Recent advances in capillary electrophoresis of proteins, peptides and amino acids

Novotný, Miloš V.; Cobb, Kelly A.; Liu, Jinping

doi:10.1002/elps.1150110911

Cited by 157 publications

(47 citation statements)

References 71 publications

(1 reference statement)

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“…Capillary electrophoresis (CE) is a newly emerging technique for rapid and high-resolution separation of biomolecules. 9 Recently, CE is widely used in the separation and determination of amine. 10 The detection methods of amine in CE include UV-VIS, 11 electrochemical detection 12 and LIF.…”

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Fluorescent Determination of Noradrenaline and Dopamine Derivatized with Cy5 in Capillary Electrophoresis

Zhang

et al. 2001

ANAL. SCI.

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Noradrenaline and dopamine are two of the most important monoamine neurotransmitters. They play important roles in learning and memory, and in regulation of cardiovascular activity and nervous system. Analysis of the monoamine neurotransmitters has conventionally used high performance liquid chromatography (HPLC) 1-3 or gas chromatography. 4,5 However, the determination of monoamine neurotransmitters in the biological matrix is very difficult because the compounds are usually present at very low concentrations, and many other compounds in biological matrix, which have also absorption in the low-wavelength region, interfere badly with the analysis of monoamine neurotransmitters. Cyanine derivative (Cy5) is a new fluorescence derivative reagent. The excitation and the emission wavelength of Cy5 are 649 and 670 nm, respectively. Thus diode lasers, which emit wavelengths from deep-red to infrared region, are chosen as the light source for laser-induced fluorescence (LIF). This is preferential for determining Cy5-labeled amine neurotransmitters, since almost all of the biological substances have no absorption band in the deep-red and infrared region, resulting in a remarkable decrease of background fluorescence noise and in an increase of detection sensitivity. Recently, LIF is widely used in the analysis of DNA sequence, oligonucleotides and proteins. 6-8But determination of Cy5-labeled monoamine neurotransmitters has not been reported. Capillary electrophoresis (CE) is a newly emerging technique for rapid and high-resolution separation of biomolecules.9 Recently, CE is widely used in the separation and determination of amine. 10 The detection methods of amine in CE include UV-VIS, 11 electrochemical detection 12 and LIF. 13 LIF is one of the most sensitive detection techniques in CE because of its low background signal and the small sample volume. In this paper, we report a rapid, sensitive method for the analysis of noradrenaline and dopamine in CE coupled diode LIF detection, in which Cy5 was chosen as the fluorescence derivative reagent.The effects of buffer concentration, pH value, and reaction time on the derivatization reaction of monoamines were investigated. Experimental ReagentsUnless stated otherwise, all chemicals were of analyticalreagent grade (Beijing Reagent Corp., Beijing, China). Noradrenaline, dopamine and sodium dodecyl sulfate (SDS) were purchased from Sigma (St. Louis, MO, USA). Cy5 was obtained from Amersham Pharmcia Biotech, USA. Water was deionized and purified by a Milli-Q Millipore purification system (Millipore, Marlborough, MA, USA). ApparatusAll analyses were performed on a Beckman P/ACE 5000 system equipped with a Beckman Laser Module 635 nm. This was used as an exciting light source (Beckman-Fullerton, CA, USA). Separations were performed in fused-silica capillaries (57 cm × 75 µm i.d.; Yong Nian Optic Fiber Factory, Hebei Province, China). Derivatization procedureNoradrenaline and dopamine were labeled with Cy5 as follows: Cy5 was dissolved in dimethylformamide. The solution with a...

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Fluorescent Determination of Noradrenaline and Dopamine Derivatized with Cy5 in Capillary Electrophoresis

Zhang

et al. 2001

ANAL. SCI.

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“…23 In the last decade, capillary-zone electrophoresis has been successfully employed in the separation of proteins and peptides, especially for those with low molecular mass. 24,25 Despite its high efficiency, capillary electrophoresis (CE) presents some limitations related to the low sensitivity of UV detectors, which are widely employed in CE. 26 In this sense, it is not unexpected that several efforts have been made to couple CE with ICP-MS in order to overcome sensitivity limitations.…”

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Identification of a Metallothionein in Synechococcus by Capillary Electrophoresis Hyphenated with Inductively Coupled Plasma Mass Spectrometry

et al. 2003

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In 1957, Margoshes and Vallée 1 discovered a new type of protein, which seemed to contain metal ions in its structure. However, the first paper confirming the presence of cadmium in an isolated protein, named metallothionein (MT), was only published in 1960 by Kagi and Vallée. 2 Metallothioneins are proteins with low molecular mass, rich in cysteine residues and extremely reactive to bivalent metals. 3 These proteins are responsible for processes related to both intracellular absorption and the regulation of biologically essential elements, such as zinc and copper. MTs are also responsible for cell protection from toxic elements, such as cadmium and mercury. 4,5 Metallothioneins are present in mammals and also in eukaryote and prokaryote organisms. 3,6 In the majority of animal species, MTs are abundant in the liver, pancreas, intestines and kidney tissues. The concentration of these proteins depends on both the species and the tissues, and reveals the diet effect in each organism. 7 Metallothioneins are usually determined by employing classical methodologies, such as metal-ligand assays with mercury 8 and cadmium. 9 Among those classical methodologies are also found electrochemical techniques based on the quantification of MT fractions via the oxidation of sulfhydryl groups, 10 and also immunology techniques, such as radioimmunoassay 11 (RIA), and enzymatic assays in sorbents 12 (ELISA). However, methods involving metal saturation or electrochemical techniques undertake interferences due to oxyreduction reactions of other species present in the samples, 12 what makes them inefficient to quantify the metal concentration in the isoforms. 13 This drawback was overcome when anionexchange high-performance liquid chromatography (HPLC) was employed in the separation of the two main MT isoforms. 14 Subsequently, reverse-phase HPLC was also employed to improve the resolution in the separation of isoforms.15,16 HPLC was coupled to atomic absorption spectrometry (AAS) by using a T-shaped interface to determine Cd, Zn and Cu in MT from rabbit liver in µg g -1 level. 17,18 Other couplings, such as RP-HPLC with inductively coupled plasma mass spectrometry 19, and RP-HPLC with electrospray mass spectrometry 21,22 (ES-MS), were also proposed for the separation and determination of Cd and Zn in MT. Nevertheless, certain demands related to the utilization of organic solvents and column specificities restrict the use of RP-HPLC as a separation method. 23 In the last decade, capillary-zone electrophoresis has been successfully employed in the separation of proteins and peptides, especially for those with low molecular mass. 24,25 Despite its high efficiency, capillary electrophoresis (CE) presents some limitations related to the low sensitivity of UV detectors, which are widely employed in CE. 26 In this sense, it is not unexpected that several efforts have been made to couple CE with ICP-MS in order to overcome sensitivity limitations. However, there are some practical aspects, such as capillary positioning in the interface, flo...

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“…Here we report two related procedures for estimating the effective charge of a protein in solution at arbitrary values of pH by using capillary electrophoresis (CE) (4)(5)(6)(7)(8). The two procedures are complementary, but both rely on the same strategy: to generate and compare the electrophoretic mobilities of a protein with a series of derivatives of the protein that differ in effective charge by simple multiples of a unit charge but that differ only minimally in hydrodynamic drag.…”

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Determination of the effective charge of a protein in solution by capillary electrophoresis.

Gao

Gomez

Härter

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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This paper describes two methods to estimate the effective charge of a protein in solution by capillary electrophoresis and demonstrates these methods by using representative proteins. In one method, a "charge ladder"-a series of derivatives of a protein differing by known increments of charge but differing only minimally in hydrodynamic drag-is generated by covalent modification of the e-amino groups of lysines with 4-sulfophenyl isothiocyanate or acetic anhydride. In the second method, the equivalent of a charge ladder is produced by noncovalent association of a protein with differently charged ligands. (1) is straightforward and can be used to estimate the net charge of a protein over a range of values of pH. This method, however, requires substantial quantities (2-10 mg) of pure protein, and the measurement may be influenced by association of the protein with ions in solution. Winzor and Ojteg (2, 3) have estimated net charges of proteins based on Donnan potential measurements. These methods permit the charge ofa protein to be determined over a range ofionic composition and values ofpH but also require significant quantities (1-10 mg) of a protein.Here we report two related procedures for estimating the effective charge of a protein in solution at arbitrary values of pH by using capillary electrophoresis (CE) (4-8). The two procedures are complementary, but both rely on the same strategy: to generate and compare the electrophoretic mobilities of a protein with a series of derivatives of the protein that differ in effective charge by simple multiples of a unit charge but that differ only minimally in hydrodynamic drag.

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Recent advances in capillary electrophoresis of proteins, peptides and amino acids

Cited by 157 publications

References 71 publications

Fluorescent Determination of Noradrenaline and Dopamine Derivatized with Cy5 in Capillary Electrophoresis

Fluorescent Determination of Noradrenaline and Dopamine Derivatized with Cy5 in Capillary Electrophoresis

Identification of a Metallothionein in Synechococcus by Capillary Electrophoresis Hyphenated with Inductively Coupled Plasma Mass Spectrometry

Determination of the effective charge of a protein in solution by capillary electrophoresis.

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