Extracting Charge and Mass Information from Highly Congested Mass Spectra Using Fourier-Domain Harmonics

  • Sean P. Cleary
  • Huilin Li
  • Dhanashri Bagal
  • Joseph A. Loo
  • Iain D. G. Campuzano
  • James S. PrellEmail author
Research Article


Native mass spectra of large, polydisperse biomolecules with repeated subunits, such as lipoprotein Nanodiscs, can often be challenging to analyze by conventional methods. The presence of tens of closely spaced, overlapping peaks in these mass spectra can make charge state, total mass, or subunit mass determinations difficult to measure by traditional methods. Recently, we introduced a Fourier Transform-based algorithm that can be used to deconvolve highly congested mass spectra for polydisperse ion populations with repeated subunits and facilitate identification of the charge states, subunit mass, charge-state-specific, and total mass distributions present in the ion population. Here, we extend this method by investigating the advantages of using overtone peaks in the Fourier spectrum, particularly for mass spectra with low signal-to-noise and poor resolution. This method is illustrated for lipoprotein Nanodisc mass spectra acquired on three common platforms, including the first reported native mass spectrum of empty “large” Nanodiscs assembled with MSP1E3D1 and over 300 noncovalently associated lipids. It is shown that overtone peaks contain nearly identical stoichiometry and charge state information to fundamental peaks but can be significantly better resolved, resulting in more reliable reconstruction of charge-state-specific mass spectra and peak width characterization. We further demonstrate how these parameters can be used to improve results from Bayesian spectral fitting algorithms, such as UniDec.

Graphical Abstract


Native mass spectrometry Nanodisc Fourier transform Deconvolution 



Research reported in this publication was supported by the National Institutes of Health under Award Number R21AI125804 (to J.S.P.) and R01GM103479 and S10RR028893 (to J.A.L.) and the American Society for Mass Spectrometry Postdoctoral Research Award (to H.L.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors would like to thank the Amgen Pharmacokinetics and Drug Metabolism group (PKDM) in South San Francisco for instrument time on the Orbitrap EMR.

Supplementary material

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ESM 1 (PDF 2515 kb)


  1. 1.
    Campuzano, I.D.G., Li, H., Bagal, D., Lippens, J.L., Svitel, J., Kurzeja, R.J.M., Xu, H., Schnier, P.D., Loo, J.A.: Native ms analysis of bacteriorhodopsin and an empty nanodisc by orthogonal acceleration time-of-flight, orbitrap and ion cyclotron resonance. Anal. Chem. 88, 12427–12436 (2016)CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Barrera, N.P., Isaacson, S.C., Zhou, M., Bavro, V.N., Welch, A., Schaedler, T.A., Seeger, M.A., Miguel, R.N., Korkhov, V.M., van Veen, H.W., Venter, H., Walmsley, A.R., Tate, C.G., Robinson, C.V.: Mass spectrometry of membrane transporters reveals subunit stoichiometry and interactions. Nat. Methods. 6, 585–587 (2009)CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Benesch, J.L.P., Ruotolo, B.T.: Mass spectrometry: come of age for structural and dynamical biology. Curr. Opin. Struct. Biol. 21, 641–649 (2011)CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Zhang, H., Cui, W.D., Gross, M.L., Blankenship, R.E.: Native mass spectrometry of photosynthetic pigment-protein complexes. FEBS Lett. 587, 1012–1020 (2013)CrossRefPubMedGoogle Scholar
  5. 5.
    Marty, M.T., Zhang, H., Cui, W.D., Blankenship, R.E., Gross, M.L., Sligar, S.G.: Native mass spectrometry characterization of intact nanodisc lipoprotein complexes. Anal. Chem. 84, 8957–8960 (2012)CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Ewing, S.A., Donor, M.T., Wilson, J.W., Prell, J.S.: Collidoscope: an improved tool for computing collisional cross-sections with the trajectory method. J. Am. Soc. Mass Spectrom. 28, 587–596 (2017)CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Donor, M.T., Ewing, S.A., Zenaidee, M.A., Donald, W.A., Prell, J.S.: Extended protein ions are formed by the chain ejection model in chemical supercharging electrospray ionization. Anal. Chem. 89, 5107–5114 (2017)CrossRefPubMedGoogle Scholar
  8. 8.
    Li, H.L., Wolff, J.J., Van Orden, S.L., Loo, J.A.: Native top-down electrospray ionization-mass spectrometry of 158 kda protein complex by high-resolution fourier transform ion cyclotron resonance mass spectrometry. Anal. Chem. 86, 317–320 (2014)CrossRefPubMedGoogle Scholar
  9. 9.
    Laganowsky, A., Reading, E., Hopper, J.T.S., Robinson, C.V.: Mass spectrometry of intact membrane protein complexes. Nat. Protoc. 8, 639–651 (2013)CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Zhou, M., Dagan, S., Wysocki, V.H.: Protein subunits released by surface collisions of noncovalent complexes: nativelike compact structures revealed by ion mobility mass spectrometry. Angew. Chem. Int. Ed. 51, 4336–4339 (2012)CrossRefGoogle Scholar
  11. 11.
    Sterling, H.J., Kintzer, A.F., Feld, G.K., Cassou, C.A., Krantz, B.A., Williams, E.R.: Supercharging protein complexes from aqueous solution disrupts their native conformations. J. Am. Soc. Mass Spectrom. 23, 191–200 (2012)CrossRefPubMedGoogle Scholar
  12. 12.
    Heck, A.J.R., van den Heuvel, R.H.H.: Investigation of intact protein complexes by mass spectrometry. Mass Spectrom. Rev. 23, 368–389 (2004)CrossRefPubMedGoogle Scholar
  13. 13.
    Loo, J.A.: Electrospray ionization mass spectrometry: a technology for studying noncovalent macromolecular complexes. Int. J. Mass Spectrom. 200, 175–186 (2000)CrossRefGoogle Scholar
  14. 14.
    Pukala, T.L., Ruotolo, B.T., Zhou, M., Politis, A., Stefanescu, R., Leary, J.A., Robinson, C.V.: Subunit architecture of multiprotein assemblies determined using restraints from gas-phase measurements. Structure. 17, 1235–1243 (2009)CrossRefPubMedGoogle Scholar
  15. 15.
    Robinson, C.V., Chung, E.W., Kragelund, B.B., Knudsen, J., Aplin, R.T., Poulsen, F.M., Dobson, C.M.: Probing the nature of noncovalent interactions by mass spectrometry. A study of protein−coa ligand binding and assembly. J. Am. Chem. Soc. 118, 8646–8653 (1996)CrossRefGoogle Scholar
  16. 16.
    Bush, M.F., Hall, Z., Giles, K., Hoyes, J., Robinson, C.V., Ruotolo, B.T.: Collision cross sections of proteins and their complexes: a calibration framework and database for gas-phase structural biology. Anal. Chem. 82, 9557–9565 (2010)CrossRefPubMedGoogle Scholar
  17. 17.
    Konijnenberg, A., Butterer, A., Sobott, F.: Native ion mobility-mass spectrometry and related methods in structural biology. Biochim. Biophys. Acta, Proteins Proteomics. 1834, 1239–1256 (2013)CrossRefGoogle Scholar
  18. 18.
    Abzalimov, R.R., Kaltashov, I.A.: Electrospray ionization mass spectrometry of highly heterogeneous protein systems: protein ion charge state assignment via incomplete charge reduction. Anal. Chem. 82, 7523–7526 (2010)CrossRefPubMedGoogle Scholar
  19. 19.
    Zhou, M., Wysocki, V.H.: Surface induced dissociation: dissecting noncovalent protein complexes in the gas phase. Acc. Chem. Res. 47, 1010–1018 (2014)CrossRefPubMedGoogle Scholar
  20. 20.
    Salbo, R., Bush, M.F., Naver, H., Campuzano, I., Robinson, C.V., Pettersson, I., Jørgensen, T.J.D., Haselmann, K.F.: Traveling-wave ion mobility mass spectrometry of protein complexes: accurate calibrated collision cross-sections of human insulin oligomers. Rapid Commun. Mass Spectrom. 26, 1181–1193 (2012)CrossRefPubMedGoogle Scholar
  21. 21.
    Pan, J., Xu, K., Yang, X., Choy, W.-Y., Konermann, L.: Solution-phase chelators for suppressing nonspecific protein−metal interactions in electrospray mass spectrometry. Anal. Chem. 81, 5008–5015 (2009)CrossRefPubMedGoogle Scholar
  22. 22.
    McKay, A.R., Ruotolo, B.T., Ilag, L.L., Robinson, C.V.: Mass measurements of increased accuracy resolve heterogeneous populations of intact ribosomes. J. Am. Chem. Soc. 128, 11433–11442 (2006)CrossRefPubMedGoogle Scholar
  23. 23.
    Trimpin, S., Plasencia, M., Isailovic, D., Clemmer, D.E.: Resolving oligomers from fully grown polymers with ims−ms. Anal. Chem. 79, 7965–7974 (2007)CrossRefPubMedGoogle Scholar
  24. 24.
    Marty, M.T., Baldwin, A.J., Marklund, E.G., Hochberg, G.K.A., Benesch, J.L.P., Robinson, C.V.: Bayesian deconvolution of mass and ion mobility spectra: from binary interactions to polydisperse ensembles. Anal. Chem. 87, 4370–4376 (2015)CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Cleary, S.P., Thompson, A.M., Prell, J.S.: Fourier analysis method for analyzing highly congested mass spectra of ion populations with repeated subunits. Anal. Chem. 88, 6205–6213 (2016)CrossRefPubMedGoogle Scholar
  26. 26.
    Hopper, J.T.S., Yu, Y.T.C., Li, D.F., Raymond, A., Bostock, M., Liko, I., Mikhailov, V., Laganowsky, A., Benesch, J.L.P., Caffrey, M., Nietlispach, D., Robinson, C.V.: Detergent-free mass spectrometry of membrane protein complexes. Nat. Methods. 10, 1206–1208 (2013)CrossRefPubMedGoogle Scholar
  27. 27.
    Trimpin, S., Clemmer, D.E.: Ion mobility spectrometry/mass spectrometry snapshots for assessing the molecular compositions of complex polymeric systems. Anal. Chem. 80, 9073–9083 (2008)CrossRefPubMedGoogle Scholar
  28. 28.
    Larriba, C., de la Mora, J.F., Clemmer, D.E.: Electrospray ionization mechanisms for large polyethylene glycol chains studied through tandem ion mobility spectrometry. J. Am. Soc. Mass Spectrom. 25, 1332–1345 (2014)CrossRefPubMedGoogle Scholar
  29. 29.
    Zhang, Y.X., Liu, L., Daneshfar, R., Kitova, E.N., Li, C.S., Jia, F., Cairo, C.W., Klassen, J.S.: Protein-glycosphingolipid interactions revealed using catch-and-release mass spectrometry. Anal. Chem. 84, 7618–7621 (2012)CrossRefPubMedGoogle Scholar
  30. 30.
    Fouquet, T., Sato, H.: Extension of the Kendrick mass defect analysis of homopolymers to low resolution and high mass range mass spectra using fractional base units. Anal. Chem. 89, 2682–2686 (2017)CrossRefPubMedGoogle Scholar
  31. 31.
    Causon, T.J., Hann, S.: Theoretical evaluation of peak capacity improvements by use of liquid chromatography combined with drift tube ion mobility-mass spectrometry. J. Chromatogr. A. 1416, 47–56 (2015)CrossRefPubMedGoogle Scholar
  32. 32.
    Arthur, K.L., Turner, M.A., Reynolds, J.C., Creaser, C.S.: Increasing peak capacity in nontargeted omics applications by combining full scan field asymmetric waveform ion mobility spectrometry with liquid chromatography - mass spectrometry. Anal. Chem. 89, 3452–3459 (2017)CrossRefPubMedGoogle Scholar
  33. 33.
    Bagal, D., Zhang, H., Schnier, P.D.: 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, 2408–2418 (2008)CrossRefPubMedGoogle Scholar
  34. 34.
    Kintzer, A.F., Thoren, K.L., Sterling, H.J., Dong, K.C., Feld, G.K., Tang, I.I., Zhang, T.T., Williams, E.R., Berger, J.M., Krantz, B.A.: The protective antigen component of anthrax toxin forms functional octameric complexes. J. Mol. Biol. 392, 614–629 (2009)CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Ruotolo, B.T., Benesch, J.L.P., Sandercock, A.M., Hyung, S.-J., Robinson, C.V.: Ion mobility-mass spectrometry analysis of large protein complexes. Nat. Protoc. 3, 1139–1152 (2008)CrossRefPubMedGoogle Scholar
  36. 36.
    Clemmer, D.E., Jarrold, M.F.: Ion mobility measurements and their applications to clusters and biomolecules. J. Mass Spectrom. 32, 577–592 (1997)CrossRefGoogle Scholar
  37. 37.
    Hoi, K.K., Robinson, C.V., Marty, M.T.: Unraveling the composition and behavior of heterogeneous lipid nanodiscs by mass spectrometry. Anal. Chem. 88, 6199–6204 (2016)CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Ewing, M.A., Glover, M.S., Clemmer, D.E.: Hybrid ion mobility and mass spectrometry as a separation tool. J. Chromatogr. A. 1439, 3–25 (2016)CrossRefPubMedGoogle Scholar
  39. 39.
    Dwivedi, P., Wu, C., Matz, L.M., Clowers, B.H., Siems, W.F., Hill, H.H.: Gas-phase chiral separations by ion mobility spectrometry. Anal. Chem. 78, 8200–8206 (2006)CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Laszlo, K.J., Munger, E.B., Bush, M.F.: Folding of protein ions in the gas phase after cation-to-anion proton-transfer reactions. J. Am. Chem. Soc. 138, 9581–9588 (2016)CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Zheng, H., Ojha, P.C., McClean, S., Black, N.D., Hughes, J.G., Shaw, C.: Heuristic charge assignment for deconvolution of electrospray ionization mass spectra. Rapid Commun. Mass Spectrom. 17, 429–436 (2003)CrossRefPubMedGoogle Scholar
  42. 42.
    Morgner, N., Robinson, C.V.: Massign: an assignment strategy for maximizing information from the mass spectra of heterogeneous protein assemblies. Anal. Chem. 84, 2939–2948 (2012)CrossRefPubMedGoogle Scholar
  43. 43.
    van Breukelen, B., Barendregt, A., Heck, A.J.R., van den Heuvel, R.H.H.: Resolving stoichiometries and oligomeric states of glutamate synthase protein complexes with curve fitting and simulation of electrospray mass spectra. Rapid Commun. Mass Spectrom. 20, 2490–2496 (2006)CrossRefPubMedGoogle Scholar
  44. 44.
    Stengel, F., Baldwin, A.J., Bush, M.F., Hilton, G.R., Lioe, H., Basha, E., Jaya, N., Vierling, E., Benesch, J.L.P.: Dissecting heterogeneous molecular chaperone complexes using a mass spectrum deconvolution approach. Chem. Biol. 19, 599–607 (2012)CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Prebyl, B.S., Cook, K.D.: Use of fourier transform for deconvolution of the unresolved envelope observed in electrospray ionization mass spectrometry of strongly ionic synthetic polymers. Anal. Chem. 76, 127–136 (2004)CrossRefGoogle Scholar
  46. 46.
    Danis, P.O., Huby, F.J.: The computer-assisted interpretation of copolymer mass spectra. J. Am. Soc. Mass Spectrom. 6, 1112–1118 (1995)CrossRefPubMedGoogle Scholar
  47. 47.
    Denisov, I.G., Sligar, S.G.: Nanodiscs in membrane biochemistry and biophysics. Chem. Rev. 117, 4669–4713 (2017)CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Ritchie, T.K., Grinkova, Y.V., Bayburt, T.H., Denisov, I.G., Zolnerciks, J.K., Atkins, W.M., Sligar, S.G.: Reconstitution of membrane proteins in phospholipid bilayer nanodiscs. Methods Enzymol. 464, 211–231 (2009)CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Cong, X., Liu, Y., Liu, W., Liang, X., Russell, D.H., Laganowsky, A.: Determining membrane protein–lipid binding thermodynamics using native mass spectrometry. J. Am. Chem. Soc. 138, 4346–4349 (2016)CrossRefPubMedGoogle Scholar
  50. 50.
    Marty, M.T., Hoi, K.K., Gault, J., Robinson, C.V.: Probing the lipid annular belt by gas-phase dissociation of membrane proteins in nanodiscs. Angew. Chem. Int. Ed. 55, 550–554 (2016)CrossRefGoogle Scholar
  51. 51.
    Bechara, C., Noell, A., Morgner, N., Degiacomi, M.T., Tampe, R., Robinson, C.V.: A subset of annular lipids is linked to the flippase activity of an abc transporter. Nat. Chem. 7, 255–262 (2015)CrossRefPubMedGoogle Scholar
  52. 52.
    Uetrecht, C., Barbu, I.M., Shoemaker, G.K., van Duijn, E., Heck, A.J.R.: Interrogating viral capsid assembly with ion mobility–mass spectrometry. Nat. Chem. 3, 126–132 (2011)CrossRefPubMedGoogle Scholar
  53. 53.
    Lange, O., Damoc, E., Wieghaus, A., Makarov, A.: Enhanced fourier transform for orbitrap mass spectrometry. Int. J. Mass Spectrom. 369, 16–22 (2014)CrossRefGoogle Scholar
  54. 54.
    Denisov, I.G., Grinkova, Y.V., Lazarides, A.A., Sligar, S.G.: Directed self-assembly of monodisperse phospholipid bilayer nanodiscs with controlled size. J. Am. Chem. Soc. 126, 3477–3487 (2004)CrossRefPubMedGoogle Scholar
  55. 55.
    Mann, M., Meng, C.K., Fenn, J.B.: Interpreting mass-spectra of multiply charged ions. Anal. Chem. 61, 1702–1708 (1989)CrossRefGoogle Scholar
  56. 56.
    Marty, M.T., Zhang, H., Cui, W.D., Gross, M.L., Sligar, S.G.: Interpretation and deconvolution of nanodisc native mass spectra. J. Am. Soc. Mass Spectrom. 25, 269–277 (2014)CrossRefPubMedGoogle Scholar
  57. 57.
    Lu, J., Trnka, M.J., Roh, S.-H., Robinson, P.J.J., Shiau, C., Fujimori, D.G., Chiu, W., Burlingame, A.L., Guan, S.: Improved peak detection and deconvolution of native electrospray mass spectra from large protein complexes. J. Am. Soc. Mass Spectrom. 26, 2141–2151 (2015)CrossRefPubMedPubMedCentralGoogle Scholar
  58. 58.
    Bayburt, T.H., Sligar, S.G.: Membrane protein assembly into nanodiscs. FEBS Lett. 584, 1721–1727 (2010)CrossRefPubMedGoogle Scholar
  59. 59.
    Dörr, J.M., Koorengevel, M.C., Schäfer, M., Prokofyev, A.V., Scheidelaar, S., van der Cruijsen, E.A.W., Dafforn, T.R., Baldus, M., Killian, J.A.: Detergent-free isolation, characterization, and functional reconstitution of a tetrameric k+ channel: the power of native nanodiscs. Proc. Natl. Acad. Sci. U. S. A. 111, 18607–18612 (2014)CrossRefPubMedPubMedCentralGoogle Scholar
  60. 60.
    Gao, Y., Cao, E., Julius, D., Cheng, Y.: Trpv1 structures in nanodiscs reveal mechanisms of ligand and lipid action. Nature. 534, 347–351 (2016)CrossRefPubMedPubMedCentralGoogle Scholar
  61. 61.
    Schultze, M., Ramasesha, K., Pemmaraju, C.D., Sato, S.A., Whitmore, D., Gandman, A., Prell, J.S., Borja, L.J., Prendergast, D., Yabana, K., Neumark, D.M., Leone, S.R.: Attosecond band-gap dynamics in silicon. Science. 346, 1348–1352 (2014)CrossRefPubMedGoogle Scholar
  62. 62.
    Forbes, A.M.G.: Fourier transform filtering - a cautionary note. J. Geophys. Res.-Oceans. 93, 6958–6962 (1988)CrossRefGoogle Scholar
  63. 63.
    Mosierboss, P.A., Lieberman, S.H., Newbery, R.: Fluorescence rejection in raman-spectroscopy by shifted-spectra, edge-detection, and fft filtering techniques. Appl. Spectrosc. 49, 630–638 (1995)CrossRefGoogle Scholar
  64. 64.
    Pandey, P.R., Roy, S.: Headgroup mediated water insertion into the dppc bilayer: a molecular dynamics study. J. Phys. Chem. B. 115, 3155–3163 (2011)CrossRefPubMedGoogle Scholar
  65. 65.
    Li, J., Richards, M.R., Bagal, D., Campuzano, I.D.G., Kitova, E.N., Xiong, Z.J., Privé, G.G., Klassen, J.S.: Characterizing the size and composition of saposin a lipoprotein picodiscs. Anal. Chem. 88, 9524–9531 (2016)CrossRefPubMedGoogle Scholar
  66. 66.
    Reid, D.J., Diesing, J.M., Miller, M.A., Perry, S.M., Wales, J.A., Montfort, W.R., Marty, M.T.: MetaUniDec: High-throughput deconvolution of native mass spectra. J. Am. Soc. Mass Spectrom. (2018). Scholar

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© American Society for Mass Spectrometry 2018

Authors and Affiliations

  1. 1.Department of Chemistry and Biochemistry1253 University of OregonEugeneUSA
  2. 2.Department of Chemistry and Biochemistry, Department of Biological Chemistry, University of CaliforniaUCLA/DOE Institute for Genomics and ProteomicsLos AngelesUSA
  3. 3.Amgen Discovery ResearchAmgen, Inc.South San FranciscoUSA
  4. 4.Molecular Structure and CharacterizationAmgen, Inc.Thousand OaksUSA
  5. 5.Materials Science Institute1252 University of OregonEugeneUSA

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