Advertisement

Surface Induced Dissociation Coupled with High Resolution Mass Spectrometry Unveils Heterogeneity of a 211 kDa Multicopper Oxidase Protein Complex

  • Mowei Zhou
  • Jing Yan
  • Christine A. Romano
  • Bradley M. Tebo
  • Vicki H. Wysocki
  • Ljiljana Paša-Tolić
Article
First Online:
Received:
Revised:
Accepted:

Abstract

Manganese oxidation is an important biogeochemical process that is largely regulated by bacteria through enzymatic reactions. However, the detailed mechanism is poorly understood due to challenges in isolating and characterizing these unknown enzymes. A manganese oxidase, Mnx, from Bacillus sp. PL-12 has been successfully overexpressed in active form as a protein complex with a molecular mass of 211 kDa. We have recently used surface induced dissociation (SID) and ion mobility-mass spectrometry (IM-MS) to release and detect folded subcomplexes for determining subunit connectivity and quaternary structure. The data from the native mass spectrometry experiments led to a plausible structural model of this multicopper oxidase, which has been difficult to study by conventional structural biology methods. It was also revealed that each Mnx subunit binds a variable number of copper ions. Becasue of the heterogeneity of the protein and limited mass resolution, ambiguities in assigning some of the observed peaks remained as a barrier to fully understanding the role of metals and potential unknown ligands in Mnx. In this study, we performed SID in a modified Fourier transform-ion cyclotron resonance (FTICR) mass spectrometer. The high mass accuracy and resolution offered by FTICR unveiled unexpected artificial modifications on the protein that had been previously thought to be iron bound species based on lower resolution spectra. Additionally, isotopically resolved spectra of the released subcomplexes revealed the metal binding stoichiometry at different structural levels. This method holds great potential for in-depth characterization of metalloproteins and protein–ligand complexes.

Graphical Abstract

ᅟ

Keywords

Native mass spectrometry High resolution mass spectrometry Protein complex Surface induced dissociation Metalloprotein Protein–ligand interaction 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgments

The authors thank Yang Song and Arpad Somogyi at The Ohio State University for helping with the SID experiments; Jeremy Wolff and Michael Easterling at Bruker Corporation, Randy Pedder at Ardara Technologies for helping with the instrument modification. This work was funded by the National Science Foundation (NSF DBI 1455654; SID development and installation) and National Institute of Health (NIH 1S10OD018507; FTICR purchase) to V.H.W., NSF CHE-1410688 to B.M.T, and an NSF Postdoctoral Research Fellowship in Biology Award ID: DBI-1202859 to C.A.R. A portion of the research was supported by the Environmental and Molecular Sciences Laboratory (EMSL), a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory.

Supplementary material

13361_2017_1882_MOESM1_ESM.pdf (1.2 mb)
ESM 1 (PDF 1227 kb)

References

  1. 1.
    Domon, B., Aebersold, R.: Mass spectrometry and protein analysis. Science. 312, 212–217 (2006)CrossRefGoogle Scholar
  2. 2.
    Robinson, C.V., Sali, A., Baumeister, W.: The molecular sociology of the cell. Nature. 450, 973–982 (2007)CrossRefGoogle Scholar
  3. 3.
    Heck, A.J.: Native mass spectrometry: a bridge between interactomics and structural biology. Nat. Methods. 5, 927–933 (2008)CrossRefGoogle Scholar
  4. 4.
    Benesch, J.L.P., Ruotolo, B.T., Simmons, D.A., Robinson, C.V.: Protein complexes in the gas phase: technology for structural genomics and proteomics. Chem. Rev. 107, 3544–3567 (2007)CrossRefGoogle Scholar
  5. 5.
    Kitova, E.N., El-Hawiet, A., Schnier, P.D., Klassen, J.S.: Reliable determinations of protein–ligand interactions by direct ESI-MS measurements. Are we there yet? J. Am. Soc. Mass Spectrom. 23, 431–441 (2012)CrossRefGoogle Scholar
  6. 6.
    Flick, T.G., Merenbloom, S.I., Williams, E.R.: Effects of metal ion adduction on the gas-phase conformations of protein ions. J. Am. Soc. Mass Spectrom. 24, 1654–1662 (2013)CrossRefGoogle Scholar
  7. 7.
    Baldwin, A.J., Lioe, H., Hilton, G.R., Baker, L.A., Rubinstein, J.L., Kay, L.E., Benesch, J.L.P.: The polydispersity of αB-crystallin is rationalized by an interconverting polyhedral architecture. Structure. 19, 1855–1863 (2011)CrossRefGoogle Scholar
  8. 8.
    Ihms, E.C., Zhou, M., Zhang, Y., Kleckner, I.R., McElroy, C.A., Wysocki, V.H., Gollnick, P., Foster, M.P.: Gene regulation by substoichiometric heterocomplex formation of undecameric TRAP and trimeric anti-TRAP. Proc. Natl. Acad. Sci. 111, 3442–447 (2014)Google Scholar
  9. 9.
    Seeger, F., Quintyn, R., Tanimoto, A., Williams, G.J., Tainer, J.A., Wysocki, V.H., Garcin, E.D.: Interfacial residues promote an optimal alignment of the catalytic center in human soluble guanylate cyclase: heterodimerization is required but not sufficient for activity. Biochemistry. 53, 2153–2165 (2014)CrossRefGoogle Scholar
  10. 10.
    Stengel, F., Baldwin, A.J., Painter, A.J., Jaya, N., Basha, E., Kay, L.E., Vierling, E., Robinson, C.V., Benesch, J.L.P.: Quaternary dynamics and plasticity underlie small heat shock protein chaperone function. Proc. Natl. Acad. Sci. 107, 2007–2012 (2010)CrossRefGoogle Scholar
  11. 11.
    Dyachenko, A., Gruber, R., Shimon, L., Horovitz, A., Sharon, M.: Allosteric mechanisms can be distinguished using structural mass spectrometry. Proc. Natl. Acad. Sci. 110, 7235–7239 (2013)CrossRefGoogle Scholar
  12. 12.
    Shaw, J.B., Lin, T.-Y., Leach, F.E., Tolmachev, A.V., Tolić, N., Robinson, E.W., Koppenaal, D.W., Paša-Tolić, L.: 21 Tesla Fourier transform ion cyclotron resonance mass spectrometer greatly expands mass spectrometry toolbox. J. Am. Soc. Mass Spectrom. 27, 1929–1936 (2016)CrossRefGoogle Scholar
  13. 13.
    Nagornov, K.O., Gorshkov, M.V., Kozhinov, A.N., Tsybin, Y.O.: High-resolution fourier transform ion cyclotron resonance mass spectrometry with increased throughput for biomolecular analysis. Anal. Chem. 86, 9020–9028 (2014)CrossRefGoogle Scholar
  14. 14.
    Rose, R.J., Damoc, E., Denisov, E., Makarov, A., Heck, A.J.R.: High-sensitivity Orbitrap mass analysis of intact macromolecular assemblies. Nat. Methods. 9, 1084–1086 (2012)CrossRefGoogle Scholar
  15. 15.
    Rosati, S., Yang, Y., Barendregt, A., Heck, A.J.R.: Detailed mass analysis of structural heterogeneity in monoclonal antibodies using native mass spectrometry. Nat. Protoc. 9, 967–976 (2014)CrossRefGoogle Scholar
  16. 16.
    Dyachenko, A., Wang, G., Belov, M., Makarov, A., de Jong, R.N., van den Bremer, E.T.J., Parren, P.W.H.I., Heck, A.J.R.: Tandem native mass-spectrometry on antibody–drug conjugates and submillion Da antibody–antigen protein assemblies on an Orbitrap EMR equipped with a high-mass quadrupole mass selector. Anal. Chem. 87, 6095–6102 (2015)CrossRefGoogle Scholar
  17. 17.
    Debaene, F., Bœuf, A., Wagner-Rousset, E., Colas, O., Ayoub, D., Corvaïa, N., Van Dorsselaer, A., Beck, A., Cianférani, S.: Innovative native MS methodologies for antibody drug conjugate characterization: high resolution native MS and IM-MS for average DAR and DAR distribution assessment. Anal. Chem. 86, 10674–10683 (2014)CrossRefGoogle Scholar
  18. 18.
    Marty, M.T., Zhang, H., Cui, W., Blankenship, R.E., Gross, M.L., Sligar, S.G.: Native mass spectrometry characterization of intact nanodisc lipoprotein complexes. Anal. Chem. 84, 8957–960 (2012)Google Scholar
  19. 19.
    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)CrossRefGoogle Scholar
  20. 20.
    Gault, J., Donlan, J.A.C., Liko, I., Hopper, J.T.S., Gupta, K., Housden, N.G., Struwe, W.B., Marty, M.T., Mize, T., Bechara, C., Zhu, Y., Wu, B., Kleanthous, C., Belov, M., Damoc, E., Makarov, A., Robinson, C.V.: High-resolution mass spectrometry of small molecules bound to membrane proteins. Nat. Methods. 13, 333–336 (2016)CrossRefGoogle Scholar
  21. 21.
    Lippens, J.L., Nshanian, M., Spahr, C., Egea, P.F., Loo, J.A., Campuzano, I.D.G.: Fourier transform-ion cyclotron resonance mass spectrometry as a platform for characterizing multimeric membrane protein complexes. J. Am. Soc. Mass Spectrom. (2017)Google Scholar
  22. 22.
    Zhou, M., Wysocki, V.H.: Surface induced dissociation: dissecting noncovalent protein complexes in the gas phase. Acc. Chem. Res. 47, 1010–1018 (2014)CrossRefGoogle Scholar
  23. 23.
    Benesch, J.L.P., Aquilina, J.A., Ruotolo, B.T., Sobott, F., Robinson, C.V.: Tandem mass spectrometry reveals the quaternary organization of macromolecular assemblies. Chem. Biol. 13, 597–605 (2006)CrossRefGoogle Scholar
  24. 24.
    Tebo, B.M., Bargar, J.R., Clement, B.G., Dick, G.J., Murray, K.J., Parker, D., Verity, R., Webb, S.M.: Biogenic manganese oxides: properties and mechanisms of formation. Annu. Rev. Earth Planet Sci. 32, 287–328 (2004)CrossRefGoogle Scholar
  25. 25.
    Butterfield, C.N., Soldatova, A.V., Lee, S.-W., Spiro, T.G., Tebo, B.M.: Mn(II,III) oxidation and MnO2 mineralization by an expressed bacterial multicopper oxidase. Proc. Natl. Acad. Sci. 110, 11731–11735 (2013)CrossRefGoogle Scholar
  26. 26.
    Romano, C.A., Zhou, M., Song, Y., Wysocki, V.H., Dohnalkova, A.C., Kovarik, L., Pasa-Tolic, L., Tebo, B.M.: Biogenic manganese oxide nanoparticle formation by a multimeric multicopper oxidase Mnx. Nat. Commun. 8, 746 (2017)CrossRefGoogle Scholar
  27. 27.
    Soldatova, A.V., Butterfield, C., Oyerinde, O.F., Tebo, B.M., Spiro, T.G.: Multicopper oxidase involvement in both Mn (II) and Mn (III) oxidation during bacterial formation of MnO2. JBIC J. Biol. Inorg. Chem. 17, 1151–1158 (2012)CrossRefGoogle Scholar
  28. 28.
    Butterfield, C.N., Tao, L., Chacón, K.N., Spiro, T.G., Blackburn, N.J., Casey, W.H., Britt, R.D., Tebo, B.M.: Multicopper manganese oxidase accessory proteins bind Cu and heme. Biochim. Biophys. Acta – Prot. Proteom. 1854, 1853–1859 (2015)CrossRefGoogle Scholar
  29. 29.
    Quintyn, R.S., Yan, J., Wysocki, V.H.: Surface-induced dissociation of homotetramers with D 2 symmetry yields their assembly pathways and characterizes the effect of ligand binding. Chem. Biol. 22, 583–592 (2015)CrossRefGoogle Scholar
  30. 30.
    Popa, V., Trecroce, D.A., McAllister, R.G., Konermann, L.: Collision-induced dissociation of electrosprayed protein complexes: an all-atom molecular dynamics model with mobile protons. J. Phys. Chem. B. 120, 5114–5124 (2016)CrossRefGoogle Scholar
  31. 31.
    Butterfield, C.: Characterizing the Mn(II) oxidizing enzyme from the marine bacillus sp. PL-12 spore. Oregon Health Science University. PhD Thesis (2014)Google Scholar
  32. 32.
    Yan, J., Zhou, M., Gilbert, J.D., Wolff, J.J., Somogyi, Á., Pedder, R.E., Quintyn, R.S., Morrison, L.J., Easterling, M.L., Paša-Tolić, L., Wysocki, V.H.: Surface-induced dissociation of protein complexes in a hybrid Fourier transform ion cyclotron resonance mass spectrometer. Anal. Chem. 89, 895–901 (2017)CrossRefGoogle Scholar
  33. 33.
    Boldin, I.A., Nikolaev, E.N.: Fourier transform ion cyclotron resonance cell with dynamic harmonization of the electric field in the whole volume by shaping of the excitation and detection electrode assembly. Rapid Commun. Mass Spectrom. 25, 122–126 (2011)CrossRefGoogle Scholar
  34. 34.
    Galhena, A.S., Dagan, S., Jones, C.M., Beardsley, R.L., Wysocki, V.H.: Surface-induced dissociation of peptides and protein complexes in a quadrupole/time-of-flight mass spectrometer. Anal. Chem. 80, 1425–1436 (2008)CrossRefGoogle Scholar
  35. 35.
    Dongré, A.R., Somogyi, Á., Wysocki, V.H.: Surface-induced dissociation: an effective tool to probe structure, energetics, and fragmentation mechanisms of protonated peptides. J. Mass Spectrom. 31, 339–350 (1996)CrossRefGoogle Scholar
  36. 36.
    Sun, W., May, J.C., Russell, D.H.: A novel surface-induced dissociation instrument for ion mobility-time-of-flight mass spectrometry. Int. J. Mass Spectrom. 259, 79–86 (2007)CrossRefGoogle Scholar
  37. 37.
    Chorush, R.A., Little, D.P., Beu, S.C., Wood, T.D., McLafferty, F.W.: Surface-induced dissociation of multiply-protonated proteins. Anal. Chem. 67, 1042–1046 (1995)CrossRefGoogle Scholar
  38. 38.
    Zhou, M.: Incorporation of surface induced dissociation into a commercial ion mobility-tandem mass spectrometer and application of mass spectrometry methods for structural analysis of non-covalent protein complexes. Thesis (2013)Google Scholar
  39. 39.
    Xu, H., Freitas, M.A.: A mass accuracy sensitive probability based scoring algorithm for database searching of tandem mass spectrometry data. BMC Bioinformatics. 8, 133 (2007)CrossRefGoogle Scholar
  40. 40.
    Xu, H., Freitas, M.A.: MassMatrix: a database search program for rapid characterization of proteins and peptides from tandem mass spectrometry data. Proteomics. 9, 1548–1555 (2009)CrossRefGoogle Scholar
  41. 41.
    Kaufman Katz, A., Shimoni-Livny, L., Navon, O., Navon, N., Bock, C.W., Glusker, J.P.: Copper-binding motifs: structural and theoretical aspects. Helv. Chim. Acta. 86, 1320–1338 (2003)CrossRefGoogle Scholar
  42. 42.
    Viles, J.H.: Metal ions and amyloid fiber formation in neurodegenerative diseases. Copper, zinc, and iron in Alzheimer's, Parkinson's, and prion diseases. Coord. Chem. Rev. 256, 2271–2284 (2012)CrossRefGoogle Scholar
  43. 43.
    Geoghegan, K.F., Dixon, H.B.F., Rosner, P.J., Hoth, L.R., Lanzetti, A.J., Borzilleri, K.A., Marr, E.S., Pezzullo, L.H., Martin, L.B., LeMotte, P.K., McColl, A.S., Kamath, A.V., Stroh, J.G.: Spontaneous α-N-6-phosphogluconoylation of a “His Tag” in Escherichia coli:the cause of extra mass of 258 or 178 Da in fusion proteins. Anal. Biochem. 267, 169–184 (1999)CrossRefGoogle Scholar
  44. 44.
    Aon, J.C., Caimi, R.J., Taylor, A.H., Lu, Q., Oluboyede, F., Dally, J., Kessler, M.D., Kerrigan, J.J., Lewis, T.S., Wysocki, L.A.: Suppressing post-translational gluconoylation of heterologous proteins by metabolic engineering of Escherichia coli. Appl. Environ. Microbiol. 74, 950–958 (2008)Google Scholar
  45. 45.
    Schuchard, M.D., Mehigh, R.J., Cockrill, S.L., Lipscomb, G.T., Stephan, J.D., Wildsmith, J., Valdes-Camin, R., Kappel, W.K., Rai, A.J., Scott, G.B.I.: Artifactual isoform profile modification following treatment of human plasma or serum with protease inhibitor, monitored by 2-dimensional electrophoresis and mass spectrometry. BioTechniques. 39, 239–247 (2005)CrossRefGoogle Scholar
  46. 46.
    Chait, B.T., Cadene, M., Olinares, P.D., Rout, M.P., Shi, Y.: Revealing higher order protein structure using mass spectrometry. J. Am. Soc. Mass Spectrom. 27, 952–965 (2016)CrossRefGoogle Scholar
  47. 47.
    Qi, Y., Witt, M., Jertz, R., Baykut, G., Barrow, M.P., Nikolaev, E.N., O'Connor, P.B.: Absorption-mode spectra on the dynamically harmonized Fourier transform ion cyclotron resonance cell. Rapid Commun. Mass Spectrom. 26, 2021–2026 (2012)CrossRefGoogle Scholar
  48. 48.
    Qi, Y., Barrow, M.P., Li, H., Meier, J.E., Van Orden, S.L., Thompson, C.J., O’Connor, P.B.: Absorption-mode: the next generation of Fourier transform mass spectra. Anal. Chem. 84, 2923–2929 (2012)CrossRefGoogle Scholar
  49. 49.
    Li, H., 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)CrossRefGoogle Scholar
  50. 50.
    Li, H., Wongkongkathep, P., Van Orden, S., Ogorzalek Loo, R., Loo, J.: Revealing ligand binding sites and quantifying subunit variants of noncovalent protein complexes in a single native top-down FTICR MS experiment. J. Am. Soc. Mass Spectrom. 25, 2060–2068 (2014)CrossRefGoogle Scholar
  51. 51.
    Valeja, S.G., Kaiser, N.K., Xian, F., Hendrickson, C.L., Rouse, J.C., Marshall, A.G.: Unit mass baseline resolution for an intact 148 kDa therapeutic monoclonal antibody by Fourier transform ion cyclotron resonance mass spectrometry. Anal. Chem. 83, 8391–8395 (2011)CrossRefGoogle Scholar
  52. 52.
    Lössl, P., Snijder, J., Heck, A.J.: Boundaries of mass resolution in native mass spectrometry. J. Am. Soc. Mass Spectrom. 25, 906–917 (2014)CrossRefGoogle Scholar
  53. 53.
    Tao, L., Stich, T.A., Liou, S.-H., Soldatova, A.V., Delgadillo, D.A., Romano, C.A., Spiro, T.G., Goodin, D.B., Tebo, B.M., Casey, W.H., Britt, R.D.: Copper binding sites in the manganese-oxidizing mnx protein complex investigated by electron paramagnetic resonance spectroscopy. J. Am. Chem. Soc. 139, 8868-8877 (2017)Google Scholar
  54. 54.
    Butterfield, C.N., Tebo, B.M.: Substrate specificity and copper loading of the manganese-oxidizing multicopper oxidase Mnx from Bacillus sp. PL-12. Metallomics. 9, 183–191 (2017)CrossRefGoogle Scholar
  55. 55.
    O’Brien, J.P., Li, W., Zhang, Y., Brodbelt, J.S.: Characterization of native protein complexes using ultraviolet photodissociation mass spectrometry. J. Am. Chem. Soc. 136, 12920–12928 (2014)CrossRefGoogle Scholar
  56. 56.
    Zhang, H., Cui, W., Wen, J., Blankenship, R.E., Gross, M.L.: Native electrospray and electron-capture dissociation in FTICR mass spectrometry provide top-down sequencing of a protein component in an intact protein assembly. J. Am. Soc. Mass Spectrom. 21, 1966–1968 (2010)CrossRefGoogle Scholar
  57. 57.
    Breuker, K., McLafferty, F.W.: Native electron capture dissociation for the structural characterization of noncovalent interactions in native cytochrome c. Angew. Chem. Int. Ed. 42, 4900–4904 (2003)Google Scholar
  58. 58.
    Lermyte, F., Konijnenberg, A., Williams, J.P., Brown, J.M., Valkenborg, D., Sobott, F.: ETD allows for native surface mapping of a 150 kDa noncovalent complex on a commercial Q-TWIMS-TOF instrument. J. Am. Soc. Mass Spectrom. 25, 343–350 (2014)CrossRefGoogle Scholar
  59. 59.
    Li, H., Sheng, Y., McGee, W., Cammarata, M., Holden, D., Loo, J.A.: Structural characterization of native proteins and protein complexes by electron ionization dissociation-mass spectrometry. Anal. Chem. 89, 2731–2738 (2017)CrossRefGoogle Scholar
  60. 60.
    Belov, M.E., Damoc, E., Denisov, E., Compton, P.D., Horning, S., Makarov, A.A., Kelleher, N.L.: From protein complexes to subunit backbone fragments: a multi-stage approach to native mass spectrometry. Anal. Chem. 85, 11163–11173 (2013)CrossRefGoogle Scholar
  61. 61.
    Skinner, O.S., Havugimana, P.C., Haverland, N.A., Fornelli, L., Early, B.P., Greer, J.B., Fellers, R.T., Durbin, K.R., Do Vale, L.H.F., Melani, R.D., Seckler, H.S., Nelp, M.T., Belov, M.E., Horning, S.R., Makarov, A.A., Leduc, R.D., Bandarian, V., Compton, P.D., Kelleher, N.L.: An informatic framework for decoding protein complexes by top-down mass spectrometry. Nat. Methods. 13, 237–240 (2016)CrossRefGoogle Scholar
  62. 62.
    Belov, A.M., Viner, R., Santos, M.R., Horn, D.M., Bern, M., Karger, B.L., Ivanov, A.R.: Analysis of proteins, protein complexes, and organellar proteomes using sheathless capillary zone electrophoresis – native mass spectrometry. J. Am. Soc. Mass Spectrom. 28, 2614-2634 (2017)Google Scholar

Copyright information

© American Society for Mass Spectrometry (outside the USA) 2018

Authors and Affiliations

  • Mowei Zhou
    • 1
  • Jing Yan
    • 2
  • Christine A. Romano
    • 3
  • Bradley M. Tebo
    • 3
  • Vicki H. Wysocki
    • 2
  • Ljiljana Paša-Tolić
    • 1
  1. 1.Environmental Molecular Sciences Laboratory, Pacific Northwest National LaboratoryRichlandUSA
  2. 2.Department of Chemistry and BiochemistryOhio State UniversityColumbusUSA
  3. 3.Division of Environmental and Biomolecular SystemsOregon Health & Science UniversityPortlandUSA

Personalised recommendations

Cite article

Buy options