Charge Reduction of Membrane Proteins in Native Mass Spectrometry Using Alkali Metal Acetate Salts

Petroff, John T.; Tong, Ailing; Chen, Lawrence J.; DeKoster, Gregory T.; Khan, Farha; Abramson, Jeff; Frieden, Carl; Cheng, Wayland W.L.

doi:10.1021/acs.analchem.0c00454

Cited by 9 publications

(11 citation statements)

References 57 publications

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“…Mixtures of AmtB in DDM with different charge-reducing molecules and at various concentrations could not generate interpretable mass spectra (Figure S2). This finding is consistent with a recent study that reports charge-reduction of membrane proteins with alkali metal acetate is dependent on detergent . However, the mechanism behind this phenomenon is not clear but a plausible explanation is that DDM may directly compete with these molecules interacting with the membrane protein.…”

Section: Resultssupporting

confidence: 92%

“…This finding is consistent with a recent study that reports chargereduction of membrane proteins with alkali metal acetate is dependent on detergent. 26 However, the mechanism behind this phenomenon is not clear but a plausible explanation is that DDM may directly compete with these molecules interacting with the membrane protein. Alternatively, the larger micelle size of DDM may prevent meaningful contacts between these molecules and the protein.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…Numerous studies have reported methods to achieve lower charge states of proteins for MS measurements. ,− Adding charge-reducing chemicals to protein samples, such as the abundant naturally occurring osmolyte − trimethylamine N -oxide (TMAO), has been shown to significantly lower the average charge state ( Z avg ) of protein complexes. ,, Imidazole and derivatives thereof have shown marginal charge reduction for membrane protein complexes (by about ∼2–3 charge states), especially in cases when noncharge-reducing detergents are used. , Besides the addition of small molecules, synthetic oligoglycerol detergents have been developed for membrane protein complexes that exhibit varying charge-reducing properties . Despite recent advances in manipulation of charge, these charge-reducing modalities have a number of limitations.…”

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confidence: 99%

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Discovery of Potent Charge-Reducing Molecules for Native Ion Mobility Mass Spectrometry Studies

et al. 2020

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There is growing interest in the characterization of protein complexes and their interactions with ligands using native ion mobility mass spectrometry. A particular challenge, especially for membrane proteins, is preserving noncovalent interactions and maintaining native-like structures. Different approaches have been developed to minimize activation of protein complexes by manipulating charge on protein complexes in solution and the gas-phase. Here, we report the utility of polyamines that have exceptionally high charge-reducing potencies with some molecules requiring 5-fold less than trimethylamine oxide to elicit the same effect. The charge-reducing molecules do not adduct to membrane protein complexes and are also compatible with ion-mobility mass spectrometry, paving the way for improved methods of charge reduction.

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Section: Resultssupporting

confidence: 92%

Section: ■ Results and Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Discovery of Potent Charge-Reducing Molecules for Native Ion Mobility Mass Spectrometry Studies

et al. 2020

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“…Here, alkylated ammonia or alkali metal acetate salts are being utilized. This furthermore prevents the undesired denaturation of labile proteins in the gas phase, as well as the loss of non-covalent interactions in native MS approaches, which could otherwise result in the loss of information on the protein structure [ 13 , 14 , 15 ].…”

Section: Top-down Methodsmentioning

confidence: 99%

Top-Down and Bottom-Up Proteomics Methods to Study RNA Virus Biology

Simanjuntak

Schamoni-Kast

Grün

et al. 2021

Viruses

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RNA viruses cause a wide range of human diseases that are associated with high mortality and morbidity. In the past decades, the rise of genetic-based screening methods and high-throughput sequencing approaches allowed the uncovering of unique and elusive aspects of RNA virus replication and pathogenesis at an unprecedented scale. However, viruses often hijack critical host functions or trigger pathological dysfunctions, perturbing cellular proteostasis, macromolecular complex organization or stoichiometry, and post-translational modifications. Such effects require the monitoring of proteins and proteoforms both on a global scale and at the structural level. Mass spectrometry (MS) has recently emerged as an important component of the RNA virus biology toolbox, with its potential to shed light on critical aspects of virus–host perturbations and streamline the identification of antiviral targets. Moreover, multiple novel MS tools are available to study the structure of large protein complexes, providing detailed information on the exact stoichiometry of cellular and viral protein complexes and critical mechanistic insights into their functions. Here, we review top-down and bottom-up mass spectrometry-based approaches in RNA virus biology with a special focus on the most recent developments in characterizing host responses, and their translational implications to identify novel tractable antiviral targets.

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“…Petroff et al used alkali metal acetate salts at low millimolar concentrations for charge reduction of membrane protein ion channels . They discovered that charge reduction was dependent on the detergent, showing the highest reduction with polyethylene glycol (PEG)-based detergents and the least effect with LDAO.…”

Section: Ionization: From Solution Into the Gas Phasementioning

confidence: 99%