Pulsed Hydrogen/Deuterium Exchange MS/MS for Studying the Relationship between Noncovalent Protein Complexes in Solution and in the Gas Phase after Electrospray Ionization
- 26 January 2006
- journal article
- Published by American Chemical Society (ACS) in Analytical Chemistry
- Vol. 78 (5), 1613-1619
- https://doi.org/10.1021/ac051687e
Abstract
Electrospray ionization mass spectrometry (ESI-MS) has become a standard method for monitoring noncovalent protein−protein interactions. Studies employing this approach tend to operate on the premise that the ionic species observed in the mass spectrum directly reflect the corresponding solution-phase protein quaternary structures. However, dissociation or clustering events taking place during ESI may lead to disparities between the ions observed in the mass spectrum and the protein binding state in bulk solution. Recognizing the occurrence of dissociation or clustering artifacts is not straightforward, leading to possible ambiguities in the interpretation of ESI-MS data. This work employs on-line pulsed hydrogen−deuterium exchange (HDX) for probing the origin of various species in the ESI mass spectrum of hemoglobin. In addition to the canonical hemoglobin tetramer, ESI-MS reveals the presence of monomers, dimers, hexamers, and octamers. Tandem mass spectrometry (MS/MS) is used for extracting HDX levels in a subunit-specific manner. Dimeric species exhibit exchange levels that are significantly above those of the tetramer. Monomeric hemoglobin subunits are labeled to an even greater extent. This HDX pattern implies that monomers and dimers do not represent dissociation artifacts generated during ESI. Instead, they are derived from preexisting solution-phase structures. In contrast, hexamers and octamers exhibit HDX levels that resemble those of the tetramer, thus identifying these larger species as nonspecific clustering artifacts. Overall, it appears that the pulsed HDX MS/MS approach introduced in this work represents a widely applicable tool for deciphering the relationship between ESI mass spectra and protein quaternary structures in solution.Keywords
This publication has 37 references indexed in Scilit:
- Determination of zinc to beta‐peptide binding constants with electrospray ionization mass spectrometryJournal of Mass Spectrometry, 2005
- Subunit Disassembly and Unfolding Kinetics of Hemoglobin Studied by Time-Resolved Electrospray Mass SpectrometryBiochemistry, 2004
- Direct Mass Spectrometric Determination of the Stoichiometry and Binding Affinity of the Complexes between Nucleocapsid Protein and RNA Stem−Loop Hairpins of the HIV-1 Ψ-Recognition ElementBiochemistry, 2003
- Highly Asymmetric Interactions between Globin Chains during Hemoglobin Assembly Revealed by Electrospray Ionization Mass SpectrometryBiochemistry, 2003
- Origin of Asymmetric Charge Partitioning in the Dissociation of Gas-Phase Protein HomodimersJournal of the American Chemical Society, 2003
- Global Analysis of Protein Activities Using Proteome ChipsScience, 2001
- The effect of the source pressure on the abundance of ions of noncovalent protein assemblies in an electrospray ionization orthogonal time‐of‐flight instrumentRapid Communications in Mass Spectrometry, 2001
- Changes in bulk solution pH caused by the inherent controlled-current electrolytic process of an electrospray ion sourceInternational Journal of Mass Spectrometry and Ion Processes, 1997
- Probing the Nature of Noncovalent Interactions by Mass Spectrometry. A Study of Protein−CoA Ligand Binding and AssemblyJournal of the American Chemical Society, 1996
- Oxidation of methionyl residues in proteins: Tools, targets, and reversalFree Radical Biology & Medicine, 1995