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Capillary electrophoresis hyphenated to inductively coupled plasma-sector field-mass spectrometry for the stoichiometric determination of Zn bound to Aeromonas hydrophila Zn beta-lactamase

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Abstract
A method was developed for the determination of the stoichiometric Zn/protein ratio for the Aeromonas hydrophila metallo beta-lactamase AE036. Capillary electrophoresis (CE) was used to separate free Zn ions from the Zn bound to the metalloprotein, the only Zn-species which is biochemically relevant. An inductively coupled plasma-sector field-mass spectrometer (ICP-SF-MS), operated at a mass resolution of 3000, was used as an element-specific detector. If the primary structure of the protein and thus the number of sulfur-containing amino acids is known, as is the case for Aeromonas hydrophila Zn beta-lactamase, monitoring of both Zn and S with ICP-SF-MS permits the Zn/protein ratio to be determined. The method was optimised in order to obtain short migration times (less than 10 min) for the metallo beta-lactamase and for standards with bare fused silica capillaries. After optimisation, a 75 mmol L-1 bis-Tris background electrolyte buffered at a pH of 6.75 was used for the separation, a voltage of 30 kV was applied over a capillary with a total length of approximately 70 cm and the sample was introduced hydrodynamically at 1000 mbar s. Quantitative results (stoichiometric Zn/protein ratio of 1 and 2, before and after saturation of the enzyme with Zn, respectively) were obtained using external calibration with albumin as a S standard and ZnCl2 as a Zn standard. The method was also demonstrated to be precise with < 5% RSD on the Zn/protein ratio for n=3. An absolute limit of detection of 8 ng of A. hydrophila Zn beta-lactamase (calculated on the basis of sulfur) was obtained under these conditions.
Keywords
METALLOTHIONEIN ISOFORMS, PHYTIC ACID, SPECIATION, PROTEINS, RESISTANCE, SEPARATION, RESOLUTION

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MLA
Van Lierde, Veerle, et al. “Capillary Electrophoresis Hyphenated to Inductively Coupled Plasma-Sector Field-Mass Spectrometry for the Stoichiometric Determination of Zn Bound to Aeromonas Hydrophila Zn Beta-Lactamase.” JOURNAL OF ANALYTICAL ATOMIC SPECTROMETRY, vol. 19, no. 7, 2004, pp. 888–93, doi:10.1039/b400962b.
APA
Van Lierde, V., Chéry, C. C., Strijckmans, K., Galleni, M., Devreese, B., Van Beeumen, J., … Vanhaecke, F. (2004). Capillary electrophoresis hyphenated to inductively coupled plasma-sector field-mass spectrometry for the stoichiometric determination of Zn bound to Aeromonas hydrophila Zn beta-lactamase. JOURNAL OF ANALYTICAL ATOMIC SPECTROMETRY, 19(7), 888–893. https://doi.org/10.1039/b400962b
Chicago author-date
Van Lierde, Veerle, Cyrille C Chéry, Karel Strijckmans, Moreno Galleni, Bart Devreese, Jozef Van Beeumen, Luc Moens, and Frank Vanhaecke. 2004. “Capillary Electrophoresis Hyphenated to Inductively Coupled Plasma-Sector Field-Mass Spectrometry for the Stoichiometric Determination of Zn Bound to Aeromonas Hydrophila Zn Beta-Lactamase.” JOURNAL OF ANALYTICAL ATOMIC SPECTROMETRY 19 (7): 888–93. https://doi.org/10.1039/b400962b.
Chicago author-date (all authors)
Van Lierde, Veerle, Cyrille C Chéry, Karel Strijckmans, Moreno Galleni, Bart Devreese, Jozef Van Beeumen, Luc Moens, and Frank Vanhaecke. 2004. “Capillary Electrophoresis Hyphenated to Inductively Coupled Plasma-Sector Field-Mass Spectrometry for the Stoichiometric Determination of Zn Bound to Aeromonas Hydrophila Zn Beta-Lactamase.” JOURNAL OF ANALYTICAL ATOMIC SPECTROMETRY 19 (7): 888–893. doi:10.1039/b400962b.
Vancouver
1.
Van Lierde V, Chéry CC, Strijckmans K, Galleni M, Devreese B, Van Beeumen J, et al. Capillary electrophoresis hyphenated to inductively coupled plasma-sector field-mass spectrometry for the stoichiometric determination of Zn bound to Aeromonas hydrophila Zn beta-lactamase. JOURNAL OF ANALYTICAL ATOMIC SPECTROMETRY. 2004;19(7):888–93.
IEEE
[1]
V. Van Lierde et al., “Capillary electrophoresis hyphenated to inductively coupled plasma-sector field-mass spectrometry for the stoichiometric determination of Zn bound to Aeromonas hydrophila Zn beta-lactamase,” JOURNAL OF ANALYTICAL ATOMIC SPECTROMETRY, vol. 19, no. 7, pp. 888–893, 2004.
@article{293910,
  abstract     = {{A method was developed for the determination of the stoichiometric Zn/protein ratio for the Aeromonas hydrophila metallo beta-lactamase AE036. Capillary electrophoresis (CE) was used to separate free Zn ions from the Zn bound to the metalloprotein, the only Zn-species which is biochemically relevant. An inductively coupled plasma-sector field-mass spectrometer (ICP-SF-MS), operated at a mass resolution of 3000, was used as an element-specific detector. If the primary structure of the protein and thus the number of sulfur-containing amino acids is known, as is the case for Aeromonas hydrophila Zn beta-lactamase, monitoring of both Zn and S with ICP-SF-MS permits the Zn/protein ratio to be determined. The method was optimised in order to obtain short migration times (less than 10 min) for the metallo beta-lactamase and for standards with bare fused silica capillaries. After optimisation, a 75 mmol L-1 bis-Tris background electrolyte buffered at a pH of 6.75 was used for the separation, a voltage of 30 kV was applied over a capillary with a total length of approximately 70 cm and the sample was introduced hydrodynamically at 1000 mbar s. Quantitative results (stoichiometric Zn/protein ratio of 1 and 2, before and after saturation of the enzyme with Zn, respectively) were obtained using external calibration with albumin as a S standard and ZnCl2 as a Zn standard. The method was also demonstrated to be precise with < 5% RSD on the Zn/protein ratio for n=3. An absolute limit of detection of 8 ng of A. hydrophila Zn beta-lactamase (calculated on the basis of sulfur) was obtained under these conditions.}},
  author       = {{Van Lierde, Veerle and Chéry, Cyrille C and Strijckmans, Karel and Galleni, Moreno and Devreese, Bart and Van Beeumen, Jozef and Moens, Luc and Vanhaecke, Frank}},
  issn         = {{0267-9477}},
  journal      = {{JOURNAL OF ANALYTICAL ATOMIC SPECTROMETRY}},
  keywords     = {{METALLOTHIONEIN ISOFORMS,PHYTIC ACID,SPECIATION,PROTEINS,RESISTANCE,SEPARATION,RESOLUTION}},
  language     = {{eng}},
  number       = {{7}},
  pages        = {{888--893}},
  title        = {{Capillary electrophoresis hyphenated to inductively coupled plasma-sector field-mass spectrometry for the stoichiometric determination of Zn bound to Aeromonas hydrophila Zn beta-lactamase}},
  url          = {{http://dx.doi.org/10.1039/b400962b}},
  volume       = {{19}},
  year         = {{2004}},
}

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