplementary material

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Copyright information

© American Society for Mass Spectrometry 2018

Authors and Affiliations

  1. 1.School of ChemistryUniversity of WollongongWollongongAustralia
  2. 2.Molecular HorizonsUniversity of WollongongWollongongAustralia
  3. 3.Discipline of ChemistryUniversity of AdelaideAdelaideAustralia

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