Capillary electrochromatography/nanoelectrospray mass spectrometry for attomole characterization of peptides

Guček, Marjan; Gaspari, Marco; Walhagen, Karin; Vreeken, Rob J.; Verheij, Elwin; Greef, J. van der

doi:10.1002/1097-0231(20000830)14:16<1448::aid-rcm44>3.0.co;2-9

Cited by 33 publications

(9 citation statements)

References 31 publications

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“…Diode array UV‐spectrometry is the method used predominantly to detect analytes in CEC, although hyphenated techniques such as CEC‐mass spectrometry (CEC‐MS) (31–33) are increasingly being applied to provide additional structural information. Because the majority of scientific publications related to the application of CEC methods have dealt with the separation of uncharged small organic molecules, a need exists to develop buffer systems specifically targeted for the isocratic separation of peptides.…”

Section: Resultsmentioning

confidence: 99%

Application of CEC procedures for the analysis of synthetic peptides: characterization of linear immunogenic peptides that mimic a HIV‐1 gp120 epitope

Walhagen

Unger

Keah

et al. 2002

The Journal of Peptide Research

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In this study, we describe the application of a new analytical procedure based on capillary electrochromatographic(CEC) techniques for the characterization of different basic and acidic peptides using isocratic eluent conditions containing acetonitrile and ammonium acetate buffers of different molarities between pH 3.8 and 5.2. In particular,10 immunogenic peptide analogs with isoelectric points ranging from 3.7 to 10.1 were investigated; nine of these peptides, 1-9, were truncated analogs of the parent peptide, 10, which is a peptidomimetic related to a HIV-1 gp120 epitope. Several of these peptides have the propensity to form alpha-helical secondary structures in solution. Electrochromatographic separations of these peptides were achieved with packed fused silica capillaries(25 cm packed length, 100 microm i.d.) containing 3 microm n-octadecylsilica particles. The influence of temperature on the CEC elution behavior of these peptides, as well as the impact of changes in the eluent composition, e.g. pH, buffer concentration and acetonitrile content, were examined. The results confirm that improvements in the resolution and analysis of synthetic peptides by CEC procedures result from the increase inelectroosmotic flow (EOF) as the temperature is increased. These findings emphasize the dominant influence of the temperature-dependent viscosity parameter, eta, on the EOF and thus on peptide resolution in CEC. Moreover, these investigations have shown that eluent properties can be specifically chosen to favor either electrophoretic mobility or chromatographic retention, with the overall CEC selectivity peptides of different sequence or composition reflecting the summated contributions from both separation mechanisms. Over the pH range 4.0-5.0, and using eluents with ionic strengths ranging from 6.2 to 15 mM ammonium acetate but containing a fixed volume fraction, psi, of acetonitrile above psi = 0.40, the CEC retention behavior of peptides 1-10 correlated with a linear relationship linking the retention coefficient, kappta(cec), and the differential frictional size-to-mass ratio parameter, Xi(fric), of these peptides. However, using eluents with a low acetonitrile content and low pH values, linear correlations were also observed between the incremental retention coefficient, Delta(Kappa)cec, and the product term [-0.66(Delta(Sigma[Xn]) log(Mi/Mj)], which links the difference in intrinsic hydrophobicities and molecular masses of two peptides, Pi and Pj. This study thus demonstrates the power of CEC procedures in the analysis of synthetic bioactive peptides and provides a general experimental framework to evaluate,using CEC procedures, the influence of the key molecular attributes of peptides on their structure-retention dependencies.Finally, these studies provide additional, practical insights into the use of CEC procedures for the analysis, resolution and biophysical characterization of closely related peptide analogs derived from solid-state peptide synthesis under conditions of different eluent composition or tempe...

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Section: Resultsmentioning

confidence: 99%

Application of CEC procedures for the analysis of synthetic peptides: characterization of linear immunogenic peptides that mimic a HIV‐1 gp120 epitope

Walhagen

Unger

Keah

et al. 2002

The Journal of Peptide Research

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“…2 shows the separation of four peptides at different salt concentrations. An experimental setup for detection of peptides by coupling CEC (containing 3 m silica particles that had either a C 8 or C 18 phase bonded to them) to an ion trap MS via nanoelectrospray interface at ambient pressure was presented by Guček et al [23]. Since the nanoelectrospray matched the flow rates in CEC no sheath flow was required.…”

Section: Capillary Electrochromatography Of Peptides and Proteins Usimentioning

confidence: 99%

Capillary electrochromatography of peptides and proteins

Bandilla

Skinner

2004

Journal of Chromatography A

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“…Due to the same instrumentation format as that used for "pure electrophoretic" capillary techniques and due to the electrodriven movement of mobile phase, CEC is included in the family of CE techniques in this article. Another advantage of CEC is that the composition of its mobile phase is more compatible with on-line coupling with MS detection than that of MEKC [104]. It is expected that CEC of peptides will benefit from the high selectivity of numerous stationary phases developed for peptide separation by RP-HPLC and from the low dispersion of electrokinetically driven movement of mobile phase.…”

Section: Electrochromatographymentioning

confidence: 99%

Recent advances in capillary electrophoresis of peptides

Kašička

2001

Electrophoresis

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The article gives a comprehensive review on the recent developments in the applications of high-performance capillary electromigration methods, including zone electrophoresis, isotachophoresis, isoelectric focusing, affinity electrophoresis, electrokinetic chromatography and electrochromatography, to analysis, preparation and physicochemical characterization of peptides. The article presents new approaches to the theoretical description and experimental verification of electromigration behavior of peptides, and covers the methodological aspects of capillary electroseparations of peptides, such as strategy and rules for the rational selection of separation mode and experimental conditions, sample treatment, suppression of peptide adsorption to the inner capillary wall, new developments in individual separation modes and new designs of detection systems. Several types of applications of capillary electromigration methods to peptide analysis are presented: conventional qualitative and quantitative analysis for determination of purity, determination in biomatrices, monitoring of physical and chemical changes and enzymatic conversions, amino acid and sequence analysis and peptide mapping of proteins. Some examples of micropreparative peptide separations are given and capabilities of capillary electromigration techniques to provide important physicochemical characteristics of peptides are demonstrated.

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Capillary electrochromatography/nanoelectrospray mass spectrometry for attomole characterization of peptides

Cited by 33 publications

References 31 publications

Application of CEC procedures for the analysis of synthetic peptides: characterization of linear immunogenic peptides that mimic a HIV‐1 gp120 epitope

Application of CEC procedures for the analysis of synthetic peptides: characterization of linear immunogenic peptides that mimic a HIV‐1 gp120 epitope

Capillary electrochromatography of peptides and proteins

Recent advances in capillary electrophoresis of peptides

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