Isolation and identification of peptide conformers by reversed-phase high-performance liquid chromatography and NMR at low temperature

Kálmán, András; Thunecke, Frank; Schmidt, Ralf; Schiller, Peter W.; Horváth, Csaba

doi:10.1016/0021-9673(95)01059-9

Cited by 45 publications

(15 citation statements)

References 47 publications

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“…It was found that proline-containing peptides isomerize during the separation process as previously described by the pioneering work of Horváth et al [30][31][32][33][34][35][36][37], who examined this phenomenon in NMR, HPLC, and CE experiments. The same group estimated isomerization rate constants and the activation barrier of some proline containing dipeptides by computer aided Damkohler analysis, a method which is commonly used in chemical engineering.…”

Section: Introductionmentioning

confidence: 78%

“…The elution order was assigned to be trans before cis for L-alanyl-L-proline, L-leucyl-L-proline and L-phenylalanyl-L-proline. This assignment was made based on the investigations of Horváth et al [32,33] and Krauss et al [41], who separated the two isomers and assigned their configuration by NMR. Surprisingly, for the two larger dipeptides L-leucyl-L-proline and L-phenylalanyl-L-proline the reasonable assumption, that the trans conformation of the L-peptidyl-L-proline dipeptides in the equilibrium is more stable than the cis conformation, and therefore giving rise to the peak with the larger area, is not valid.…”

Section: Separationmentioning

confidence: 99%

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Determination of thecis-trans isomerization barrier of severalL-peptidyl-L-proline dipeptides by dynamic capillary electrophoresis and computer simulation

2001

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Dynamic capillary electrophoresis (DCE) and computer simulation of the elution profiles with the theoretical plate and the stochastic model has been applied to determine the isomerization barriers of the three dipeptides L-alanyl-L-proline, L-leucyl-L-proline, and L-phenylalanyl-L-proline. The separation of the rotational cis-trans isomers has been performed in an aqueous 70 mM borate buffer at pH 9.5. Interconversion profiles featuring plateau formation and peak broadening were observed. To determine the rate constants k1 and k(-1) of the cis-trans isomerization in dynamic capillary electrophoresis, equations have been derived for the theoretical plate model and stochastic model. The electropherograms were simulated with the ChromWin software which uses the experimental data plateau height h(plateau), peak width at half height Wh, the total migration times of the cis-trans isomers tR and the electroosmotic break-through time t0 as well as the peak ratio [cis]/[trans]. From temperature-dependent measurements, the rate constants k1 and k(-1) and the kinetic activation parameters deltaG#, deltaH# and deltaS# of the cis-trans isomerization of the three dipeptides were obtained.

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Section: Introductionmentioning

confidence: 78%

Section: Separationmentioning

confidence: 99%

Determination of thecis-trans isomerization barrier of severalL-peptidyl-L-proline dipeptides by dynamic capillary electrophoresis and computer simulation

2001

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“…Two conformations of aplidine (aplidine A and B) can be observed. The two conformers are the cis and trans isomers at the pyruvoyl–proline amide bond and the plateau between the peaks is caused by the interconversion of the aplidine isomers during the chromatographic run11–14. The trans conformer of aplidine is eluted before the cis conformer13.…”

Section: Resultsmentioning

confidence: 99%

Structure elucidation of aplidine metabolites formed in vitro by human liver microsomes using triple quadrupole mass spectrometry

Brandon¹,

Ooijen²,

Sparidans³

et al. 2005

J. Mass Spectrom.

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The cyclic depsipeptide aplidine is a new anti-cancer drug of marine origin. Four metabolites of this compound were found after incubation with pooled human microsomes using gradient high-performance liquid chromatography with ultraviolet detection. After chromatographic isolation, the metabolites have been identified using nano-electrospray triple quadrupole mass spectrometry. A highly specific sodium-ion interaction with the cyclic structure opens the depsipeptide ring, and cleavage of the amino acid residues gives sequence information when activated by collision-induced dissociation in the second quadrupole. The aplidine molecule could undergo the following metabolic reactions: hydroxylation at the isopropyl group (metabolites apli-h 1 and apli-h 2); C-dealkylation at the N(Me)-leucine group (metabolite apli-da); hydroxylation at the isopropyl group and C-dealkylation at the N(Me)-leucine group (metabolite apli-da/h), and C-demethylation at the threonine group (metabolite apli-dm). The identification of these metabolites formed in vitro may greatly aid the elucidation of the metabolic pathways of aplidine in humans.

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“…Chromatography at temperatures below the freezing point of water has been used extensively within the fields of cryo-enzymology 10 , and biochemistry 11 where the reduced temperature is used to slow down enzyme function or reaction kinetics and provide a platform for the study of dynamic systems. To our knowledge, the benefits of sub-zero temperature chromatography have never been demonstrated in the context of HDX MS.…”

Section: Introductionmentioning

confidence: 99%

“…Ethylene glycol in particular, has seen previous use as modifier in low temperature chromatography. 11a The impacts of these modifiers on various aspects of a typical HDX MS experiment are evaluated and the potential benefits and feasibility of using these modifiers to perform sub-zero temperature chromatography with HDX MS are discussed.…”

Section: Introductionmentioning

confidence: 99%

Subzero Temperature Chromatography for Reduced Back-Exchange and Improved Dynamic Range in Amide Hydrogen/Deuterium Exchange Mass Spectrometry

et al. 2012

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Amide hydrogen/deuterium exchange is a commonly used technique for studying the dynamics of proteins and their interactions with other proteins or ligands. When coupled with liquid chromatography and mass spectrometry, hydrogen/deuterium exchange provides several unique advantages over other structural characterization techniques including very high sensitivity, the ability to analyze proteins in complex environments, and a large mass range. A fundamental limitation of the technique arises from the loss of deuterium label (back-exchange) during the course of the analysis. A method to limit loss of label during the separation stage of the analysis using sub-zero temperature reversed-phase chromatography is presented. The approach is facilitated by the use of buffer modifiers that prevent freezing. We evaluated ethylene glycol, dimethyl formamide, formamide, and methanol for their freezing point suppression capabilities, effects on peptide retention, and their compatibilities with electrospray ionization. Ethylene glycol was used extensively because of its good electrospray ionization compatibility; however formamide has potential to be a superior modifier if detrimental effects on ionization can be overcome. It is demonstrated using suitable buffer modifiers that separations can be performed at temperatures as low as −30°C with negligible loss of deuterium label, even during long chromatographic separations. The reduction in back-exchange is shown to increase the dynamic range of HDX MS in terms of mixture complexity, and the magnitude with which changes in deuteration level can be quantified.

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Isolation and identification of peptide conformers by reversed-phase high-performance liquid chromatography and NMR at low temperature

Cited by 45 publications

References 47 publications

Determination of thecis-trans isomerization barrier of severalL-peptidyl-L-proline dipeptides by dynamic capillary electrophoresis and computer simulation

Determination of thecis-trans isomerization barrier of severalL-peptidyl-L-proline dipeptides by dynamic capillary electrophoresis and computer simulation

Structure elucidation of aplidine metabolites formed in vitro by human liver microsomes using triple quadrupole mass spectrometry

Subzero Temperature Chromatography for Reduced Back-Exchange and Improved Dynamic Range in Amide Hydrogen/Deuterium Exchange Mass Spectrometry

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