Structural Effects of Solvation by 18-Crown-6 on Gaseous Peptides and TrpCage after Electrospray Ionization

Bonner, J.G.; Hendricks, Nathan; Julian, Ryan R.

doi:10.1007/s13361-016-1456-3

Cited by 7 publications

(13 citation statements)

References 38 publications

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“…We predict that these conditions will lower the extent of protein charging in the presence of SCAs. 18-crown-6 (18C6) is of particular interest due to its ability to accommodate ESI-relevant species (Na + , NH 4 + , and H 3 O + ) in solution and in the gas phase. ,− Previous studies explored 18C6 binding to Lys + and N + -termini of gaseous peptides or proteins, − but the consequences of crown ethers for the ESI process remain largely unexplored . The MD simulations of this work, as well as experiments on proteins and dendrimers, support the proposed hypothesis.…”

supporting

confidence: 62%

“…At this point, residual Na + and [18C6 + Na] + underwent ion pairing with protein carboxylates, while nonsodiated 18C6 bound to Lys + (Figure S6). − These MD-predicted 18C6 adducts were experimentally observable under gentle ESI conditions (Figure b). The number of MD-adducted 18C6 (10 ± 1) was consistent with the experimental data of Figure b, where the most intense signal corresponded to hMb 6+ attached to 10 crown ethers.…”

Section: Resultsmentioning

confidence: 92%

“…Our use of 18C6 as a mechanistic ESI probe expands on previous studies, where this remarkable molecule was applied to examine protein structures in solution 68 and in the gas phase. 69,70 In future work, we hope to apply strategies similar to those used here for uncovering the mechanistic basis of supercharging under denaturing conditions, where the charge states formed are even higher than under the native ESI conditions examined here. 32,44,54 ■ ASSOCIATED CONTENT * S Supporting Information…”

Section: ■ Conclusionmentioning

confidence: 98%

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Crown Ether Effects on the Location of Charge Carriers in Electrospray Droplets: Implications for the Mechanism of Protein Charging and Supercharging

Metwally

Konermann

2018

Anal. Chem.

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"Native" electrospray ionization (ESI) mass spectrometry (MS) aims to transfer proteins from solution into the gas phase while maintaining solution-like structures and interactions. The ability to control the charge states of protein ions produced in these experiments is of considerable importance. Supercharging agents (SCAs) such as sulfolane greatly elevate charge states without significantly affecting the protein structure in bulk aqueous solution. The origin of native ESI supercharging remains contentious. According to one model, SCAs trigger unfolding within ESI droplets. In contrast, the "charge trapping model" envisions that SCAs impede the ejection of charge carriers (e.g., NH or Na) from the droplet. We addressed this controversy experimentally and computationally by employing 18C6 crown ether as a mechanistic probe in native ESI-MS experiments on holo-myoglobin. Remarkably, 18C6 suppressed the supercharging capability of sulfolane. Molecular dynamics (MD) simulations reproduced the experimental charge states. The MD data revealed that 18C6 altered the location of charge carriers in the ESI droplets. Without 18C6, sulfolane covered the droplets in an ionophobic layer that impeded charge carrier access to the surface. In contrast, 18C6 complexation caused charge carrier enrichment in this surface layer, thereby promoting charge ejection. For late droplets, all the water had left and the protein was encapsulated in sulfolane; charge ejection at this stage continued only in the presence of 18C6. As a result, evaporation to dryness of charge-depleted water/sulfolane/18C6 droplets produced low protein charge states, whereas charge-abundant water/sulfolane droplets generated high charge states. Our data support the view that native ESI supercharging is caused by charge trapping. Unfolding within the droplet may play an ancillary role under some conditions, but for the cases examined here, protein structural changes are not a causative factor for supercharging. Our conclusions are bolstered by dendrimer supercharging experiments.

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supporting

confidence: 62%

Section: Resultsmentioning

confidence: 92%

Section: ■ Conclusionmentioning

confidence: 98%

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Crown Ether Effects on the Location of Charge Carriers in Electrospray Droplets: Implications for the Mechanism of Protein Charging and Supercharging

Metwally

Konermann

2018

Anal. Chem.

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“…Given that RDD is a structurally sensitive technique that is able to differentiate peptide diastereomers on the basis of l / d isomerization, RDD should have been able to produce differences in fragmentation patterns if the proline cis/trans isomers were able to survive ESI without scrambling and radical migration occurred prior to scrambling once in the gas phase. To further dissect out these possibilities, experiments were conducted with the addition of 18-crown-6 (18C6), which has been shown to solvate charges and aid in the preservation of native-like structure during ESI. , Using the same source conditions as in the previous protocol with the addition of 18C6 to the isolated fractions prior to direct infusion yielded the PD spectra shown in Figure a,b. One of the primary losses corresponds to the loss of iodine, as expected.…”

Section: Resultsmentioning

confidence: 99%

Probing the Stability of Proline Cis/Trans Isomers in the Gas Phase with Ultraviolet Photodissociation

Silzel

Murphree

Paranji

et al. 2020

J. Am. Soc. Mass Spectrom.

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Although most peptide bonds in proteins exist in the trans configuration, when cis peptide bonds do occur, they can have major impact on protein structure and function. The rapid identification of cis peptide bonds is therefore an important task. Peptide bonds containing proline are more likely to adopt the cis configuration because the ring connecting the side chain and backbone in proline flattens the energetic landscape relative to amino acids with free side chains. Examples of cis proline isomers have been identified in both solution and in the gas phase by a variety of structure-probing methods. Mass spectrometry is an attractive potential method for identifying cis proline due to its speed and sensitivity; however, the question remains of whether cis/ trans proline isomers originating in solution are preserved during ionization and manipulation within a mass spectrometer. Herein, we investigate the gas-phase stability of isolated solution-phase cis and trans proline isomers using a synthetic peptide sequence with a Tyr-Pro-Pro motif. A variety of dissociation methods were explored to evaluate their potential to distinguish cis/trans configuration, including collision-induced dissociation, radical-directed dissociation, and photodissociation. Only photodissociation employed in conjunction with extremely gentle electrospray and charge solvation by 18-crown-6 ether was able to distinguish cis/ trans isomers for our model peptide, suggesting that any thermal activation during transfer or while in the gas phase leads to isomer scrambling. Furthermore, the necessity for 18-crown-6 suggests that intramolecular charge solvation taking place during electrospray ionization can override cis/trans isomer homogeneity. Overall, the results suggest that solution-phase cis/trans proline isomers are fragile and easily lost during electrospray, requiring careful selection of instrument parameters and consideration of charge solvation to prevent cis/trans scrambling.

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“…The sites of adduction do not appear to be random, but occur at preferred locations. Crown ethers have also been shown to replace solvent in peptides and proteins sprayed into a mass spectrometer, [42][43][44] helping maintain their liquid phase structure. Diradical covalent coupling may provide useful insights into the sites of solvation that help retain tertiary structure as proteins are transferred from liquid to the gas phase.…”

mentioning

confidence: 99%

Synthesis of new S S and C C bonds by photoinitiated radical recombination reactions in the gas phase

Talbert

Zhang

Hendricks

et al. 2019

International Journal of Mass Spectrometry

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Photoinitiated radical chemistry has proven to be useful for breaking covalent bonds within many biomolecules in the gas phase. Herein, we demonstrate that radical chemistry is useful for bond synthesis in the gas phase. Single peptides containing two cysteine residues capped with propylmercaptan (PM) often form disulfide bonds following ultraviolet excitation at 266 nm and loss of both PM groups. Similarly, noncovalently bound peptide pairs where each peptide contains a single cysteine residue can be induced to form disulfide bonds. Comparison with disulfide bound species sampled directly from solution yields identical collisional activation spectra, suggesting that native disulfide bonds have been recapitulated in the gas phase syntheses. Another approach utilizing radical chemistry for covalent bond synthesis involves creation of a reactive diradical that can first abstract hydrogen from a target peptide, creating a new radical site, and then recombine the second radical with the new radical to form a covalent bond. This chemistry is illustrated with 2-(hydroxymethyl-3,5-diiodobenzoate)-18-crown-6 ether, which attaches noncovalently to protonated primary amines in peptides and proteins. Following photoactivation and crosslinking, the site of noncovalent adduct attachment can frequently be determined. The ramifications of these observations on peptide structure and noncovalent attachment of 18-crown-6-based molecules is discussed. of cases, radical chemistry is utilized to modulate dissociation when applied to biomolecular study rather than to initiate bond formation. 12-Herein we demonstrate that radicals can be used to efficiently create covalent bonds. New S-S bonds that recapitulate native disulfide links can be generated in highly efficient gas phase reactions in both intramolecular and intermolecular systems. New C-C bonds can be created from diradical, noncovalently bound species, allowing for the location of noncovalent adducts to be investigated. The details and potential applications of these reactions are discussed.

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Structural Effects of Solvation by 18-Crown-6 on Gaseous Peptides and TrpCage after Electrospray Ionization

Cited by 7 publications

References 38 publications

Crown Ether Effects on the Location of Charge Carriers in Electrospray Droplets: Implications for the Mechanism of Protein Charging and Supercharging

Crown Ether Effects on the Location of Charge Carriers in Electrospray Droplets: Implications for the Mechanism of Protein Charging and Supercharging

Probing the Stability of Proline Cis/Trans Isomers in the Gas Phase with Ultraviolet Photodissociation

Synthesis of new S S and C C bonds by photoinitiated radical recombination reactions in the gas phase

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