Article

Capillary array isoelectric focusing with laser-induced fluorescence detection: milli-pH unit resolution and yoctomole mass detection limits in a 32-channel system

Chemistry Department, University of Washington, Seattle, WA 98195-1700, USA.
Analytical and Bioanalytical Chemistry (Impact Factor: 3.58). 03/2010; 397(8):3305-10. DOI: 10.1007/s00216-010-3595-x
Source: PubMed

ABSTRACT We report a multiplexed capillary electrophoresis system employing an array of 32 capillaries with a micromachined sheath-flow cuvette as the detection chamber. The sample streams were simultaneously excited with a 473-nm laser beam, and the fluorescence emission was imaged on a CCD camera with a pair of doublet achromat lens. The instrument produced mass detection limits of 380 +/- 120 yoctomoles for fluorescein in zone electrophoresis. Capillary isoelectric focusing of fluorescent standards produced peaks with an average width of 0.0029 +/- 0.0008 pH. Capillary coating stability limits the reproducibility of the analysis.

0 Followers
 · 
99 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: The on-line hyphenation of Capillary IsoElectric Focusing (CIEF) with ElectroSpray Ionization Mass Spectrometry (ESI/MS) has been carried out in a non-denaturing detection mode at the CIEF-MS interface. This CIEF-MS coupling methodology relied on the use of 40% glycerol-water medium as anti-convective agent in the CE capillary and the addition of 10 mM ammonium acetate buffer, pH 5, as a volatile aqueous sheath liquid. These CIEF-MS coupling conditions allowed the characterization of the highly basic cytokine human interferon-gamma (IFN-γ) and its detection as a non-covalent homodimer (33 814.3 g mol(-1)) corresponding to the active form of this immune-regulatory protein. An experimental pI value of 9.95 was determined for the human IFN-γ homodimer in these conditions. The CIEF-MS analysis of several variants bearing punctual or deletion mutations within the two D1 and D2 basic clusters at the C-terminal end of IFN-γ revealed the different contribution of these domains to the charge properties of this heparan sulfate-binding protein.
    The Analyst 11/2014; 140(2). DOI:10.1039/c4an01305k · 3.91 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The review brings a comprehensive survey of the recent developments of high-performance electroseparation methods in capillary and microchip formats: zone electrophoresis, isotachophoresis, isoelectric focusing, affinity electrophoresis, electrokinetic chromatography and electrochromatography. Applications of these techniques to analysis, isolation, purification and physicochemical and biochemical characterization of peptides are described. Advances in the investigation of electromigration properties of peptides, and in the methodology of their analysis, such as sample preparation, adsorption suppression, EOF control and detection, are presented. New developments, in particular, CE and CEC modes are reported and several types of their applications to peptide analysis are described: conventional qualitative and quantitative analysis, determination in complex (bio)matrices, monitoring of chemical and enzymatical reactions and physical changes, amino acid, sequence and chiral analysis, and peptide mapping of proteins. Some micropreparative peptide separations are shown and capabilities of CE and CEC techniques to provide relevant physicochemical characteristics of peptides are demonstrated.
    Electrophoresis 01/2012; 33(1):48-73. DOI:10.1002/elps.201100419 · 3.16 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The review presents a comprehensive survey of recent developments and applications of capillary and microchip electroseparation methods (zone electrophoresis, ITP, IEF, affinity electrophoresis, EKC, and electrochromatography) for analysis, isolation, purification, and physicochemical and biochemical characterization of peptides. Advances in the investigation of electromigration properties of peptides, in the methodology of their analysis, including sample preseparation, preconcentration and derivatization, adsorption suppression and EOF control, as well as in detection of peptides, are presented. New developments in particular CE and CEC modes are reported and several types of their applications to peptide analysis are described: conventional qualitative and quantitative analysis, determination in complex (bio)matrices, monitoring of chemical and enzymatical reactions and physical changes, amino acid, sequence and chiral analysis, and peptide mapping of proteins. Some micropreparative peptide separations are shown and capabilities of CE and CEC techniques to provide relevant physicochemical characteristics of peptides are demonstrated.
    Electrophoresis 01/2014; 35(1). DOI:10.1002/elps.201300331 · 3.16 Impact Factor

Preview

Download
1 Download
Available from