Conformational changes in proteins probed by hydrogen‐exchange electrospray‐ionization mass spectrometry

Katta, Viswanatham; Chait, Brian T.; Carr, Steven A.

doi:10.1002/rcm.1290050415

Cited by 442 publications

(346 citation statements)

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“…Currently, we have no definitive explanation for this charge-shifting behavior. Denaturation has been shown to lead to higher charging (Katta & Chait, 1991aLeBlanc et al, 1991;Loo et al, 1991Loo et al, , 1993a. However, we consider the possibility of further denaturation to be unlikely under these conditions because the myoglobin should already be completely denatured in a 4% acetic acid/50% acetonitrile solution and this same effect is also observed with peptides as small as 6 residues, which are unlikely to have higher-order structure (data not shown).…”

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confidence: 88%

Surfactant effects on protein structure examined by electrospray ionization mass spectrometry

1994

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Electrospray ionization mass spectrometry (ESI-MS) has proven to be a useful tool for examining noncovalent complexes between proteins and a variety of ligands. It has also been used to distinguish between denatured and refolded forms of proteins. Surfactants are frequently employed to enhance solubilization or to modify the tertiary or quaternary structure of proteins, but are usually considered incompatible with mass spectrometry. A broad range of ionic, nonionic, and zwitterionic surfactants was examined to characterize their effects on ESI-MS and on protein structure under ESI-MS conditions. Solution conditions studied include 4% acetic acid/50% acetonitrile/46% H 2 0 and 100% aqueous. Of the surfactants examined, the nonionic saccharides, such as n-dodecyl-P-D-glucopyranoside, at 0.1 Vo to 0.01% (w/v) concentrations, performed best, with limited interference from chemical background and adduct formation. Under the experimental conditions used, ESI-MS performance in the presence of surfactants was found to be unrelated to critical micelle concentration. It is demonstrated that surfactants can affect both the tertiary and quaternary structures of proteins under conditions used for ESI-MS. However, several of the surfactants caused significant shifts in the charge-state distributions, which appeared to be independent of conformational effects. These observations suggest that surfactants, used in conjunction with ESI-MS, can be useful for protein structure studies, if care is used in the interpretation of the results.Keywords: conformation; denaturation; detergent; electrospray; mass spectrometry; surfactant Surfactants play a significant role in protein chemistry with primary applications ranging from solubilization and stabilization of proteins to disaggregation of protein complexes and denaturation (Neugebauer, 1990(Neugebauer, , 1992Bollag & Edelstein, 1991). Some surfactants can disrupt protein higher order structure, whereas others are used as aids in protein refolding. They are employed in both gel and capillary electrophoresis and enhance protein and peptide recoveries from synthetic membranes employed in electroblotting and in electroelution of proteins from gels (Fernandez et al., 1994). Membrane-bound proteins, in particular, require surfactant treatment for solubilization. Abbreviotiom: CMC, critical micelle concentration; ESI, electrospray ionization; ESI-MS, electrospray ionization mass spectrometry; MALDI, matrix-assisted laser desorption/ionization; NP40, Nonidet P a , CHAPS, 3-(3-cholamidopropyl)dimethylammonio-I-propane sulfonate; CTAB, cetyl trimethylammonium bromide; LDAO, laurel dimethylamine oxide; n-dodecyl glucoside, n-dodecyl-0-D-glucopyranoside; n-hexylglucoside, n-hexyl-0-D-glucopyranoside; octyl glucoside, octyl-fl-D-glucopyranoside; octyl thioglucoside, I-S-octyl-fi-~-thioglucopyranoside.Matrix-assisted laser desorption/ionization or electrospray ionization have demonstrated molecular weight determinations for proteins greater than 100 kDa (Fenn et al., 1989;Smith et al., 19...

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confidence: 88%

Surfactant effects on protein structure examined by electrospray ionization mass spectrometry

1994

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“…H /D exchange (HDX) is one of the most popular techniques for the structural elucidation of biomolecules in mass spectrometry and has been widely implemented for both solution [1][2][3][4] and gas phase studies [5][6][7][8][9][10][11][12][13][14][15][16][17][18]. In solution, the extent of HDX can be directly related to the solvent accessibility of amino acid residues in proteins.…”

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confidence: 99%

“…Beauchamp and coworkers [9] suggested that lower basicity reagents, such as D 2 O and CH 3 OD, proceed via a "relay" mechanism [20] for protonated peptides, while the higher basicity ND 3 reagent gives rise to an "onium" mechanism [21]. In the case of amino acids cationized by an alkali metal ion rather than a proton, polar deuterating ligands such as D 2 O can exchange via a similar relay mechanism involving charge solvation-zwitterion (CS-ZW) isomerization [18]. Modeling of observed HDX processes and quantum chemical calculations of the potential surfaces have made a strong circumstantial case that ZW structures are traversed in many cases during HDX with basic partners like D 2 O, CH 3 OD, and ND 3 [14,16,18].…”

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Observation of zwitterion formation in the gas-phase H/D-exchange with CH₃OD: Solution-phase structures in the gas phase

Polfer

Dunbar

2007

J. Am. Soc. Mass Spectrom.

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Infrared spectroscopy of gas-phase singly deuterated [Trp ϩ K] ϩ (formed by H/D exchange with CH 3 OD) shows that some (ϳ20%) kinetically stable zwitterionic (ZW) conformer is formed, based on the diagnostic antisymmetric CO stretch of the deprotonated carboxylate moiety, as (CO 2 Ϫ ), at 1680 cm Ϫ1 . A majority of the deuterated [Trp ϩ K] ϩ is found to be in the charge solvation (CS) conformation, with deuterium exchange occurring on both the acid and amino groups, which is consistent with H/D scrambling. Interestingly, H/D exchange with the more basic ND 3 reagent did not result in the stabilization of a kinetically stable zwitterion, although it is not clear yet what causes this observation. The result for CH 3 OD shows that H/D exchange can in fact alter the structure of the analyte and, hence, care needs to be taken when interpreting gas-phase H/D exchange studies. Moreover, this result shows the possibility of forming solution-phase structures that are thermodynamically disfavored in the gas phase, thus opening a new area of study. (HDX) is one of the most popular techniques for the structural elucidation of biomolecules in mass spectrometry and has been widely implemented for both solution [1][2][3][4] and gas phase studies [5][6][7][8][9][10][11][12][13][14][15][16][17][18]. In solution, the extent of HDX can be directly related to the solvent accessibility of amino acid residues in proteins. However, in the gas phase there are many structural parameters apart from the surface availability that affect the HDX rates, as illustrated in a recent HDX study on ion-mobilityselected charge states of bovine ubiquitin [17]. Systematic studies on small peptide systems showed that the difference in proton affinity of the biomolecules and deuterated donor reagent plays a crucial role in the HDX mechanism [7][8][9][10]19]. Beauchamp and coworkers [9] suggested that lower basicity reagents, such as D 2 O and CH 3 OD, proceed via a "relay" mechanism [20] for protonated peptides, while the higher basicity ND 3 reagent gives rise to an "onium" mechanism [21]. In the case of amino acids cationized by an alkali metal ion rather than a proton, polar deuterating ligands such as D 2 O can exchange via a similar relay mechanism involving charge solvation-zwitterion (CS-ZW) isomerization [18]. Modeling of observed HDX processes and quantum chemical calculations of the potential surfaces have made a strong circumstantial case that ZW structures are traversed in many cases during HDX with basic partners like D 2 O, CH 3 OD, and ND 3 [14,16,18]. Nevertheless, computations also generally indicate that the bare ZW is sufficiently disfavored energetically that its equilibrium presence in the M ϩ /amino acid would be negligible [22,23].Here, we show unambiguous spectroscopic evidence that bare ZW can in fact be kinetically trapped as a result of the HDX of [Trp ϩ K] ϩ with CH 3 OD. Infrared multiple-photon dissociation (IR-MPD) spectroscopy of metal-tagged amino acids and peptides has already been shown to be a powerful probe for CS ...

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“…(J Am Soc Mass Spectrom 2007, 18, 1525-1532) © 2007 American Society for Mass Spectrometry E lectrospray ionization mass spectrometry (ESI-MS) has emerged as a powerful tool to investigate the conformation, dynamics, and interactions of proteins (recently reviewed in [1][2][3][4][5]). ESI-MS has been used to probe protein conformation in solution either by measuring mass changes in response to hydrogendeuterium exchange, or by monitoring the charge state distribution (CSD) of protein ions in the gas-phase [6,7]. Advantages of ESI-MS as a conformational probe include the preservation of native conformation and noncovalent protein-protein interactions, as well as its sensitivity and speed of analysis, applicability to large proteins, and compatibility with rapid mixing approaches to examine kinetic events [3].…”

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Electrospray ionization mass spectra of acyl carrier protein are insensitive to its solution phase conformation

Murphy

Rowland

Byers

2007

J. Am. Soc. Mass Spectrom.

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Electrospray ionization mass spectrometry (ESI-MS) can be used to monitor conformational changes of proteins in solution based on the charge state distribution (CSD) of the corresponding gas-phase ions, although relatively few studies of acidic proteins have been reported. Here, we have compared the CSD and solution structure of recombinant Vibrio harveyi acyl carrier protein (rACP), a small acidic protein whose secondary and tertiary structure can be manipulated by pH, fatty acylation, and site-directed mutagenesis. Circular dichroism and intrinsic fluorescence demonstrated that apo-rACP adopts a folded helical conformation in aqueous solution below pH 6 or in 50% acetonitrile/0.1% formic acid, but is unfolded at neutral and basic pH values. A rACP mutant, in which seven conserved acidic residues were replaced with their corresponding neutral amides, was folded over the entire pH range of 5 to 9. However, under the same solvent conditions, both wild type and mutant ACPs exhibited similar CSDs (6 ϩ -9 ϩ species) at all pH values. Covalent attachment of myristic acid to the phosphopantetheine prosthetic group of rACP, which is known to stabilize a folded conformation in solution, also had little influence on its CSD in either positive or negative ion modes. Overall, our results are consistent with ACP as a "natively unfolded" protein in a dynamic conformational equilibrium, which allows access to (de)protonation events during the electrospray process. (J Am Soc Mass Spectrom 2007, 18, 1525-1532

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Conformational changes in proteins probed by hydrogen‐exchange electrospray‐ionization mass spectrometry

Cited by 442 publications

References 17 publications

Surfactant effects on protein structure examined by electrospray ionization mass spectrometry

Surfactant effects on protein structure examined by electrospray ionization mass spectrometry

Observation of zwitterion formation in the gas-phase H/D-exchange with CH₃OD: Solution-phase structures in the gas phase

Electrospray ionization mass spectra of acyl carrier protein are insensitive to its solution phase conformation

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Conformational changes in proteins probed by hydrogen‐exchange electrospray‐ionization mass spectrometry

Cited by 442 publications

References 17 publications

Surfactant effects on protein structure examined by electrospray ionization mass spectrometry

Surfactant effects on protein structure examined by electrospray ionization mass spectrometry

Observation of zwitterion formation in the gas-phase H/D-exchange with CH3OD: Solution-phase structures in the gas phase

Electrospray ionization mass spectra of acyl carrier protein are insensitive to its solution phase conformation

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Observation of zwitterion formation in the gas-phase H/D-exchange with CH₃OD: Solution-phase structures in the gas phase