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Insights into the Mechanism of Protein Electrospray Ionization From Salt Adduction Measurements

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Journal of The American Society for Mass Spectrometry

Abstract

The mechanisms whereby protein ions are liberated from charged droplets during electrospray ionization (ESI) remain under investigation. Compact conformers electrosprayed from aqueous solution in positive ion mode likely follow the charged residue model (CRM), which envisions analyte release after solvent evaporation to dryness. The concentration of nonvolatile salts such as NaCl increases sharply within vanishing CRM droplets, promoting nonspecific pairing of Cl- and Na+ with charged groups on the protein surface. For unfolded proteins, it has been proposed that ion formation occurs via the chain ejection model (CEM). During the CEM proteins are expelled from the droplet long before complete solvent evaporation has taken place. Here we examine whether salt adduction levels support the view that folded and unfolded proteins follow different ESI mechanisms. Solvent evaporation during the CEM is expected to be less extensive and, hence, the salt concentration at the point of protein release should be substantially lower than for the CRM. CEM ions should therefore exhibit lower adduction levels than CRM species. We explore the adduction behavior of several proteins that were chosen to allow comparative studies on folded and unfolded structures in the same solution. In-source activation eliminates chloride adducts via HCl release, generating protein ions that are heterogeneously charged because of sodiation and protonation. Sodiation levels measured under such conditions provide estimates of the salt adduction behavior experienced by the “nascent” analyte ions. Sodiation levels are significantly reduced for unfolded proteins, supporting the view that these species are indeed formed via the CEM.

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Acknowledgments

Funding was provided by the Natural Sciences and Engineering Research Council of Canada, the Canada Foundation for Innovation, and the Canada Research Chairs Program.

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Authors and Affiliations

  1. Department of Chemistry, The University of Western Ontario, London, ON, N6A 5B7, Canada

    Xuanfeng Yue, Siavash Vahidi & Lars Konermann

  2. Key Laboratory of Analytical Chemistry for Life Science of the Shaanxi Province, Shaanxi Normal University, Xi’an, 710062, China

    Xuanfeng Yue

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  1. Xuanfeng Yue
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Correspondence to Lars Konermann.

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Xuanfeng Yue and Siavash Vahidi contributed equally to this work.

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Yue, X., Vahidi, S. & Konermann, L. Insights into the Mechanism of Protein Electrospray Ionization From Salt Adduction Measurements. J. Am. Soc. Mass Spectrom. 25, 1322–1331 (2014). https://doi.org/10.1007/s13361-014-0905-0

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