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Using nanoelectrospray ion mobility spectrometry (GEMMA) to determine the size and relative molecular mass of proteins and protein assemblies: a comparison with MALLS and QELS

Analytical and Bioanalytical Chemistry volume 399, pages 2421–2433 (2011)Cite this article

Abstract

The determination of protein assembly size and relative molecular mass is currently of great importance in biochemical analysis. In particular, the technique of nanoelectrospray (nES) with a gas-phase electrophoretic mobility molecular analyzer (GEMMA) has received increased attention for such measurements. However, in order for the GEMMA technique to gain broader acceptance in protein analysis, it must be further evaluated and compared with other established bioanalytical techniques. In the present study, nES-GEMMA was evaluated for the analysis of a set of protein and protein complexes involved in the Sec and the bacterial type III secretion pathway of enteropathogenic Escherichia coli bacteria. The same set of proteins, isolated and purified using standard biochemical protocols, were also analyzed using multi-angle laser light scattering (MALLS) and quasi-elastic light scattering (QELS), following size exclusion chromatography. This allowed for direct comparisons between the three techniques. It was found that nES-GEMMA, in comparison to the more established MALLS and QELS techniques, offers several complementary advantages. It requires considerably less amount of material, i.e., nanogram vs. milligram amounts, and time per sample analysis, i.e., few minutes vs. tens of minutes. Whereas the determined size and relative molecular mass are similar between the compared methods, the electrophoretic diameters determined using nES-GEMMA seem to be systematically smaller compared to the hydrodynamic diameter derived by QELS. Some of the GEMMA technique disadvantages include its narrow dynamic range, limited by the fact that at elevated protein concentrations there is increased potential for the occurrence of nES-induced oligomers. Thus, it is preferred to analyze dilute protein solutions because non-specific oligomers are less likely to occur whereas biospecific oligomers remain detected. To further understand the formation of nES-oligomers, the effect of buffer concentration on their formation was evaluated. Also, nES-GEMMA is not compatible with all the buffers commonly used with MALLS and QELS. Overall, however, the nES-GEMMA technique shows promise as a high-throughput proteomics/protein structure tool.

NanoES-GEMMA electropherogram of SecA protein dimer

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References

  1. Robinson CV, Sali A, Baumeister W (2007) The molecular sociology of the cell. Nature 450(7172):973–982

    CAS  Article  Google Scholar 

  2. Devos D, Russell RB (2007) A more complete, complexed and structured interactome. Curr Opin Struct Biol 17(3):370–377

    CAS  Article  Google Scholar 

  3. Kocher T, Superti-Furga G (2007) Mass spectrometry-based functional proteomics: from molecular machines to protein networks. Nat Methods 4(10):807–815

    Article  Google Scholar 

  4. Bacher G, Szymanski WW, Kaufman SL, Zollner P, Blaas D, Allmaier G (2001) Charge-reduced nano electrospray ionization combined with differential mobility analysis of peptides, proteins, glycoproteins, noncovalent protein complexes and viruses. J Mass Spectrom 36(9):1038–1052

    CAS  Article  Google Scholar 

  5. de la Mora JF, Ude S, Thomson BA (2006) The potential of differential mobility analysis coupled to MS for the study of very large singly and multiply charged proteins and protein complexes in the gas phase. Biotechnol J 1(9):988–997

    Article  Google Scholar 

  6. Kaufman SL, Skogen JW, Dorman FD, Zarrin F, Lewis KC (1996) Macromolecule analysis based on electrophoretic mobility in air: globular proteins. Anal Chem 68(11):1895–1904

    CAS  Article  Google Scholar 

  7. Sharon M, Robinson CV (2007) The role of mass spectrometry in structure elucidation of dynamic protein complexes. Annu Rev Biochem 76:167–193

    CAS  Article  Google Scholar 

  8. Loo JA (1997) Studying noncovalent protein complexes by electrospray ionization mass spectrometry. Mass Spectrom Rev 16(1):1–23

    CAS  Article  Google Scholar 

  9. Kaufman SL, Kuchumov AR, Kazakevich M, Vinogradov SN (1998) Analysis of a 3.6-MDa hexagonal bilayer hemoglobin from Lumbricus terrestris using a gas-phase electrophoretic mobility molecular analyzer. Anal Biochem 259(2):195–202

    CAS  Article  Google Scholar 

  10. Benesch JL, Robinson CV (2006) Mass spectrometry of macromolecular assemblies: preservation and dissociation. Curr Opin Struct Biol 16(2):245–251

    CAS  Article  Google Scholar 

  11. Ruotolo BT, Robinson CV (2006) Aspects of native proteins are retained in vacuum. Curr Opin Chem Biol 10(5):402–408

    CAS  Article  Google Scholar 

  12. Kaddis CS, Loo JA (2007) Native protein MS and ion mobility: large flying proteins with ESI. Anal Chem 79(5):1778–1784

    CAS  Article  Google Scholar 

  13. Allmaier G, Laschober C, Szymanski WW (2008) Nano ES GEMMA and PDMA, new tools for the analysis of nanobioparticles–protein complexes, lipoparticles, and viruses. J Am Soc Mass Spectrom 19(8):1062–1068

    CAS  Article  Google Scholar 

  14. Laschober C, Wruss J, Blaas D, Szymanski WW, Allmaier G (2008) Gas-phase electrophoretic molecular mobility analysis of size and stoichiometry of complexes of a common cold virus with antibody and soluble receptor molecules. Anal Chem 80(6):2261–2264

    CAS  Article  Google Scholar 

  15. Scalf M (1999) Controlling charge states of large ions. Science 283(5410):2105

    Google Scholar 

  16. Reischl GP, Makela JM, Karch R, Necid J (1996) Bipolar charging of ultrafine particles in the size range below 10 nm. J Aerosol Sci 27(6):931–949

    CAS  Article  Google Scholar 

  17. Fuchs NA (1963) On the stationary charge distribution on aerosol particles in bipolar ionic atmosphere. Pure Appl Geophys 56(1):185–193

    Article  Google Scholar 

  18. Agarwal JK, Sem GJ (1980) Continuous-flow, single-particle-counting condensation nucleus counter. J Aerosol Sci 11(4):343

    CAS  Article  Google Scholar 

  19. Kesten J, Reineking A, Porstendorfer J (1991) Calibration of a TSI-model-3025 ultrafine condensation particle counter. Aerosol sci tech 15(2):107–111

    CAS  Article  Google Scholar 

  20. Carazzone C, Raml R, Pergantis SA (2008) Nanoelectrospray ion mobility spectrometry online with inductively coupled plasma–mass spectrometry for sizing large proteins, DNA, and nanoparticles. Anal Chem 80(15):5812–5818

    CAS  Article  Google Scholar 

  21. Thomas JJ, Bothner B, Traina J, Benner WH, Siuzdak G (2004) Electrospray ion mobility spectrometry of intact viruses. Spectrosc Int J 18(1):31–36

    CAS  Google Scholar 

  22. Loo JA, Berhane B, Kaddis CS, Wooding KM, Xie Y, Kaufman SL, Chernushevich IV (2005) Electrospray ionization mass spectrometry and ion mobility analysis of the 20S proteasome complex. J Am Soc Mass Spectrom 16(7):998–1008

    CAS  Article  Google Scholar 

  23. Kaddis CS, Lomeli SH, Yin S, Berhane B, Apostol MI, Kickhoefer VA, Rome LH, Loo JA (2007) Sizing large proteins and protein complexes by electrospray ionization mass spectrometry and ion mobility. J Am Soc Mass Spectrom 18(7):1206–1216

    CAS  Article  Google Scholar 

  24. Hogan CJ Jr, Kettleson EM, Ramaswami B, Chen DR, Biswas P (2006) Charge reduced electrospray size spectrometry of mega- and gigadalton complexes: whole viruses and virus fragments. Anal Chem 78(3):844–852

    CAS  Article  Google Scholar 

  25. Wick CH, McCubbin PE (1999) Characterization of purified MS2 bacteriophage by the physical counting methodology used in the integrated virus detection system (IVDS). Toxicol methods 9(4):245–252

    CAS  Article  Google Scholar 

  26. Muller R, Laschober C, Szymanski WW, Allmaier G (2007) Determination of molecular weight, particle size, and density of high number generation PAMAM dendrimers using MALDI-TOF-MS and nES-GEMMA. Macromolecules 40(15):5599–5605

    Article  Google Scholar 

  27. Saucy DA, Ude S, Lenggoro IW, de la Mora JF (2004) Mass analysis of water-soluble polymers by mobility measurement of charge-reduced ions generated by electrosprays. Anal Chem 76(4):1045–1053

    CAS  Article  Google Scholar 

  28. Poderycki MJ, Kickhoefer VA, Kaddis CS, Raval-Fernandes S, Johansson E, Zink JI, Loo JA, Rome LH (2006) The vault exterior shell is a dynamic structure that allows incorporation of vault-associated proteins into its interior. Biochemistry 45(39):12184–12193

    CAS  Article  Google Scholar 

  29. Karamanou S, Vrontou E, Sianidis G, Baud C, Roos T, Kuhn A, Politou AS, Economou A (1999) A molecular switch in SecA protein couples ATP hydrolysis to protein translocation. Mol Microbiol 34(5):1133–1145

    CAS  Article  Google Scholar 

  30. Schenkman L, Koukaki M, Karamanou S, Economou A. The P. CÉZANNE Project: Innovative approaches to continuous glucose monitoring. Conference Proceedings of the IEEE Engineering in Medicine and Biology Society, Tel Aviv, 22–26 Aug. 2007, Tel Aviv 2007, pp 6060–6063

  31. Sardis MF, Economou A (2010) SecA: a tale of two protomers. Mol Microbiol 76(5):1070–1081

    CAS  Article  Google Scholar 

  32. Tian Y, Cuneo MJ, Changela A, Hocker B, Beese LS, Hellinga HW (2007) Structure-based design of robust glucose biosensors using a Thermotoga maritima periplasmic glucose-binding protein. Protein Sci 16(10):2240–2250

    CAS  Article  Google Scholar 

  33. Wachter RM, Elsliger MA, Kallio K, Hanson GT, Remington SJ (1998) Structural basis of spectral shifts in the yellow-emission variants of green fluorescent protein. Structure 6(10):1267–1277

    CAS  Article  Google Scholar 

  34. Liu J, Walsh CT (1990) Peptidyl-prolyl cis-trans-isomerase from Escherichia coli: a periplasmic homolog of cyclophilin that is not inhibited by cyclosporin A. Proc Natl Acad Sci USA 87(11):4028–4032

    CAS  Article  Google Scholar 

  35. Yip CK, Finlay BB, Strynadka NC (2005) Structural characterization of a type III secretion system filament protein in complex with its chaperone. Nat Struct Mol Biol 12(1):75–81

    CAS  Article  Google Scholar 

  36. Gelis I, Bonvin AM, Keramisanou D, Koukaki M, Gouridis G, Karamanou S, Economou A, Kalodimos CG (2007) Structural basis for signal-sequence recognition by the translocase motor SecA as determined by NMR. Cell 131(4):756–769

    CAS  Article  Google Scholar 

  37. Wyttenbach T, Bowers MT (2009) Hydration of biomolecules. Chem phys lett 480(1–3):1–16

    CAS  Article  Google Scholar 

  38. Sianidis G, Karamanou S, Vrontou E, Boulias K, Repanas K, Kyrpides N, Politou AS, Economou A (2001) Cross-talk between catalytic and regulatory elements in a DEAD motor domain is essential for SecA function. EMBO J 20(5):961–70

    CAS  Article  Google Scholar 

  39. Heck AJ, Van Den Heuvel RH (2004) Investigation of intact protein complexes by mass spectrometry. Mass Spectrom Rev 23(5):368–389

    CAS  Article  Google Scholar 

  40. McKay AR, Ruotolo BT, Ilag LL, Robinson CV (2006) Mass measurements of increased accuracy resolve heterogeneous populations of intact ribosomes. J Am Chem Soc 128(35):11433–11442

    CAS  Article  Google Scholar 

  41. Woodbury RL, Hardy SJ, Randall LL (2002) Complex behavior in solution of homodimeric SecA. Protein Sci 11(4):875–882

    CAS  Article  Google Scholar 

  42. Xu Z, Knafels JD, Yoshino K (2000) Crystal structure of the bacterial protein export chaperone secB. Nat Struct Biol 7(12):1172–1177

    CAS  Article  Google Scholar 

  43. Goosney DL, Gruenheid S, Finlay BB (2000) Gut feelings: enteropathogenic E. coli (EPEC) interactions with the host. Annu Rev Cell Dev Biol 16:173–189

    CAS  Article  Google Scholar 

  44. Spears KJ, Roe AJ, Gally DL (2006) A comparison of enteropathogenic and enterohaemorrhagic Escherichia coli pathogenesis. FEMS Microbiol Lett 255(2):187–202

    CAS  Article  Google Scholar 

  45. Kemptner J, Marchetti-Deschmann M, Siekmann J, Turecek PL, Schwarz HP, Allmaier G (2010) GEMMA and MALDI-TOF MS of reactive PEGs for pharmaceutical applications. J Pharm Biomed Anal 52(4):432–437

    CAS  Article  Google Scholar 

  46. Pease LF 3rd, Elliott JT, Tsai DH, Zachariah MR, Tarlov MJ (2008) Determination of protein aggregation with differential mobility analysis: application to IgG antibody. Biotechnol Bioeng 101(6):1214–1222

    CAS  Article  Google Scholar 

  47. Gamero-Castano M, de la Mora JF (2000) Mechanisms of electrospray ionization of singly and multiply charged salt clusters. Anal Chim Acta 406(1):67–91

    CAS  Article  Google Scholar 

  48. Dole M, Mack LL, Hines RL, Mobley RC, Ferguson LD, Alice MB (1968) Molecular beams of macroions. J Chem Phys 49:2240–2249

    CAS  Article  Google Scholar 

  49. Mouradian S, Skogen JW, Dorman FD, Zarrin F, Kaufman SL, Smith LM (1997) DNA analysis using an electrospray scanning mobility particle sizer. Anal Chem 69(5):919–925

    CAS  Article  Google Scholar 

  50. Kaufman SL (1998) Analysis of biomolecules using electrospray and nanoparticle methods: the gas-phase electrophoretic mobility molecular analyzer (GEMMA). J Aerosol Sci 29(5–6):537–552

    CAS  Article  Google Scholar 

  51. de la Mora JF (2000) Electrospray ionization of large multiply charged species proceeds via Dole’s charged residue mechanism. Anal Chim Acta 406(1):93–104

    Article  Google Scholar 

  52. Iribarne JV, Thomson BA (1976) Evaporation of small ions from charged droplets. J Chem Phys 64(6):2287–2294

    CAS  Article  Google Scholar 

  53. Thomson BA, Iribarne JV, Dziedzic PJ (1982) Liquid ion evaporation/mass spectrometry/mass spectrometry for the detection of polar and labile molecules. Anal Chem 54(13):2219–2224

    CAS  Article  Google Scholar 

  54. Chen DR, Pui DYH (1997) Experimental investigation of scaling laws for electrospraying: dielectric constant effect. Aerosol sci tech 27(3):367–380

    CAS  Article  Google Scholar 

  55. TSI Inc. (2006) Operation and service manual of macroion mobility spectrometer. P/N 1930110, RevA ed.

  56. Juraschek R, Dulcks T, Karas M (1999) Nanoelectrospray—more than just a minimized-flow electrospray ionization source. J Am Soc Mass Spectrom 10(4):300–308

    CAS  Article  Google Scholar 

  57. Karamanou S, Sianidis G, Gouridis G, Pozidis C, Papanikolau Y, Papanikou E, Economou A (2005) Escherichia coli SecA truncated at its termini is functional and dimeric. FEBS Lett 579(5):1267–1271

    CAS  Article  Google Scholar 

  58. Zacharias DA, Violin JD, Newton AC, Tsien RY (2002) Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296(5569):913–916

    CAS  Article  Google Scholar 

  59. Clubb RT, Ferguson SB, Walsh CT, Wagner G (1994) Three-dimensional solution structure of Escherichia coli periplasmic cyclophilin. Biochemistry 33(10):2761–2772

    CAS  Article  Google Scholar 

  60. Li H, Yang F, Kang X, Xia B, Jin C (2008) Solution structures and backbone dynamics of Escherichia coli rhodanese PspE in its sulfur-free and persulfide-intermediate forms: implications for the catalytic mechanism of rhodanese. Biochemistry 47(15):4377–4385

    CAS  Article  Google Scholar 

Download references

Acknowledgments

We are grateful to Dr. M. Papanastasiou for discussions, to Dr. G. Gouridis for help with the MALLS instrument, and to members of the Economou laboratory for protein samples. The research leading to these results has received funding from the European Commission (EC) through the funding of a Marie Curie Excellence Grant MEXT-CT-2003-002788 (to S.A.P.) and from the EC’s Sixth Framework Programme agreement no. LSHC-CT-2006-037834 “Streptomics” (to A.E.), the Greek General Secretariat of Research and the European Regional Development Fund (PENED03ED623) (to A.E.), and from the European Community’s Sixth Framework Programme agreement 031867–Integrated Project P.Cezanne (to A.E.). The proteomics facility of the IMBB was established through the European Community’s Seventh Framework Programme agreement 229823 Capacities-FP7-REGPOT-2008-1/project “ProFI”. M.F.S. is an Onassis foundation pre-doctoral fellow. Also, E.A.K. thanks the Greek Ministry of Education, Lifelong Learning and Religious Affairs for funding an Hrakleitos II PhD studentship.

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Authors and Affiliations

  1. Environmental Chemical Processes Laboratory, Department of Chemistry, University of Crete, Voutes Campus, 71003, Heraklion, Crete, Greece

    E. A. Kapellios & S. A. Pergantis

  2. IMBB-FORTH, Vassilika Vouton, 71110, Heraklion, Crete, Greece

    S. Karamanou, M. F. Sardis, M. Aivaliotis & A. Economou

  3. Department of Biology, University of Crete, Voutes Campus, 71003, Heraklion, Crete, Greece

    M. F. Sardis & A. Economou

Authors
  1. E. A. Kapellios
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  2. S. Karamanou
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  3. M. F. Sardis
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  4. M. Aivaliotis
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  5. A. Economou
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  6. S. A. Pergantis
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Correspondence to A. Economou or S. A. Pergantis.

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Kapellios, E.A., Karamanou, S., Sardis, M.F. et al. Using nanoelectrospray ion mobility spectrometry (GEMMA) to determine the size and relative molecular mass of proteins and protein assemblies: a comparison with MALLS and QELS. Anal Bioanal Chem 399, 2421–2433 (2011). https://doi.org/10.1007/s00216-010-4634-3

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Keywords

  • Bioanalytical methods
  • Mass spectrometry/ICP-MS
  • Aerosols/particulates