Reaction of fluorogenic reagents with proteins

Wojcik, Roza; Swearingen, Kristian E.; Dickerson, Jane A.; Turner, Emily H.; Ramsay, Lauren M.; Dovic̀hi, Norman J.

doi:10.1016/j.chroma.2008.04.042

Cited by 29 publications

(32 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This reagent, developed by Wolfbeis, reacts with the unblocked N-terminus and the e-amines of lysine residues, converting the cationic amine into a cationic pyridinium, which should preserve the pI of the protein [27,28]. We have demonstrated sub-zeptomole detection limits, high efficiency, and high resolution generated by Chromeo P503-labeled proteins in sieving and sub-micellar electrophoresis [29,30]. This paper exploits the reagent for CIEF.…”

Section: Introductionmentioning

confidence: 94%

“…2). The background signal was also roughly four orders of magnitude more noisy than the background signal generated by a CHES-Tris-Dextran buffer used for sieving electrophoresis [29,30].…”

Section: Calibration Curve and Detection Limits For Labeled Proteinmentioning

confidence: 99%

“…Each day, a new sample was taken from the freezer and thawed at room temperature. Proteins were then labeled with Chromeo P503 [26][27][28][29][30]. To label proteins, samples were diluted to 5 mg/mL in ddH 2 O.…”

Section: Sample and Ampholyte Preparationmentioning

confidence: 99%

See 2 more Smart Citations

Attomole protein analysis by CIEF with LIF detection

2009

Self Cite

View full text Add to dashboard Cite

We have coupled CIEF with an LIF detector that is based on a post-column sheath flow cuvette. We employed Chromeo P503 as a fluorogenic reagent to label proteins before analysis. This reagent reacts with the epsilon-amine of lysine residues, preserving the cationic nature of the residue; labeled proteins generate extremely sharp peaks in CIEF. A set of four standard proteins generated a linear relationship between migration time and pI. A protein homogenate prepared from a Barrett's esophagus cell line resolved over 100 components in a 40 min separation. Detection limits for Chromeo P503-labeled beta-lactoglobulin were 5 amol injected into the capillary. Fluorescent impurities present in the ampholytes generated a large background signal that degraded the detection limit by four orders of magnitude compared with other forms of capillary electrophoresis with this detector.

show abstract

Section: Introductionmentioning

confidence: 94%

“…2). The background signal was also roughly four orders of magnitude more noisy than the background signal generated by a CHES-Tris-Dextran buffer used for sieving electrophoresis [29,30].…”

Section: Calibration Curve and Detection Limits For Labeled Proteinmentioning

confidence: 99%

See 1 more Smart Citation

Attomole protein analysis by CIEF with LIF detection

2009

Self Cite

View full text Add to dashboard Cite

show abstract

“…Fluorogenic reagents, which undergo a large increase in fluorescence signal upon reacting with a primary amine, are particularly useful for the analysis of very low concentration protein samples. 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].…”

Section: Introductionmentioning

confidence: 99%

Reaction of fluorogenic reagents with proteins

Swearingen

Dickerson

Turner

et al. 2008

Journal of Chromatography A

Self Cite

View full text Add to dashboard Cite

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.

show abstract

“…Therefore, the amounts of labeling reagent must be controlled carefully in precolumn and oncolumn derivatizations so as not to produce multiply labeled fluorescent derivatives. However, more recently, multiple labeling of a protein was characterized by MS, in which it has been shown that most labeling reactions produce a mixture of products with different number of labels attached to the protein [17]. Non-covalent precolumn derivatization is one of promising techniques for LIF detection of proteins.…”

Section: Introductionmentioning

confidence: 99%

Postcolumn derivatization of proteins in capillary sieving electrophoresis/laser‐induced fluorescence detection

Kaneta¹,

Yamamoto²,

Imasaka³

2009

Electrophoresis

View full text Add to dashboard Cite

The separation methods for proteins with high resolution and sensitivity are absolutely important in the field of biological sciences. Capillary sieving electrophoresis (CSE) is an excellent separation technique for DNA and proteins with high resolution, while LIF permits the most sensitive detection in CSE. Therefore, proteins have to be labeled with fluorescent or fluorogenic reagent to produce fluorescent derivatives. Both precolumn and oncolumn derivatization have been employed for the labeling of proteins in CSE. However, there is no report on the postcolumn derivatization due to the limitation in the use of a standard migration buffer, despite it being a promising method for sensitive detection of proteins. Here, we show a novel postcolumn derivatization method for protein separation by CSE, using a tertiary amine as a buffer component in the running buffer. Tris, which is commonly used as a base in CSE separation buffers, was substituted by tertiary amines, 2-(diethylamino)ethanol and triethanolamine. A buffer solution containing 2-(diethylamino)ethanol or triethanolamine can be used for the CSE separation followed by the postcolumn derivatization of proteins, since both reagents are unreactive toward a fluorogenic labeling reagent, naphthalene-2,3-dicarbaldehyde. Thus, LIF detection using the postcolumn derivatization permits significant reduction in the LOD (by a factor of 2.4-28) of proteins, compared with conventional absorbance detection.

show abstract

Reaction of fluorogenic reagents with proteins

Cited by 29 publications

References 31 publications

Attomole protein analysis by CIEF with LIF detection

Attomole protein analysis by CIEF with LIF detection

Reaction of fluorogenic reagents with proteins

Postcolumn derivatization of proteins in capillary sieving electrophoresis/laser‐induced fluorescence detection

Contact Info

Product

Resources

About