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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

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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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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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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  • DOI: https://doi.org/10.1007/s00216-010-4634-3

Keywords

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