Picomolar Assay of Native Proteins by Capillary Electrophoresis Precolumn Labeling, Submicellar Separation, and Laser-Induced Fluorescence Detection

Pinto, Devanand M.; Arriaga, Edgar A.; Craig, Doug; Angelova, Jordanka; Sharma, Neepun; Ahmadzadeh, Hossein; Dovic̀hi, Norman J.; Boulet, Camille A.

doi:10.1021/ac9611677

Cited by 123 publications

(114 citation statements)

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“…These products can have dramatically different electrophoretic behavior, leading to an envelope of peaks producing complex electropherograms [4][5][6]. Most of our work has been with FQ, which converts cationic lysine residues into neutral products, which can be detected with exquisite sensitivity [7][8][9][10]. More importantly, we have shown that the incorporation of an anionic surfactant, such as SDS, results in the collapse of the multiple labeling envelope into a single, sharp peak [7].…”

Section: Introductionmentioning

confidence: 99%

Reaction of fluorogenic reagents with proteins

Swearingen

Dickerson

Turner

et al. 2008

Journal of Chromatography A

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The fluorogenic reagent Chromeo P465 is considered for analysis of proteins by capillary electrophoresis with laser-induced fluorescence detection. The reagent was first used to label α-lactalbumin; the product was analyzed by capillary zone electrophoresis in a sub-micellar sodium dodecyl sulfate (SDS) buffer. The product generated a set of equally spaced but poorly resolved peaks that formed a broad envelope with a net mobility of 4 × 10 −4 cm 2 V −1 s −1 . The components of the envelope were presumably protein that had reacted with different numbers of labels. The mobility of these components decreased by roughly 1 % with the addition of each label. The signal increased linearly from 1.0 nM to 100 nM α-lactalbumin (r 2 = 0.99), with a 3σ detection limit of 70 pM. We then considered the separation of a mixture of ovalbumin, α-chymotrypsinogen A, and αlactalbumin labeled with Chromeo P465; unfortunately, baseline resolution was not achieved with a borax/SDS buffer. Better resolution was achieved with N-cyclohexyl-2-aminoethanesulfonic acid/ Tris/SDS/dextran capillary sieving electrophoresis; however, dye interactions with this buffer system produced a less than ideal blank.

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Section: Introductionmentioning

confidence: 99%

Reaction of fluorogenic reagents with proteins

Swearingen

Dickerson

Turner

et al. 2008

Journal of Chromatography A

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“…These disadvantages make this method inappropriate for large volumes; (ii) the use of the laser-induced fluorescence detection system would be another way to improve the detectability and sensitivity of the method. 17 For the phytohormones, which do not have a natural fluorescence, derivatization reactions are widely described in the literature and can be used, 18 however, derivatization steps are always susceptible to error and involve cost and are time consuming.…”

Section: Resultsmentioning

confidence: 99%

Direct determination of plant-growth related metabolites by capillary electrophoresis with spectrophotometric UV detection

Assunção¹,

Arruda²,

Martinelli³

et al. 2009

J. Braz. Chem. Soc.

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A detecção de hormônios e reguladores de crescimento em plantas é de grande interesse em uma enorme variedade de estudos biológicos. Visando este propósito nós desenvolvemos um método simples baseado em eletroforese capilar (CE) para a separação de diferentes classes de reguladores de crescimento de plantas como as auxinas, citocinina, ácidos giberélico e abscíssico. Foi utilizada CE, com detecção na região de UV; as condições de separação foram: fosfato de potássio como tampão, em concentração de 25 mmol L -1 , pH 12 ou 2,5, tempo de injeção 5 s, elevando por sinfonamento o capilar, à altura de 10 cm e voltagem de separação de 22 kV. A absorbância foi fixada a 220 nm ou 270 nm, dependendo da classe do fitohormônio. Nestas condições os fitohormônios (IAA, GA 3 , ABA, picloram, zeatina e BAP) foram separados em um tempo de 3 a 5 min. O material oriundo de planta para verificar a possibilidade de detecção de hormônio/reguladores de crescimento de plantas foi a citro (Citrus sinensis L. Osbeck) callus no estágio de multiplicação. Na amostra de tecido de planta, a zeatina foi detectada com sucesso. Os resultados confirmaram a pontencialidade do uso de CE como um método eficiente, de baixo custo e alternativo para a detecção dessa classe de compostos em tecidos de plantas.The detection of plant hormones and growth regulators is of great interest for many biological studies especially in the determination of metabolites related to plan growth and differentiation. In this work, we propose a simple method based on capillary electrophoresis (CE) for the separation of different classes of plant growth regulators such as auxins, cytokinins, gibberelic acid and abscisic acid. CE with UV detection was used and the analytical conditions were as follows: phosphate buffer 25 mmol L -1 , for all the measurements and the separation conditions pH 12 or 2.5, by hydrodynamic injection 5 s at 10 cm and separation voltage of 22 kV. The absorbance detection was fixed at either 220 nm or 270 nm depending on a given phytohormone class. Under these conditions, phytohormones (Indole-3-acetic acid (IAA), Gibberellic acid (GA 3 ), Abscisic acid (ABA), picloram, zeatin and 6-Benzylaminopurine (BAP) were separated in approximately 3 to 5 min. The plant material used to verify the possibility of detection of hormone/plant growth regulators was citro (Citrus sinensis L. Osbeck) callus in the multiplication stage. In the plant tissue sample, zeatin was successfully detected. The results confirmed the potential use of CE as an efficient alternative and simple method to the classical procedures used for phytohormone detection in plant tissues.Keywords: plant metabolites, capillary electrophoresis, phytohormones, Citrus spp IntroductionThe determination and characterization of metabolites involved in plant growth play an important role in plant morphogenesis studies. Among the substances involved in this process are the plant hormones, which are traditionally classified in the following groups: auxins, cytokinins, gibberellins, abscisic acid, and et...

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“…by adopting a frequency-doubled Ar laser operating at 257 nm and a Kr laser pumping at 284 nm [55]. Alternatively, peptides can be pre-or post-column derivatized with a number of fluorescent dyes, as reviewed in [56,57], among which: fluorescein isothiocyanate, fluorescamine and the near-infrared fluorescent dye NN382 [58] and the fluorogenic dye 5-furoyl-quinoline-3-carboxyaldehyde [59]. Mass spectrometry, in addition, represents perhaps the ultimate in peptide detection, since it couples good sensitivities (low femtomoles) to the ability of identifying the peptide via the possibility of obtaining its sequencing in the post-source decay mode in MALDI-TOF instrumentation [60,61].…”

Section: Permanent and Dynamic Coatings For Proper Peptide/protein Sementioning

confidence: 99%

Capillary electrophoretic analysis of proteins and peptides of biomedical and pharmacological interest

Righetti

2001

Biopharm & Drug Disp

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Capillary electrophoresis (CE) is an automated approach to electrokinetic separations that has had a deep impact in all fields of life sciences, including biomedical and biotechnological research and clinical and forensic practice. The present review highlights aspects of peptides and proteins separations, with particular emphasis on macromolecular analytes of biomedical interest. Among the various CE techniques available, a novel methodology is here illustrated consisting in separations in acidic, isoelectric buffers, which have the advantage of protonating the silica wall, thus minimizing interactions of proteinaceous material with the siliceous surface, while allowing delivery of high voltage gradients, due to their low conductivities. The review ends with applications of CE to the analysis of folding/unfolding/refolding/misfolding of proteins, a field which has deep implications in the biomedical arena, since it is connected to a host of disorders, such as prion protein diseases.

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Picomolar Assay of Native Proteins by Capillary Electrophoresis Precolumn Labeling, Submicellar Separation, and Laser-Induced Fluorescence Detection

Cited by 123 publications

References 42 publications

Reaction of fluorogenic reagents with proteins

Reaction of fluorogenic reagents with proteins

Direct determination of plant-growth related metabolites by capillary electrophoresis with spectrophotometric UV detection

Capillary electrophoretic analysis of proteins and peptides of biomedical and pharmacological interest

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