Fluorescence reagents for high-sensitivity chromatographic measurements of primary amines

Beale, Stephen C.; Savage, Joseph C.; Wiesler, Donald; Wietstock, Shawn M.; Novotný, Miloš V.

doi:10.1021/ac00168a025

Cited by 65 publications

(24 citation statements)

References 22 publications

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“…The preceding paper in this issue considered the reaction kinetics of three fluorogenic reagents, 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ), Chromeo P465, and Chromeo P503 with α-lactalbumin [1]. These reagents were introduced by Novotny and Wolfbeis [2,3]. We observed that the reaction of FQ was sluggish compared with the Chromeo reagents but that the FQ reaction ultimately incorporated more labels at room temperature.…”

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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“…The ophthaldehyde (OPA) that produces fluorescent isoindoles is detected in the fentomolar range [34]. Naphtalene-2,3-dicarboxaldehyde (NDA), an analog of OPA, with higher quantum yield, and 3-benzoyl-2-quinolinecarboxaldehyde (BQCA) have more stable derivatives than OPA [35,36]. 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CNBQCA) forms highly fluorescent isoindoles, just as OPA and NDA, but with advantages such as detection limits in nanomolar range, fast reaction time and good product stability [37].…”

Section: Detection Techniques Coupled To Cementioning

confidence: 99%

Biomedical applications of capillary electrophoresis with laser‐induced fluorescence detection

Páez

Hernández

2001

Biopharm & Drug Disp

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Capillary electrophoresis (CE) is a high-efficiency analytical technique that has had a great impact as a tool in biomedical research, clinical and forensic practice in the last ten years. Only in one of the applications, the DNA analysis, it has had an explosive exponential growth in the last few years. This impact is expressed in an enormous amount of CE articles and many reviews. The CE advantages with respect to other analytical techniques: the required very small sample volume, rapid analysis, great resolution power and low costs, have made this technique ideal for the analysis of a numerous endogenous and exogenous substances present in biological fluids. The different modes of CE have been coupled to different detection techniques such as UV-absorbance, electrochemical, mass spectrometry and laser-induced fluorescence detection (LIFD) to detect different nature and molecular size separated analytes. This review focuses mostly on the applications of CE-LIFD, to measure drugs and endogenous neuroactive substances such as amino acids and monoamines, especially in microdialysis samples from experimental animals and humans. CE-LIFD trends are discussed: automated faster analysis with capillary array systems, resolution power improvement, higher detection sensitivity, and CE systems miniaturization for extremely small sample volume, in order to make CE easier and affordable to the lab bench or the clinical bed.

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“…Fluorogenic reagents are weakly fluorescent until they react with a primary amine to produce a highly fluorescent product. Novotny developed a set of fluorogenic reagents that react with primary amines in the presence of a nucleophile, such as cyanide, to produce isoindoles with excitation maxima in the visible portion of the spectrum [13,14].We find 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ) to be particularly useful for the ultrasensitive detection of proteins by capillary electrophoresis [15][16][17][18]. The products of this reagent with primary amines can be excited efficiently by relatively inexpensive and reliable 473 nm or 488 nm lasers, and labeled proteins have good electrophoretic properties in buffers containing anionic surfactants, such as sodium dodecyl sulfate (SDS) [15,17].…”

Section: Introductionmentioning

confidence: 99%

Reaction of fluorogenic reagents with proteins

Wojcik

Swearingen

Dickerson

et al. 2008

Journal of Chromatography A

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Abstract3-(2-Furoyl)quinoline-2-carboxaldehyde (FQ), Chromeo P465, and Chromeo P503 are weakly fluorescent reagents that react with primary amines to produce fluorescent products. We studied the reaction of these reagents with α-lactalbumin by mass spectrometry. The reaction generated a set of products by the addition of one or more labels to the protein. At room temperature, the reaction was an order of magnitude faster with the Chromeo reagents than with FQ; however, the steady-state labeling efficiency was a factor of two higher for FQ compared with the Chromeo reagents. The relative abundance of the products with FQ usually followed a binomial distribution, which suggests that the labeling sites were uniformly accessible to this reagent. In contrast, the distribution of reaction products with the Chromeo reagents did not follow a binomial distribution for reactions performed in the absence of sodium dodecyl sulfate (SDS); it appears that the protein labeled with the Chromeo reagents refolded into a relatively stable secondary structure that hid some reactive sites. The reaction with the Chromeo reagent did follow the binomial distribution if the protein underwent treatment with 1% SDS at 95 °C for 5 min, which apparently disrupts the protein's secondary structure and allowed uniform access to all labeling sites. Chromeo 503 labeled seven of 17 the 13 primary amines in denatured α-lactalbumin.

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Fluorescence reagents for high-sensitivity chromatographic measurements of primary amines

Abstract: Model fluorogenlc reagents have been synthesized and evaluated toward the goal of ultrahlgh-sensltlvlty chromatographic analysis of primary amines with laser-based detection.

Cited by 65 publications

References 22 publications

Reaction of fluorogenic reagents with proteins

Reaction of fluorogenic reagents with proteins

Biomedical applications of capillary electrophoresis with laser‐induced fluorescence detection

Reaction of fluorogenic reagents with proteins

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