An Analytical Perspective on Protein Analysis and Discovery Proteomics by Ion Mobility-Mass Spectrometry

  • Johannes P. C. VissersEmail author
  • Michael McCullagh
Part of the Methods in Molecular Biology book series (MIMB, volume 2084)


Ion mobility combined with mass spectrometry (IM-MS) is a powerful technique for the analysis of biomolecules and complex mixtures. This chapter reviews the current state-of-the-art in ion mobility technology and its application to biology, protein analysis, and quantitative discovery proteomics in particular, from an analytical perspective. IM-MS can be used as a technique to separate mixtures, to determine structural information (rotationally averaged cross-sectional area) and to enhance MS duty cycle and sensitivity. Moreover, IM-MS is ideally suited for hyphenating with liquid chromatography, or other front-end separation techniques such as, GC, microcolumn LC, capillary electrophoresis, and direct analysis, including MALDI and DESI, providing an semiorthogonal layer of separation, which affords the more unambiguous and confident detection of a wide range of analytes. To illustrate these enhancements, as well as recent developments, the principle of in-line IM separation and hyphenation to orthogonal acceleration time-of-flight mass spectrometers are discussed, in addition to the enhancement of biophysical MS-based analysis using typical proteomics and related application examples.

Key words

Ion mobility-mass spectrometry Proteomics Protein analysis Cyclic TWIMS 



The Scientific Operations, MS Research, and Advanced Mass Spectrometry Technology groups of Waters Corporation are kindly acknowledged for their support and help in obtaining experimental data and information. Martin Palmer is acknowledged for providing example cyclic IM-MS data.


  1. 1.
    Valentine SJ et al (2006) Toward plasma proteome profiling with ion mobility-mass spectrometry. J Proteome Res 5:2977–2984PubMedCrossRefGoogle Scholar
  2. 2.
    McLean JA, Ruotolo BT, Gillig KJ, Russell DH (2005) Ion mobility–mass spectrometry: a new paradigm for proteomics. Int J Mass Spectrom 240:301–315CrossRefGoogle Scholar
  3. 3.
    Gabryelski W, Froese KL (2003) Rapid and sensitive differentiation of anomers, linkage, and position isomers of disaccharides using high-field asymmetric waveform ion mobility spectrometry (FAIMS). J Am Soc Mass Spectrom 14:265–277PubMedCrossRefGoogle Scholar
  4. 4.
    Bruno VM et al (2010) Comprehensive annotation of the transcriptome of the human fungal pathogen Candida albicans using RNA-seq. Genome Res 20:1451–1458PubMedPubMedCentralCrossRefGoogle Scholar
  5. 5.
    Jin L, Barran PE, Deakin JA, Lyon M, Uhrín D (2005) Conformation of glycosaminoglycans by ion mobility mass spectrometry and molecular modelling. Phys Chem Chem Phys 7:3464–3471PubMedCrossRefGoogle Scholar
  6. 6.
    Hoaglund CS, Valentine SJ, Clemmer DE (1997) An ion trap interface for ESI−ion mobility experiments. Anal Chem 69:4156–4161CrossRefGoogle Scholar
  7. 7.
    Dwivedi P et al (2008) Metabolic profiling by ion mobility mass spectrometry (IMMS). Metabolomics 4:63–80CrossRefGoogle Scholar
  8. 8.
    Kanu AB, Dwivedi P, Tam M, Matz L, Hill HH (2008) Ion mobility–mass spectrometry. J Mass Spectrom 43:1–22PubMedCrossRefGoogle Scholar
  9. 9.
    Lanucara F, Holman SW, Gray CJ, Eyers CE (2014) The power of ion mobility-mass spectrometry for structural characterization and the study of conformational dynamics. Nat Chem 6:281PubMedPubMedCentralCrossRefGoogle Scholar
  10. 10.
    Gabelica V et al (2019) Recommendations for reporting ion mobility mass spectrometry measurements. Mass Spectrom Rev 38:291–230Google Scholar
  11. 11.
    Clemmer DE, Hudgins RR, Jarrold MF (1995) Naked protein conformations: cytochrome C in the gas phase. J Am Chem Soc 117:10141–10142CrossRefGoogle Scholar
  12. 12.
    Pringle SD et al (2007) An investigation of the mobility separation of some peptide and protein ions using a new hybrid quadrupole/travelling wave IMS/oa-ToF instrument. Int J Mass Spectrom 261:1–12CrossRefGoogle Scholar
  13. 13.
    Baker ES et al (2007) Ion mobility spectrometry–mass spectrometry performance using electrodynamic ion funnels and elevated drift gas pressures. J Am Soc Mass Spectrom 18:1176–1187PubMedPubMedCentralCrossRefGoogle Scholar
  14. 14.
    Fernandez-Lima F, Kaplan DA, Suetering J, Park MA (2011) Gas-phase separation using a trapped ion mobility spectrometer. Int J Ion Mobil Spectrom 14. Scholar
  15. 15.
    Dear GJ et al (2010) Sites of metabolic substitution: investigating metabolite structures utilising ion mobility and molecular modelling. Rapid Commun Mass Spectrom 24:3157–3162PubMedCrossRefPubMedCentralGoogle Scholar
  16. 16.
    Chalet C, Hollebrands B, Janssen HG, Augustijns P, Duchateau G (2018) Identification of phase-II metabolites of flavonoids by liquid chromatography–ion-mobility spectrometry–mass spectrometry. Anal Bioanal Chem 410(2):471–482. Scholar
  17. 17.
    Kliman M, May JC, McLean JA (2011) Lipid analysis and lipidomics by structurally selective ion mobility-mass spectrometry. Biochim Biophys Acta Mol Cell Biol Lipids 1811:935–945CrossRefGoogle Scholar
  18. 18.
    Eckers C, Laures AM-F, Giles K, Major H, Pringle S (2007) Evaluating the utility of ion mobility separation in combination with high-pressure liquid chromatography/mass spectrometry to facilitate detection of trace impurities in formulated drug products. Rapid Commun Mass Spectrom 21:1255–1263PubMedCrossRefPubMedCentralGoogle Scholar
  19. 19.
    Vakhrushev SY, Langridge J, Campuzano I, Hughes C, Peter-Katalinić J (2008) Ion mobility mass spectrometry analysis of human glycourinome. Anal Chem 80:2506–2513PubMedCrossRefPubMedCentralGoogle Scholar
  20. 20.
    Schenauer MR, Meissen JK, Seo Y, Ames JB, Leary JA (2009) Heparan sulfate separation, sequencing, and isomeric differentiation: ion mobility spectrometry reveals specific iduronic and glucuronic acid-containing hexasaccharides. Anal Chem 81:10179–10185PubMedPubMedCentralCrossRefGoogle Scholar
  21. 21.
    Fasciotti M et al (2012) Separation of isomeric disaccharides by traveling wave ion mobility mass spectrometry using CO2 as drift gas. J Mass Spectrom 47(12):1643–1647. Scholar
  22. 22.
    Ruotolo BT et al (2005) Evidence for macromolecular protein rings in the absence of bulk water. Science (80) 310:1658–1661CrossRefGoogle Scholar
  23. 23.
    Bereszczak JZ et al (2014) Sizing up large protein complexes by electrospray ionisation-based electrophoretic mobility and native mass spectrometry: morphology selective binding of Fabs to hepatitis B virus capsids. Anal Bioanal Chem 406:1437–1446PubMedCrossRefPubMedCentralGoogle Scholar
  24. 24.
    Helm D et al (2014) Ion mobility tandem mass spectrometry enhances performance of bottom-up proteomics. Mol Cell Proteomics 13:3709–3715PubMedPubMedCentralCrossRefGoogle Scholar
  25. 25.
    Meier F et al (2015) Parallel accumulation–serial fragmentation (PASEF): multiplying sequencing speed and sensitivity by synchronized scans in a trapped ion mobility device. J Proteome Res 14:5378–5387PubMedCrossRefPubMedCentralGoogle Scholar
  26. 26.
    Göth M, Pagel K (2017) Ion mobility–mass spectrometry as a tool to investigate protein–ligand interactions. Anal Bioanal Chem 409:4305–4310PubMedCrossRefPubMedCentralGoogle Scholar
  27. 27.
    Williams JP et al (2009) Isomer separation and gas-phase configurations of organoruthenium anticancer complexes: ion mobility mass spectrometry and modeling. J Am Soc Mass Spectrom 20:1119–1122PubMedCrossRefPubMedCentralGoogle Scholar
  28. 28.
    Bagal D, Zhang H, Schnier PD (2008) Gas-phase proton-transfer chemistry coupled with TOF mass spectrometry and ion mobility-MS for the facile analysis of poly(ethylene glycols) and PEGylated polypeptide conjugates. Anal Chem 80:2408–2418PubMedCrossRefPubMedCentralGoogle Scholar
  29. 29.
    Towers MW, Karancsi T, Jones EA, Pringle SD, Claude E (2018) Optimised desorption electrospray ionisation mass spectrometry imaging (DESI-MSI) for the analysis of proteins/peptides directly from tissue sections on a travelling wave ion mobility Q-ToF. J Am Soc Mass Spectrom 29:2456–2466PubMedPubMedCentralCrossRefGoogle Scholar
  30. 30.
    Sans M, Feider CL, Eberlin LS (2018) Advances in mass spectrometry imaging coupled to ion mobility spectrometry for enhanced imaging of biological tissues. Curr Opin Chem Biol 42:138–146PubMedCrossRefPubMedCentralGoogle Scholar
  31. 31.
    Giles K et al (2004) Applications of a travelling wave-based radio-frequency-only stacked ring ion guide. Rapid Commun Mass Spectrom 18:2401–2414CrossRefGoogle Scholar
  32. 32.
    Giles K, Williams JP, Campuzano I (2011) Enhancements in travelling wave ion mobility resolution. Rapid Commun Mass Spectrom 25:1559–1566PubMedCrossRefGoogle Scholar
  33. 33.
    Giles K et al (2017) Design and performance of a second-generation cyclic ion mobility enabled Q-ToF. In 65th ASMS conference on mass spectrometry and allied topicsGoogle Scholar
  34. 34.
    Ujma J et al (2019) Cyclic ion mobility mass spectrometry distinguishes anomers and open-ring forms of pentasaccharides. J Am Soc Mass Spectrom 30(6):1028–1037. Scholar
  35. 35.
    Shvartsburg AA, Smith RD (2008) Fundamentals of traveling wave ion mobility spectrometry. Anal Chem 80:9689–9699PubMedPubMedCentralCrossRefGoogle Scholar
  36. 36.
    Richardson K, Langridge D, Giles K (2018) Fundamentals of travelling wave ion mobility revisited: I. Smoothly moving waves. Int J Mass Spectrom 428:71–80CrossRefGoogle Scholar
  37. 37.
    Eldrid C et al (2018) Gas phase stability of protein ions in a cyclic ion mobility spectrometry travelling wave device. Anal Chem 91(12):7554–7561. Scholar
  38. 38.
    Deng L et al (2016) Ultra-high resolution ion mobility separations utilizing traveling waves in a 13 m serpentine path length structures for lossless ion manipulations module. Anal Chem 88(18):8957–8964. Scholar
  39. 39.
    Deng L et al (2017) Serpentine ultralong path with extended routing (SUPER) high resolution traveling wave ion mobility-MS using structures for lossless ion manipulations. Anal Chem 89(8):4628–4634. Scholar
  40. 40.
    Graves DB (2018) Transport properties of ions in gases by Edward A. Mason and Earl W. McDaniel, John Wiley and Sons, New York, NY, 1988, 560 + xvi pp. AIChE J 35:701CrossRefGoogle Scholar
  41. 41.
    Bowers MT, Marshall AG, McLafferty FW (1996) Mass spectrometry: recent advances and future directions. J Phys Chem 100:12897–12910CrossRefGoogle Scholar
  42. 42.
    Henderson SC, Valentine SJ, Counterman AE, Clemmer DE (1999) ESI/ion trap/ion mobility/time-of-flight mass spectrometry for rapid and sensitive analysis of biomolecular mixtures. Anal Chem 71:291–301PubMedCrossRefPubMedCentralGoogle Scholar
  43. 43.
    Verbeck GF, Ruotolo BT, Sawyer HA, Gillig KJ, Russell DH (2002) A fundamental introduction to ion mobility mass spectrometry applied to the analysis of biomolecules. J Biomol Tech 13:56–61PubMedPubMedCentralGoogle Scholar
  44. 44.
    Zhong Y, Hyung S-J, Ruotolo BT (2012) Ion mobility-mass spectrometry for structural proteomics. Expert Rev Proteomics 9:47–58PubMedPubMedCentralCrossRefGoogle Scholar
  45. 45.
    Uetrecht C, Rose RJ, van Duijn E, Lorenzen K, Heck AJR (2010) Ion mobility mass spectrometry of proteins and protein assemblies. Chem Soc Rev 39:1633–1655PubMedCrossRefGoogle Scholar
  46. 46.
    Snijder J, Heck AJR (2014) Analytical approaches for size and mass analysis of large protein assemblies. Annu Rev Anal Chem 7:43–64CrossRefGoogle Scholar
  47. 47.
    Stojko J et al (2015) Ion mobility coupled to native mass spectrometry as a relevant tool to investigate extremely small ligand-induced conformational changes. Analyst 140(21):7234–7245. Scholar
  48. 48.
    Hoffmann W, von Helden G, Pagel K (2017) Ion mobility-mass spectrometry and orthogonal gas-phase techniques to study amyloid formation and inhibition. Curr Opin Struct Biol 46:7–15PubMedCrossRefGoogle Scholar
  49. 49.
    Eschweiler JD, Kerr R, Rabuck-Gibbons J, Ruotolo BT (2017) Sizing up protein–ligand complexes: the rise of structural mass spectrometry approaches in the pharmaceutical sciences. Annu Rev Anal Chem 10:25–44CrossRefGoogle Scholar
  50. 50.
    Eyers CE, Vonderach M, Ferries S, Jeacock K, Eyers PA (2018) Understanding protein–drug interactions using ion mobility–mass spectrometry. Curr Opin Chem Biol 42:167–176. Scholar
  51. 51.
    Hopper JTS, Oldham NJ (2009) Collision induced unfolding of protein ions in the gas phase studied by ion mobility-mass spectrometry: The effect of ligand binding on conformational stability. J Am Soc Mass Spectrom 20:1851–1858PubMedCrossRefPubMedCentralGoogle Scholar
  52. 52.
    Migas LG, France A, Bellina B, Barran P (2017) ORIGAMI: a software suite for activated ion mobility mass spectrometry (aIM-MS) applied to multimeric protein assemblies. bioRxiv 427:20–28Google Scholar
  53. 53.
    Beveridge R et al (2016) Mass spectrometry locates local and allosteric conformational changes that occur on cofactor binding. Nat Commun 7:12163PubMedPubMedCentralCrossRefGoogle Scholar
  54. 54.
    Valentine SJ, Counterman AE, Clemmer DE (1999) A database of 660 peptide ion cross sections: use of intrinsic size parameters for bona fide predictions of cross sections. J Am Soc Mass Spectrom 10:1188–1211PubMedCrossRefPubMedCentralGoogle Scholar
  55. 55.
    Liu X et al (2007) Mapping the human plasma proteome by SCX-LC-IMS-MS. J Am Soc Mass Spectrom 18:1249–1264PubMedPubMedCentralCrossRefGoogle Scholar
  56. 56.
    Valentine SJ, Kulchania M, Barnes CAS, Clemmer DE (2001) Multidimensional separations of complex peptide mixtures: a combined high-performance liquid chromatography/ion mobility/time-of-flight mass spectrometry approach. Int J Mass Spectrom 212:97–109CrossRefGoogle Scholar
  57. 57.
    Thalassinos K et al (2012) Design and application of a data-independent precursor and product ion repository. J Am Soc Mass Spectrom 23:1808–1820PubMedCrossRefPubMedCentralGoogle Scholar
  58. 58.
    Paglia G et al (2014) Ion mobility derived collision cross sections to support metabolomics applications. Anal Chem 86:3985–3993PubMedPubMedCentralCrossRefGoogle Scholar
  59. 59.
    Shah AR et al (2010) Machine learning based prediction for peptide drift times in ion mobility spectrometry. Bioinformatics 26:1601–1607PubMedPubMedCentralCrossRefGoogle Scholar
  60. 60.
    Rodriguez-Suarez E et al (2013) An ion mobility assisted data independent LC-MS strategy for the analysis of complex biological samples. Curr Anal Chem 9:199–211Google Scholar
  61. 61.
    Geromanos SJ, Hughes C, Ciavarini S, Vissers JPC, Langridge JI (2012) Using ion purity scores for enhancing quantitative accuracy and precision in complex proteomics samples. Anal Bioanal Chem 404:1127–1139PubMedCrossRefPubMedCentralGoogle Scholar
  62. 62.
    Distler U et al (2014) Drift time-specific collision energies enable deep-coverage data-independent acquisition proteomics. Nat Methods 11:167–170PubMedCrossRefPubMedCentralGoogle Scholar
  63. 63.
    Yang WS et al (2014) Regulation of ferroptotic cancer cell death by GPX4. Cell 156:317–331PubMedPubMedCentralCrossRefGoogle Scholar
  64. 64.
    Distler U, Kuharev J, Tenzer S (2014) Biomedical applications of ion mobility-enhanced data-independent acquisition-based label-free quantitative proteomics. Expert Rev Proteomics 11:675–684PubMedCrossRefPubMedCentralGoogle Scholar
  65. 65.
    Kedia K et al (2018) Application of multiplexed ion mobility spectrometry towards the identification of host protein signatures of treatment effect in pulmonary tuberculosis. Tuberculosis 112:52–61. Scholar
  66. 66.
    MacLean BX et al (2018) Using skyline to analyze data-containing liquid chromatography, ion mobility spectrometry, and mass spectrometry dimensions. J Am Soc Mass Spectrom 29:2182–2188PubMedPubMedCentralCrossRefGoogle Scholar
  67. 67.
    Iacob RE, Murphy JP, Engen JR (2008) Ion mobility adds an additional dimension to mass spectrometric analysis of solution-phase hydrogen/deuterium exchange. Rapid Commun Mass Spectrom 22:2898–2904PubMedCrossRefPubMedCentralGoogle Scholar
  68. 68.
    James JMB, Cryar A, Thalassinos K (2019) An optimization workflow for the analysis of cross-linked peptides using a quadrupole time-of-flight mass spectrometer. Anal Chem 91(3):1808–1814. Scholar
  69. 69.
    Meier F et al (2018) Online parallel accumulation − serial fragmentation (PASEF) with a novel trapped ion mobility mass spectrometer. bioRxiv 17(12):2534–2545. Scholar
  70. 70.
    Domanski D et al (2012) MRM-based multiplexed quantitation of 67 putative cardiovascular disease biomarkers in human plasma. Proteomics 12:1222–1243PubMedCrossRefGoogle Scholar
  71. 71.
    Mbasu RJ et al (2016) Advances in quadrupole and time-of-flight mass spectrometry for peptide MRM based translational research analysis. Proteomics 16:2206–2220PubMedCrossRefGoogle Scholar
  72. 72.
    Waldrop MM (2016) The chips are down for Moore’s law. Nature 530(7589):144–147. Scholar
  73. 73.
    McCullagh M, Douce D, Van Hoeck E, Goscinny S (2018) Exploring the complexity of steviol glycosides analysis using ion mobility mass spectrometry. Anal Chem 90(7):4585–4595. Scholar
  74. 74.
    Righetti L et al (2018) High resolution-ion mobility mass spectrometry as an additional powerful tool for structural characterization of mycotoxin metabolites. Food Chem 245:768–774PubMedCrossRefGoogle Scholar
  75. 75.
    Hernández-Mesa M, Le Bizec B, Monteau F, García-Campaña AM, Dervilly-Pinel G (2018) Collision cross section (CCS) database: an additional measure to characterize steroids. Anal Chem 90(7):4616–4625. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Waters CorporationWilmslowUK

Personalised recommendations