High-sensitivity Orbitrap mass analysis of intact macromolecular assemblies

Abstract

The analysis of intact protein assemblies in native-like states by mass spectrometry offers a wealth of information on their biochemical and biophysical properties. Here we show that the Orbitrap mass analyzer can be used to measure protein assemblies of molecular weights approaching one megadalton with sensitivity down to the detection of single ions. Minor instrumental modifications enabled the measurement of various protein assemblies with outstanding mass-spectral resolution.

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Figure 1: Orbitrap-based mass spectra of intact proteins and protein assemblies.
Figure 2: Orbitrap analyzer allows detection at the sensitivity of individual GroEL ions.
Figure 3: The mass resolution of the Orbitrap analyzer allows small molecules binding to large assemblies to be monitored.

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Acknowledgements

This work was supported in part by the PRIME-XS project, Grant Agreement Number 262067, and by the PROSPECTS network (grant HEALTH-F4-2008-201648) both funded by the European Union Seventh Framework Program. The Netherlands Proteomics Centre, embedded in The Netherlands Genomics Initiative, is acknowledged for funding. The authors thank Michael Gröll (Technische Universitat Munchen) for his kind donation of the 20S proteasome sample, Jack Johnson (Scripps Research Institute) for the HK97 capsomer sample, Genmab for the IgG antibody, and Uwe Rickens (Thermo Fisher Scientific) for developing IonDetectionConverter software for the single ion experiments.

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R.J.R., E.Da., E.De., A.M. and A.J.R.H. performed the experiments and wrote the manuscript. A.M. supervised the modifications on the Orbitrap mass analyzer. A.M. and A.J.R.H. conceptually designed the work.

Corresponding author

Correspondence to Albert J R Heck.

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Competing interests

E.Da., E.De. and A.M. are employees of Thermo Fisher Scientific, the manufacturer of the Exactive Plus instrument used in this research.

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Rose, R., Damoc, E., Denisov, E. et al. High-sensitivity Orbitrap mass analysis of intact macromolecular assemblies. Nat Methods 9, 1084–1086 (2012). https://doi.org/10.1038/nmeth.2208

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