Mass Spectrometry and Its Applications

Jovcevski, Blagojce; Pukala, Tara L.

doi:10.1002/9781119483977.ch10

Cited by 3 publications

(3 citation statements)

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“…Native mass spectrometry (MS) has emerged as a powerful tool for characterizing biomolecular complexes. By preserving noncovalent interactions during electrospray ionization (ESI), native MS enables quantitation of protein complex stoichiometry, ligand binding, and lipid interactions. − It has been applied to a range of different complexes, including soluble proteins, membrane proteins, nucleic acids, , and lipid nanoparticles. , However, even under nondenaturing ESI conditions, labile interactions can be disrupted. Because lowering the charge of ions reduces electrostatic repulsions that drive dissociation in the gas phase, charge reduction is widely used to mitigate gas-phase dissociation and unfolding. − Charge reducing reagents may also improve stability of complexes by reducing the internal energy of ions through evaporative cooling as weakly bound adducts are released in the gas phase. , In addition to improving stability, charge reduction can also aid data analysis by reducing overlap between charge states and narrowing the charge state distribution, which improves the signal-to-noise ratio.…”

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confidence: 99%

Imidazole Derivatives Improve Charge Reduction and Stabilization for Native Mass Spectrometry

et al. 2019

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Noncovalent interactions between biomolecules are critical to their activity. Native mass spectrometry (MS) has enabled characterization of these interactions by preserving noncovalent assemblies for mass analysis, including protein–ligand and protein–protein complexes for a wide range of soluble and membrane proteins. Recent advances in native MS of lipoprotein nanodiscs have also allowed characterization of antimicrobial peptides and membrane proteins embedded in intact lipid bilayers. However, conventional native electrospray ionization (ESI) can disrupt labile interactions. To stabilize macromolecular complexes for native MS, charge reducing reagents can be added to the solution prior to ESI, such as triethylamine, trimethylamine oxide, and imidazole. Lowering the charge acquired during ESI reduces Coulombic repulsion that leads to dissociation, and charge reduction reagents may also lower the internal energy of the ions through evaporative cooling. Here, we tested a range of imidazole derivatives to discover improved charge reducing reagents and to determine how their chemical properties influence charge reduction efficacy. We measured their effects on a soluble protein complex, a membrane protein complex in detergent, and lipoprotein nanodiscs with and without embedded peptides, and used computational chemistry to understand the observed charge-reduction behavior. Together, our data revealed that hydrophobic substituents at the 2 position on imidazole can significantly improve both charge reduction and gas-phase stability over existing reagents. These new imidazole derivatives will be immediately beneficial for a range of native MS applications and provide chemical principles to guide development of novel charge reducing reagents.

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Imidazole Derivatives Improve Charge Reduction and Stabilization for Native Mass Spectrometry

et al. 2019

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“…3A). Native IM‐MS is a high‐resolution analytical technique capable of identifying the quaternary state and conformation of multimeric protein assemblies with high mass accuracy [26,27]. Here, native MS shows that both A. fumigatus unbound‐NDK and ADP‐NDK predominately forms a species with a molecular mass of 108.4 kDa (theoretical hexameric mass 108.1 kDa), corresponding to hexameric NDK (Fig.…”

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confidence: 95%

Nucleoside selectivity of Aspergillus fumigatus nucleoside‐diphosphate kinase

et al. 2020

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“…From the most fundamental perspective, an MS experiment involves the conversion of molecules into gas-phase ions, where they are separated in a mass analyzer according to their mass-to-charge ratios ( m/z ) [ 17 ]. For protein structural analysis, this technology was revolutionized with the advent of soft ionization techniques such as electrospray ionization (ESI), which enable large biomolecules to be carefully transferred into the gas phase while retaining solution phase relevance [ 18 ].…”

Section: Significance Of Protein Aggregationmentioning

confidence: 99%

Mass spectrometric insights into protein aggregation

Pukala

2023

Essays in Biochemistry

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Protein aggregation is now recognized as a generic and significant component of the protein energy landscape. Occurring through a complex and dynamic pathway of structural interconversion, the assembly of misfolded proteins to form soluble oligomers and insoluble aggregates remains a challenging topic of study, both in vitro and in vivo. Since the etiology of numerous human diseases has been associated with protein aggregation, and it has become a field of increasing importance in the biopharmaceutical industry, the biophysical characterization of protein misfolded states and their aggregation mechanisms continues to receive increased attention. Mass spectrometry (MS) has firmly established itself as a powerful analytical tool capable of both detection and characterization of proteins at all levels of structure. Given inherent advantages of biological MS, including high sensitivity, rapid timescales of analysis, and the ability to distinguish individual components from complex mixtures with unrivalled specificity, it has found widespread use in the study of protein aggregation, importantly, where traditional structural biology approaches are often not amenable. The present review aims to provide a brief overview of selected MS-based approaches that can provide a range of biophysical descriptors associated with protein conformation and the aggregation pathway. Recent examples highlight where this technology has provided unique structural and mechanistic understanding of protein aggregation.

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Mass Spectrometry and Its Applications

Cited by 3 publications

References 97 publications

Imidazole Derivatives Improve Charge Reduction and Stabilization for Native Mass Spectrometry

Imidazole Derivatives Improve Charge Reduction and Stabilization for Native Mass Spectrometry

Nucleoside selectivity of Aspergillus fumigatus nucleoside‐diphosphate kinase

Mass spectrometric insights into protein aggregation

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