Protocol for resolving protein mixtures in capillary zone electrophoresis

Gordon, Manuel J.; Lee, Kong Joo.; Arias, Angela A.; Zare, Richard N.

doi:10.1021/ac00001a012

Cited by 118 publications

(29 citation statements)

References 22 publications

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“…[18]). Addition of ethylene glycol to samples (20-30% (v/v)) improves resolution of polypeptides in capillary electrophoresis [2,23]. However, a drawback is the broad LrV absorption of ethylene glycol resulting in a high background and poor detectability.…”

Section: Resultsmentioning

confidence: 99%

Micropreparation of peptides by capillary electrophoresis for matrix assisted laser desorption mass spectrometry

Bergman

1996

FEBS Letters

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In the separation of peptides by capillary electrophoresis and analysis by matrix assisted laser desorption mass spectrometry, strong suppression of the mass spectrometric signals is a problem with many common electrolytes used in the separation step such as sodium phosphate. We describe an approach employing individual electrolytes selected for highest performance in each process. Suppression with samples collected into phosphate buffers is avoided when citrate, trifluoroacetic acid or hydrochloric acid is used for collection, while phosphate still provides excellent resolution in the capillary. Low concentrations of hydrochloric acid added to the sample/matrix mixture generate essentially adduct-free mass spectra with better signalto-noise ratios and detection limits (fmol range) than those obtained with citrate or trifluoroacetic acid. Addition of 0.25% ethylene glycol to both the phosphate electrolyte and the sample improves peak shape and resolution, and is crucial for preparative separations in large diameter capillaries.

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Section: Resultsmentioning

confidence: 99%

Micropreparation of peptides by capillary electrophoresis for matrix assisted laser desorption mass spectrometry

Bergman

1996

FEBS Letters

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“…The purity of the electroeluted 27-kDa Fasciola antigen was assessed using gel silver staining (32) and capillary zone electrophoresis (CZE) with a modification of the method described by Gordon et al (33) Production of specific anti-27-kDa Fasciola antigen IgG antibody. Specific IgG antibodies were produced in 4 New Zealand White rabbits immunized subcutaneously in three different inoculation sites with the purified 27-kDa Fasciola antigen according to the method described by Attallah et al (22).…”

Section: Methodsmentioning

confidence: 99%

Immunodetection of Fasciola gigantica Circulating Antigen in Sera of Infected Individuals for Laboratory Diagnosis of Human Fascioliasis

Attallah¹,

Bughdadi

El-Shazly

et al. 2013

Clin. Vaccine Immunol.

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c Currently, the laboratory diagnosis of human fascioliasis is based on the parasitological examination of parasite eggs in stool specimens and serological detection of specific antibodies in serum samples, which are often unreliable diagnostic approaches. Ideally, a sensitive and specific diagnostic test for Fasciola infection should be based on the detection of circulating Fasciola antigen, which implies active infection. Here, a 27-kDa-molecular-mass antigen was identified in a Fasciola gigantica adult worm antigen preparation, excretory-secretory products, and sera from F. gigantica-infected individuals, and it was not detected in antigenic extracts of other parasites and sera from noninfected individuals. The target antigen was isolated and partially characterized as a protein. Immunoperoxidase staining located the target epitope within teguments and guts of F. gigantica adult worms. The performance characteristics of a newly developed enzyme-linked immunosorbent assay (ELISA) based on F. gigantica circulating antigen detection in serum (FgCA-27 ELISA) were investigated using sera of 120 parasitologically diagnosed F. gigantica-infected individuals and 80 noninfected individuals. The area under the receiving operating characteristic (ROC) curve (AUC) for ELISA was significantly high (AUC ‫؍‬ 0.961, P < 0.0001) for discriminating Fasciola-infected and noninfected individuals. The developed assay showed high degrees of sensitivity, specificity, and efficiency (>93%), and a significant correlation (r ‫؍‬ 0.715, P < 0.0001) between antigen level and parasite egg count was shown. In conclusion, a 27-kDa Fasciola antigen was identified in sera of F. gigantica-infected individuals. A highly sensitive and specific Fasciola antigen detection assay, FgCA-27 ELISA, was developed for laboratory diagnosis of human fascioliasis.

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“…Net charge and state of the inner capillary surface [149,150] have been revealed to represent essential factors in affecting adsorption of proteins, which is governed by hydrogen bonding, Van der Waals forces and hydrophobic interactions [151][152][153]. Low diffusion coefficients in combination with slow sorption/desorption kinetics of proteins will entail pronounced peak-tailing and impair protein resolution even if contacts with the capillary wall are limited [154,155]. Once adsorbed, proteins might undergo (partial) unfolding and thus can serve as "hot spots" for further protein attachment [156].…”

Section: Cze Separation Of Proteins In Esi-ms Modementioning

confidence: 99%

Advances in the analysis of proteins and peptides by capillary electrophoresis with matrix‐assisted laser desorption/ionization and electrospray‐mass spectrometry detection

2005

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High throughput, outstanding certainty in peptide/protein identification, exceptional resolution, and quantitative information are essential pillars in proteome research. Capillary electrophoresis (CE) coupled to mass spectrometry (MS) has proven to meet these requirements. Soft ionization techniques, such as matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI), have paved the way for the story of success of CE-MS in the analysis of biomolecules and both approaches are subject of discussion in this article. Meanwhile, CE-MS is far away from representing a homogeneous field. Therefore the review will cover a vast area including the coupling of different modes of CE (capillary zone electrophoresis, capillary isoelectric foscusing, capillary electrochromatography, micellar electrokinetic chromatography, nonaqueous capillary electrophoresis) to MS as well as on-line preconcentration techniques (transient capillary isotachophoresis, solid-phase extraction, membrane preconcentration) applied to compensate for restricted detection sensitivity. Special attention is given to improvements in interfacing, namely addressing nanospray and coaxial sheath liquid design. Peptide mapping, collision-induced dissociation with subsequent tandem MS, and amendments in mass accuracy of instruments improve information validity gained from MS data. With 2-D on-line coupling of liquid chromatography (LC) and CE a further topic will be discussed. A special section is dedicated to recent attempts in establishing CE-ESI-MS in proteomics, in the clinical and diagnostic field, and in the food sector.

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Protocol for resolving protein mixtures in capillary zone electrophoresis

Cited by 118 publications

References 22 publications

Micropreparation of peptides by capillary electrophoresis for matrix assisted laser desorption mass spectrometry

Micropreparation of peptides by capillary electrophoresis for matrix assisted laser desorption mass spectrometry

Immunodetection of Fasciola gigantica Circulating Antigen in Sera of Infected Individuals for Laboratory Diagnosis of Human Fascioliasis

Advances in the analysis of proteins and peptides by capillary electrophoresis with matrix‐assisted laser desorption/ionization and electrospray‐mass spectrometry detection

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