Manipulation of protein fingerprints during on-column fluorescent labeling: Protein fingerprinting of sixStaphylococcus species by capillary electrophoresis

Abstract

Bacterial proteomes were analyzed by use of electrophoretically mediated microanalysis (EMMA) and field-enhanced stacking. A water-soluble protein fraction was injected onto a capillary. Next, a fluorogenic reagent was injected and allowed to react with the protein mixture, producing fluorescent products that were separated by submicellar capillary electrophoresis and detected by laser-induced fluorescence. By use of a low-ionic strength sample buffer and a brief electrophoretic step, slow moving anionic proteins were stacked at the reagent-sample interface and were preferentially labeled. By reversing the order of sample injection and labeling reagent, fast moving cationic proteins were preferentially labeled. By adjustment of the sample buffer pH, proteins with different isoelectric points were selectively labeled. Electrophoresis fingerprints were generated for the water-soluble protein fraction from six Staphylococcus species. The protein patterns produced were species-specific and were used to construct a phylogenetic tree.

Methods for samples preparation in proteomic research

“…Therefore, in order to remove the contaminants, TCA/acetone precipitation or other saltexcluding techniques are effectively performed. Alternatively, high-speed centrifugation [234,235] can be applied.…”

Separation methods applicable to the evaluation of enzyme–inhibitor and enzyme–substrate interactions

“…Enzyme-substrate reactions are model systems for analysis using EMMA [109][110][111][112][113][114][115][116][117][118][119]. For example, the Glatz group has used EMMA to analyze the activity of rhodanese, the enzyme which catalyzes the reaction of thiosulfate with cyanide to form thiocyanate and sulfite [120][121][122].…”

Capillary sodium dodecyl sulfate-DALT electrophoresis of proteins in a single human cancer cell

“…SDS is used to denature the proteins and make them negativecharged for the subsequent separation. As we have demonstrated, the order of introduction of protein and labeling reagent affects the labeling efficiency [38]. For example, slow moving proteins are labeled more efficiently if their injection is followed by that of the labeling reagent; this order of injection places the slow moving proteins in intimate contact with the derivatization reagent.…”