Using covalent modifications to distinguish protein electrospray mechanisms: Charged residue model (CRM) vs. chain ejection model (CEM)

Pimlott, Douglas J.D.; Konermann, Lars

doi:10.1016/j.ijms.2021.116678

Cited by 7 publications

(14 citation statements)

References 86 publications

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“… These findings clearly illustrate that ESI charging is independent of surface properties, but rather reflects key features of protein solution structure. This interpretation is in good agreement with reports that chemical modifications of side-chain charges do not affect ESI charges of compactly folded protein ions. , To understand further the role of ionizable residues, we tested whether we could reduce the charge of EXG WT and EXG QQQW ions in the gas phase. Trimethylamine- N -oxide (TMAO) is a basic osmolyte that strips protons off ionized proteins through collisions in the gas phase. , After addition of 50 mM TMAO to the ESI solution, both proteins shift to lower charge states (Figure S3).…”

Section: Resultssupporting

confidence: 87%

“…This interpretation is in good agreement with reports that chemical modifications of side-chain charges do not affect ESI charges of compactly folded protein ions. 28 , 40 To understand further the role of ionizable residues, we tested whether we could reduce the charge of EXG WT and EXG QQQW ions in the gas phase. Trimethylamine- N -oxide (TMAO) is a basic osmolyte that strips protons off ionized proteins through collisions in the gas phase.…”

Section: Resultsmentioning

confidence: 99%

“…This interpretation is supported by recent observations from chemically modified proteins. 28 For native-like proteins, however, a lack of ionizable sites does not appear to alter the conformational landscape. An excess of ionizable side chains, on the other hand, has a direct impact on the conformational stability, as shown for three GFP variants.…”

Section: Discussionmentioning

confidence: 99%

“… 20 , 24 , 25 In positive-mode ESI, the number of charges acquired by native proteins scales with their solvent-accessible surface area (SASA), as predicted by the charge residue model (CRM), and is in virtually all cases lower than the number of basic surface residues. 26 − 28 Protein stability in solution depends on a delicate balance of surface electrostatics. 29 Therefore, the charging process can distort protein stability by adding Coulombic repulsion between ESI charges.…”

mentioning

confidence: 99%

“…Common to all of these applications is a reliance on the generation of native-like protein ions through electrospray ionization (ESI), where the protein is desolvated and charges are added to facilitate its detection in the mass spectrometer. A significant body of work over many decades has been dedicated to understanding the mechanism of protein charging in ESI and protein structures in the gas phase. − However, while the three-dimensional structures of protein complexes can be preserved for the time frame of an MS experiment, their surface charges inevitably differ between solution and gas phase. ,, In positive-mode ESI, the number of charges acquired by native proteins scales with their solvent-accessible surface area (SASA), as predicted by the charge residue model (CRM), and is in virtually all cases lower than the number of basic surface residues. − Protein stability in solution depends on a delicate balance of surface electrostatics . Therefore, the charging process can distort protein stability by adding Coulombic repulsion between ESI charges. − This discrepancy raises the question to what extent the surface properties of a protein can affect its behavior in the gas phase and thus in IM-MS. For example, drug conjugation of antibodies can alter their surface charges, and membrane proteins have a lower percentage and a more uneven distribution of ionizable residues on their surface than soluble proteins .…”

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confidence: 99%

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Charge Engineering Reveals the Roles of Ionizable Side Chains in Electrospray Ionization Mass Spectrometry

Abramsson

Sahin

Hopper

et al. 2021

JACS Au

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In solution, the charge of a protein is intricately linked to its stability, but electrospray ionization distorts this connection, potentially limiting the ability of native mass spectrometry to inform about protein structure and dynamics. How the behavior of intact proteins in the gas phase depends on the presence and distribution of ionizable surface residues has been difficult to answer because multiple chargeable sites are present in virtually all proteins. Turning to protein engineering, we show that ionizable side chains are completely dispensable for charging under native conditions, but if present, they are preferential protonation sites. The absence of ionizable side chains results in identical charge state distributions under native-like and denaturing conditions, while coexisting conformers can be distinguished using ion mobility separation. An excess of ionizable side chains, on the other hand, effectively modulates protein ion stability. In fact, moving a single ionizable group can dramatically alter the gas-phase conformation of a protein ion. We conclude that although the sum of the charges is governed solely by Coulombic terms, their locations affect the stability of the protein in the gas phase.

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Section: Resultssupporting

confidence: 87%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Charge Engineering Reveals the Roles of Ionizable Side Chains in Electrospray Ionization Mass Spectrometry

Abramsson

Sahin

Hopper

et al. 2021

JACS Au

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Mass spectrometry of polymers: A tutorial review

Wesdemiotis

Williams-Pavlantos

Keating

et al. 2023

Mass Spectrometry Reviews

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Ever since the inception of synthetic polymeric materials in the late 19th century, the number of studies on polymers as well as the complexity of their structures have only increased. The development and commercialization of new polymers with properties fine‐tuned for specific technological, environmental, consumer, or biomedical applications requires powerful analytical techniques that permit the in‐depth characterization of these materials. One such method with the ability to provide chemical composition and structure information with high sensitivity, selectivity, specificity, and speed is mass spectrometry (MS). This tutorial review presents and exemplifies the various MS techniques available for the elucidation of specific structural features in a synthetic polymer, including compositional complexity, primary structure, architecture, topology, and surface properties. Key to every MS analysis is sample conversion to gas‐phase ions. This review describes the fundamentals of the most suitable ionization methods for synthetic materials and provides relevant sample preparation protocols. Most importantly, structural characterizations via one‐step as well as hyphenated or multidimensional approaches are introduced and demonstrated with specific applications, including surface sensitive and imaging techniques. The aim of this tutorial review is to illustrate the capabilities of MS for the characterization of large, complex polymers and emphasize its potential as a powerful compositional and structural elucidation tool in polymer chemistry.

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Desalting strategies for native mass spectrometry

Li,

Zheng

et al. 2025

Talanta

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Using covalent modifications to distinguish protein electrospray mechanisms: Charged residue model (CRM) vs. chain ejection model (CEM)

Cited by 7 publications

References 86 publications

Charge Engineering Reveals the Roles of Ionizable Side Chains in Electrospray Ionization Mass Spectrometry

Charge Engineering Reveals the Roles of Ionizable Side Chains in Electrospray Ionization Mass Spectrometry

Mass spectrometry of polymers: A tutorial review

Desalting strategies for native mass spectrometry

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