Reaction of fluorogenic reagents with proteins. III. Spectroscopic and electrophoretic behavior of proteins labeled with Chromeo P503

Department of Chemistry, University of Washington, Seattle, WA 98195, USA.
Journal of Chromatography A (Impact Factor: 4.17). 07/2008; 1194(2):253-6. DOI: 10.1016/j.chroma.2008.04.046
Source: PubMed


The spectroscopic and electrophoretic properties of proteins labeled with Chromeo P503 were investigated. Its photobleaching characteristics were determined by continually infusing Chromeo P503-labeled alpha-lactalbumin into a sheath-flow cuvette and monitored fluorescence as a function of laser power. The labeled protein is relatively photo-labile with an optimum excitation power of about 2 mW. The unreacted reagent is weakly fluorescent but present at much higher concentration than the labeled protein. The unreacted reagent undergoes photobleaching at a laser power more than an order of magnitude higher than the labeled protein. One-dimensional capillary electrophoresis analysis of Chromeo P503-labeled alpha-lactalbumin produced concentration detection limits (3sigma) of 12 pM and mass detection limits of 0.7 zmol, but with modest theoretical plate counts of 17,000. The reagent was employed for the two-dimensional capillary electrophoresis analysis of a homogenate prepared from a Barrett's esophagus cell line; the separation quality is similar to that produced by 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ), a more commonly used reagent.

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    ABSTRACT: The fluorogenic reagent Chromeo P465 is considered for the analysis of proteins by capillary electrophoresis with laser-induced fluorescence detection. The reagent was first used to label alpha-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 x 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 alpha-lactalbumin (r(2)=0.99), with a 3sigma detection limit of 70 pM. We then considered the separation of a mixture of ovalbumin, alpha-chymotrypsinogen A, and alpha-lactalbumin 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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    ABSTRACT: 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.
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