Gas-Phase Collisions with Trimethylamine-<i>N</i>-Oxide Enable Activation-Controlled Protein Ion Charge Reduction

Kaldmäe, Margit; Österlund, Nicklas; Lianoudaki, Danai; Sahin, Cagla; Bergman, Peter; Nyman, T.; Kronqvist, Nina; Ilag, Leopold L.; Allison, Timothy M.; Marklund, Erik; Landreh, Michael

doi:10.1007/s13361-019-02177-8

Cited by 17 publications

(27 citation statements)

References 18 publications

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“…To elucidate the mechanism of charge reduction by alkali metals in ELIC, we analyzed the effect of other charge-reducing agents, including triethylamine (TEA), imidazole, and trimethylamine N -oxide (TMAO). ,,, Additionally, we examined the possibility that charge reduction is occurring through a specific interaction with the protein by comparing the aforementioned charge-reducing agents with the effect of a known agonist of ELIC, cysteamine . Imidazole, TEA, TMAO, and cysteamine were separately introduced, and the respective samples were analyzed by native MS.…”

Section: Resultsmentioning

confidence: 99%

“…Altering the detergent to polyethylene glycol (PEG)-based or amine N -oxide amphiphiles affords meaningful charge reduction; even so, the degree of charge reduction may be insufficient to prevent the deleterious effects of excessive Coulombic repulsion . To further reduce charge, small molecules are added to the protein solution including triethylamine (TEA), imidazole, and trimethylamine N -oxide (TMAO). ,,, Recently, Townsend et al reported a new host of imidazole analogues that aid in charge reduction and afford peptide–nanodisc stability . However, these small amine-containing molecules may interact with the protein and result in undesired effects.…”

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

et al. 2020

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Native mass spectrometry paired with ion mobility (IM-MS) provides the capacity to monitor the structure of protein complexes and simultaneously assess small molecule binding to the protein. Native IM-MS typically utilizes positive mode electrospray ionization producing a distribution of multiply charged protein species. For membrane proteins, these charge states are often too high resulting in protein gas phase unfolding or loss of noncovalent interactions. In an effort to reduce the charge of membrane proteins, the utility of alkali metal salts as a charge reducing additive was explored. Low concentrations of alkali metal salts caused marked charge reduction in the membrane protein, ELIC. The charge reducing effect was only present in membrane proteins, and could not be accounted for by conformational changes in ELIC structure. Charge reduction by alkali metal salts was also detergent dependent, and was most pronounced in long PEG-based detergents such as C10E5 and C12E8. Based on these results, a mechanism was posited for alkali metal charge reduction of membrane proteins. Addition of low concentration of alkali metals may provide an advantageous approach for charge reduction of detergent solubilized membrane proteins by native MS. File list (2) download file view on ChemRxiv Manuscript Petroff and Cheng.pdf (872.09 KiB) download file view on ChemRxiv SI Petroff and Cheng.pdf (1.33 MiB)

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Section: Resultsmentioning

confidence: 99%

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

et al. 2020

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“…The most commonly used method is to add charge reducing reagents to solution prior to ESI. Imidazole and triethylamine (TEA) are commonly used, but recent work has shown that trimethylamine oxide (TMAO) can be a potent charge reducing reagent. − However, TMAO tends to form adducts with protein complexes, which is an important component of its activity …”

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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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“…Trimethylamine- N -oxide (TMAO) is a basic osmolyte that strips protons off ionized proteins through collisions in the gas phase. 41 , 42 After addition of 50 mM TMAO to the ESI solution, both proteins shift to lower charge states ( Figure S3 ). However, we also observed that the intensity of the EXG QQQW peaks was significantly reduced.…”

Section: Resultsmentioning

confidence: 99%

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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Gas-Phase Collisions with Trimethylamine-N-Oxide Enable Activation-Controlled Protein Ion Charge Reduction

Cited by 17 publications

References 18 publications

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

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

Imidazole Derivatives Improve Charge Reduction and Stabilization for Native Mass Spectrometry

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

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