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bibo:Article rdac:C10001

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Dynamic Labeling during Capillary or MicrochipElectrophoresis for Laser-Induced FluorescenceDetection of Protein−SDS Complexes without Pre-or Postcolumn Labeling

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The analysis of proteins under denaturing conditions isroutinely performed with SDS−polyacrylamide gel electrophoresis. The automated capabilities of CE, use ofnongel sieving matrixes, and on-line optical detection byeither ultraviolet (UV) absorption or laser-induced fluorescence (LIF) promise to revolutionize this method.While direct on-line detection of proteins is possible as aresult of their intrinsic ability to absorb light in the UVpart of the spectrum (detection sensitivity comparable toCoomassie Blue staining of gels), LIF provides morepowerful detection but requires pre- or postcolumn fluorescence labeling of the proteins. The development of aprotocol analogous to that used for double-stranded DNAanalysis, where fluorescent intercalating dyes are simplyincluded in the separation medium, would simplify size-based protein analysis immensely. This would avoid thecomplications associated with covalent modification of theproteins but still exploit the sensitivity of LIF detection.We demonstrate that this is possible with CE and microchip detection by incorporating, into the run buffer, afluorescent dye that interacts hydrophobically with protein−SDS complexes. Key to this is a dye that fluorescessignificantly when bound to protein−SDS complexes butnot when bound to SDS micelles. Comparison of electropherograms from CE-based denaturing protein analysiswith UV and LIF detection indicates that the presence ofthe fluor does not alter separation of the proteins. Moreover, comparison with electropherograms generated frommicrochip electrophoresis with LIF detection shows thatequivalent patterns can be obtained. Despite the unoptimized nature of this separation system, a dynamic labelingprotocol that allows for LIF detection for proteins isattractive and has the potential to circumvent the tediouslabeling steps typically required.

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