ArticleAnalytical chemistry2020
Probing Protein Denaturation during Size-Exclusion Chromatography Using Native Mass Spectrometry.
Article in Analytical chemistry, 2020. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 26 papers.
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Who cites it
26 citing papers in PubMed, 66 citations in OpenAlex.
- Parallel Analysis of Acidic and Basic Proteoforms in Cell Lysates via Native Cation and Anion Exchange Chromatography-Native Mass Spectrometry.Proteomics · 2026Article
- Single-Ion Imaging Native Mass Spectrometry: Unraveling the Structural Features and Dissociation Energetics of Macromolecular Assemblies.Journal of the American Society for Mass Spectrometry · 2026Article
- Top-Down Mass Spectrometry and Its Current Applications in Biomarker Discovery in Aging and Age-Related Diseases.International journal of molecular sciences · 2026Review
- Development and Validation of SEC-UV/HRMS Procedure for Simultaneous Determination of BSA and Its Association Products.Molecules (Basel, Switzerland) · 2026Article
- Multiple Heart-Cut Ion-Exchange Chromatography-Reversed-Phase Liquid Chromatography Platform for Online Desalting and Fractionation of Monoclonal Antibody Charge Variants.Journal of separation science · 2026Article
- IscS Kinetics in Native Mass Spectrometry Buffers Reveal Key Physiochemical Properties that Influence Enzyme Activity.Journal of the American Society for Mass Spectrometry · 2026Article
- Stabilizing Proteins by Chemical Cross-Linking: Insights into Conformation, Unfolding, and Aggregation Using Native Ion Mobility Mass Spectrometry.Analytical chemistry · 2025Article
- Intraparticle diffusion of proteins in diol group-functionalized porous silica particle revealed by fluorescence correlation spectroscopy.Analytical sciences : the international journal of the Japan Society for Analytical Chemistry · 2025Article
- Developments in Ion Exchange Chromatography-Mass Spectrometry for the Characterization of Intact Proteins and Proteoforms.Journal of separation science · 2025Review
- Analytical strategies for identification and quantitation of heterodimers in co-formulated monoclonal antibody cocktails by native SEC-MS.Scientific reports · 2025Article
- Buffer 4-Ethylmorpholinium/Acetate: Exploring a New Alternative Buffer for Native Mass Spectrometry.Rapid communications in mass spectrometry : RCM · 2025Article
- Characterization of insulin and bile acid complexes in liposome by different mass spectrometry techniques.Analytical and bioanalytical chemistry · 2025Article
- In situ characterization of amine-forming enzymes shows altered oligomeric state.Protein science : a publication of the Protein Society · 2025Article
- [Applications of high performance liquid chromatography-mass spectrometry in proteomics].Se pu = Chinese journal of chromatography · 2024Review
- Characterization of Complex Proteoform Mixtures by Online Nanoflow Ion-Exchange Chromatography-Native Mass Spectrometry.Analytical chemistry · 2024Article
- Micro simulated moving bed chromatography-mass spectrometry as a continuous on-line process analytical tool.Analytical and bioanalytical chemistry · 2024Article
- Multidimensional Separations in Top-Down Proteomics.Analytical science advances · 2023Article
- Characterizing Non-covalent Protein Complexes Using Asymmetrical Flow Field-Flow Fractionation On-Line Coupled to Native Mass Spectrometry.Analytical chemistry · 2023Article
- Protein Interactome Profiling of Stable Molecular Complexes in Biomaterial Lysate.International journal of molecular sciences · 2022Review
- Studying protein structure and function by native separation-mass spectrometry.Nature reviews. Chemistry · 2022Review
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Authors and funding
7 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Size-exclusion chromatography employing aqueous mobile phases with volatile salts at neutral pH combined with electrospray-ionization mass spectrometry (SEC-ESI-MS) is a useful tool to study proteins in their native state. However, whether the applied eluent conditions actually prevent protein-stationary phase interactions, and/or protein denaturation, often is not assessed. In this study, the effects of volatile mobile phase additives on SEC retention and ESI of proteins were thoroughly investigated. Myoglobin was used as the main model protein, and eluents of varying ionic strength and pH were applied. The degree of interaction between protein and stationary phase was evaluated by calculating the SEC distribution coefficient. Protein-ion charge state distributions obtained during offline and online native ESI-MS were used to monitor alterations in protein structure. Interestingly, most of the supposedly mild eluent compositions induced nonideal SEC behavior and/or protein unfolding. SEC experiments revealed that the nature, ionic strength, and pH of the eluent affected protein retention. Protein-stationary phase interactions were effectively avoided using ammonium acetate at ionic strengths above 0.1 M. Direct-infusion ESI-MS showed that the tested volatile eluent salts seem to follow the Hofmeister series: no denaturation was induced using ammonium acetate (kosmotropic), whereas ammonium formate and bicarbonate (both chaotropic) caused structural changes. Using a mobile phase of 0.2 M ammonium acetate (pH 6.9), several proteins (i.e., myoglobin, carbonic anhydrase, and cytochrome c) could be analyzed by SEC-ESI-MS using different column chemistries without compromising their native state. Overall, with SEC-ESI-MS, the effect of nonspecific interactions between protein and stationary phase on the protein structure can be studied, even revealing gradual structural differences along a peak.
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