Optimization of peptide and protein separation with a monolithic reversed-phase column and application to arsenic-binding studies

Schmidt, Anne-Christine; Mickein, Kathleen

doi:10.1016/j.chroma.2010.11.031

Cited by 5 publications

(19 citation statements)

References 22 publications

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“…Different conformers that arise from changing chemical environment during gradient elution occur in the form of multiple peaks . In agreement with the former RPLC‐UV analyses in which the water–acetonitrile gradient elution was supported by an ion pairing reagent, the phenylarsenic‐substituted peptide forms elute later than the unsubstituted forms owing to a larger molecular size.…”

Section: Resultsmentioning

confidence: 99%

“…b). In agreement with this, in RPLC separations using another eluent system, both the reduced and the phenylarsenic‐containing Apr eluted over a wide time interval …”

Section: Resultsmentioning

confidence: 99%

“…The Lys type used in the current study originating from hen egg white ( M = 14.3 kDa, p I = 10.7) contains four disulfide bridges, which hold the native protein in a globular conformation. In contrast to former studies, one phenylarsenic substitution was discovered for Lys using RPLC‐ESI‐MS that contains four potential phenylarsenic‐binding sites. The accessibility of the corresponding cysteine pairs for TCEP has been graduated in the following order: Cys30–Cys115 > Cys6–Cys127 > Cys64–Cys80 > Cys76–Cys94 .…”

Section: Resultsmentioning

confidence: 99%

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Qualitative and quantitative characterization of the arsenic‐binding behaviour of sulfur‐containing peptides and proteins by the coupling of reversed phase liquid chromatography to electrospray ionization mass spectrometry

Schmidt

Mickein

2012

J. Mass. Spectrom.

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Phenylarsenic-substituted cysteine-containing peptides and proteins were completely differentiated from their unbound original forms by the coupling of reversed phase liquid chromatography with electrospray ionization mass spectrometry. The analysis of biomolecules possessing structure-stabilizing disulfide bridges after reduction provides new insights into requirements concerning the accessibility of cysteine residues for reducing agents as well as for arsenic compounds in a spatial protein structure. Complementary binding studies performed using direct ESI-MS without chromatographic coupling in different solvent systems demonstrated that more than one binding site were activated for aprotinin and lysozyme in denaturing solvents because of a stronger defolding. From the intensities of the different charge states occurring in the mass spectra as well as from the LC elution behaviour, it can be deduced that the folding state of the arsenic-bound protein species resembles the native, oxidized conformation. In contrast, although the milk protein α-lactalbumin has several disulfide bridges, only one phenylarsenic moiety was bound under strongly denaturing conditions. Because of the charge state distribution in the ESI mass spectra, a conformational change to a molten globule structure is assumed. For the second considered milk protein ß-lactoglobulin, a noncovalent interaction with phenylarsine oxide was detected. In general, smaller apparent binding constants for the condensation reactions of the biomolecules with phenylarsine oxide leading to covalent arsenic-sulfur bindings were determined from direct injection ESI-MS measurements than from LC-ESI-MS coupling. The following order of binding affinities for one phenylarsenic group can be assumed from both ESI-MS and LC-ESI-MS: nonapeptide vasopressin > nonapeptide vasotocin > lysozyme > aprotinin > α-lactalbumin > thioredoxin. Kinetic investigations by LC-ESI-MS yielded a partial reaction order of 2 for vasopressin, Lys and α-lactalbumin and corresponding half-lives of 0.93, 2.56 and 123.5 min, respectively.

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Section: Resultsmentioning

confidence: 99%

“…b). In agreement with this, in RPLC separations using another eluent system, both the reduced and the phenylarsenic‐containing Apr eluted over a wide time interval …”

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Qualitative and quantitative characterization of the arsenic‐binding behaviour of sulfur‐containing peptides and proteins by the coupling of reversed phase liquid chromatography to electrospray ionization mass spectrometry

Schmidt

Mickein

2012

J. Mass. Spectrom.

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show abstract

“…Thus, in the further separation process, a content of 0.1% (m/v) TFA was selected in the mobile phase. Moreover, because very strong interactions existed between the individual variables such as eluent strength [15], flow rate and gradient time that influence the peak capacity of gradient elution, multivariate optimization strategies can help to find optimum separation procedures.…”

Section: Chromatographic Properties Of Pst Reversed Phase Columnmentioning

confidence: 99%

“…silica based, polymer based, silica based with a polymer coating). Most of the peptides are recovered from the columns in high yield, particularly from those employing wide-pore silica packing materials [15]. Although reversed-phase HPLC can be used for the purification of peptides with a range of physicochemical properties, the technique has some limitations.…”

Section: Introductionmentioning

confidence: 99%

Rapid octreotide separation from synthetic peptide crude mixtures by chromatography on poly(styrene–co-divinylbenzene)-based reversed phases

Zhang

et al. 2015

Separation and Purification Technology

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Characterization and quantification of racemic and meso-ethylenediamine-N,N′-bis(2-hydroxy-5-sulfophenylacetic) acid/iron (III) by ion-pair ultra-high performance liquid chromatography coupled with diode array detector and electrospray tandem mass spectrometry

Biasone¹,

Cianci²,

Tommaso³

et al. 2013

Journal of Chromatography A

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Optimization of peptide and protein separation with a monolithic reversed-phase column and application to arsenic-binding studies

Cited by 5 publications

References 22 publications

Qualitative and quantitative characterization of the arsenic‐binding behaviour of sulfur‐containing peptides and proteins by the coupling of reversed phase liquid chromatography to electrospray ionization mass spectrometry

Qualitative and quantitative characterization of the arsenic‐binding behaviour of sulfur‐containing peptides and proteins by the coupling of reversed phase liquid chromatography to electrospray ionization mass spectrometry

Rapid octreotide separation from synthetic peptide crude mixtures by chromatography on poly(styrene–co-divinylbenzene)-based reversed phases

Characterization and quantification of racemic and meso-ethylenediamine-N,N′-bis(2-hydroxy-5-sulfophenylacetic) acid/iron (III) by ion-pair ultra-high performance liquid chromatography coupled with diode array detector and electrospray tandem mass spectrometry

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