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Collision-Induced Release, Ion Mobility Separation, and Amino Acid Sequence Analysis of Subunits from Mass-Selected Noncovalent Protein Complexes

  • Research Article
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Journal of The American Society for Mass Spectrometry

Abstract

In recent years, mass spectrometry has become a valuable tool for detecting and characterizing protein–protein interactions and for measuring the masses and subunit stoichiometries of noncovalent protein complexes. The gas-phase dissociation of noncovalent protein assemblies via tandem mass spectrometry can be useful in confirming subunit masses and stoichiometries; however, dissociation experiments that are able to yield subunit sequence information must usually be conducted separately. Here, we furnish proof of concept for a method that allows subunit sequence information to be directly obtained from a protein aggregate in a single gas-phase analysis. The experiments were carried out using a quadrupole time-of-flight mass spectrometer equipped with a traveling-wave ion mobility separator. This instrument configuration allows for a noncovalent protein assembly to be quadrupole selected, then subjected to two successive rounds of collision-induced dissociation with an intervening stage of ion mobility separation. This approach was applied to four model proteins as their corresponding homodimers: glucagon, ubiquitin, cytochrome c, and β-lactoglobulin. In each case, b- and y-type fragment ions were obtained upon further collisional activation of the collisionally-released subunits, resulting in up to 50% sequence coverage. Owing to the incorporation of an ion mobility separation, these results also suggest the intriguing possibility of measuring complex mass, complex collisional cross section, subunit masses, subunit collisional cross sections, and sequence information for the subunits in a single gas-phase experiment. Overall, these findings represent a significant contribution towards the realization of protein interactomic analyses, which begin with native complexes and directly yield subunit identities.

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Acknowledgments

Funding from the University of Nebraska, which was provided in part by the Nebraska Tobacco Settlement Biomedical Research Development Fund, is gratefully acknowledged. The authors also thank Yuting Huang for a critical reading of the manuscript.

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  1. Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE, 68588-0304, USA

    Deepali Rathore & Eric D. Dodds

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  1. Deepali Rathore
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Correspondence to Eric D. Dodds.

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Rathore, D., Dodds, E.D. Collision-Induced Release, Ion Mobility Separation, and Amino Acid Sequence Analysis of Subunits from Mass-Selected Noncovalent Protein Complexes. J. Am. Soc. Mass Spectrom. 25, 1600–1609 (2014). https://doi.org/10.1007/s13361-014-0946-4

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