Heterogeneity of protein labeling with a fluorogenic reagent, 3-(2-furoyl)quinoline-2-carboxaldehyde

Stoyanov, Alexander V.; Ahmadzadeh, Hossein; Krylov, Sergey N.

doi:10.1016/s1570-0232(02)00535-4

Cited by 24 publications

(23 citation statements)

References 14 publications

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“…Two peaks, migrating earlier than oxidized BSA, appeared only in the Alexa 488 hydrazide trace (top trace) and are attributed to residual Alexa 488 hydrazide. Figure 2, lower trace, also shows that the new peak has fluorescence resulting from the FQ-labeling of primary amino groups of BSA [17][18][19][20]. It is clear that oxidized BSA is detected in both the Alexa 488 and the FQ detectors, confirming that Alexa 488 hydrazide is capable of labeling oxidized proteins.…”

Section: Labeling Of Carbonyl Groups With Alexa 488 Hydrazidementioning

confidence: 73%

“…Because carbonyls may also be found in lipids, carbohydrates, and nucleic acids [16], the proteins were labeled with the fluorogenic reagent 3-(2-furoyl) quinoline-2-carboxaldehyde (FQ), which forms a fluorescent product that shows little spectral overlap with the Alexa 488 fluorescence [17][18][19][20]. In addition, the resulting separation of the labeled mixture in the CSE system suggested that the detected carbonyl-containing proteins have molecular weights in the range of ,26-30 kDa.…”

Section: Introductionmentioning

confidence: 99%

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Quantification of carbonylated proteins in rat skeletal muscle mitochondria using capillary sieving electrophoresis with laser‐induced fluorescence detection

Feng

Arriaga

2008

Electrophoresis

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Carbonyl-modified proteins are markers of oxidative damage. Here, we report a new method for detecting and quantifying carbonylated proteins by capillary sieving electrophoresis (CSE) with LIF detection (CSE-LIF). Alexa 488 hydrazide is used for the specific labeling of carbonyls while 3-(2-furoyl) quinoline-2-carboxaldehyde (FQ) is used for protein labeling. BSA subjected to metal-catalyzed oxidation is used to optimize the labeling reactions, confirm the separation power of CSE, and characterize the response of the LIF detector. The method is capable of detecting femtomole (fmol) amounts of carbonyls in proteins with molecular masses ranging from 26 to 30 kDa. Using this method, we determined that mitochondrial proteins isolated from skeletal muscle contains 2.1 +/- 0.1 (average +/- SD; n = 3) nmol carbonyl/mg protein. The methodology described here should be compatible with the analysis of single cells and needle biopsies taken from oxidative stress animal models.

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Section: Labeling Of Carbonyl Groups With Alexa 488 Hydrazidementioning

confidence: 73%

Section: Introductionmentioning

confidence: 99%

Quantification of carbonylated proteins in rat skeletal muscle mitochondria using capillary sieving electrophoresis with laser‐induced fluorescence detection

Feng

Arriaga

2008

Electrophoresis

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“…Each of these products can have different electrophoretic or chromatographic properties, and labeled proteins can produce complex electropherograms during separation [26][27][28].…”

Section: Discussionmentioning

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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“…The block's temperature dropped from 95 to 60-707C during the 5-min incubation. Previous research on the reaction kinetics of FQ labeling demonstrated that the amount of labeled product reached a plateau after heating a standard protein with an excess amount of FQ and KCN for 5 min at 657C [26,27]. …”

Section: -De Single-cell Analysismentioning

confidence: 99%

Repeatability of chemical cytometry: 2‐DE analysis of single RAW 264.7 macrophage cells

2007

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This report presents the use of 2-DE with ultrasensitive fluorescence detection as a chemical cytometry tool to characterize the protein and biogenic amine content of single cells from the RAW 264.7 murine macrophage cell line. Cells were sorted by cell cycle prior to 2-DE analysis. Cells in the G2/M phase of the cell cycle were aspirated into the first-dimensional capillary and lysed. The cellular contents were fluorescently labeled and first separated by capillary sieving electrophoresis (CSE). Over 380 fractions were transferred from the first-dimensional capillary to the second-dimensional capillary, where components were further separated by MEKC and detected by laser-induced fluorescence. Twenty-five spots common to the four electropherograms were fit with a 2-D Gaussian surface to determine spot position, width, and amplitude. The RSD in CSE mobility was 1.0 +/- 0.6%. The mean uncertainty in spot position was 1.3 times larger than the mean spot width in the CSE dimension. The average SD in MEKC migration time was 0.37 +/- 0.13 s, which is smaller than the average spot size in this dimension. Spot capacity was 200. The RSD in spot amplitude was 50%, reflecting a large cell-to-cell variation in component expression.

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Heterogeneity of protein labeling with a fluorogenic reagent, 3-(2-furoyl)quinoline-2-carboxaldehyde

Cited by 24 publications

References 14 publications

Quantification of carbonylated proteins in rat skeletal muscle mitochondria using capillary sieving electrophoresis with laser‐induced fluorescence detection

Quantification of carbonylated proteins in rat skeletal muscle mitochondria using capillary sieving electrophoresis with laser‐induced fluorescence detection

Reaction of fluorogenic reagents with proteins

Repeatability of chemical cytometry: 2‐DE analysis of single RAW 264.7 macrophage cells

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