Real-time monitoring of tryptic digests using the fluorophore epicocconone

H. Choi, A. S. Crawford, D. E. Chen, R. J. Meheigh, J. Wildsmith, K. M. Ray, G. B. I. Scott, J. Mackintosh, D. A. Veal, P. H. Karuso

    Research output: Contribution to journalMeeting abstract


    Mass Spectrometry is a powerful tool for protein identification following proteolytic digestion using a protease such as trypsin. One problem inherent to the use of proteases is ensuring that digestion is as complete as possible. SDS-PAGE is often used to evaluate the totality of digestion, but this technique is laborious and semi-quantitative at best. Here, we demonstrate that epicocconone, a fluorophore currently being used as part of FluoroProfile Protein Quantification Kit (Sigma Aldrich), can be used to successfully monitor the degree of digestion in real time. Specifically, it is observed that fluorescence drops exponentially during the proteolysis reaction until it reaches a low baseline steady state level, indicating that the reaction has been driven to completion. With suitable controls, it is also possible to extract kinetic data from the fluorescence progress curves, making this a novel method for following protease kinetics. The in situ monitoring of proteolysis using epicocconone was confirmed by SDS-PAGE, HPLC and LC mass spectrometry. Additionally, it was demonstrated that this fluorophore does not create adducts that interfere with the downstream mass spectrometric analysis.
    Original languageEnglish
    Pages (from-to)67
    Number of pages1
    JournalJournal of biomolecular techniques : ABRF 2006 poster abstracts
    Issue number1
    Publication statusPublished - 2006
    EventABRF 2006 integrating science, tools and technologies with systems biology - Long Beach, CA
    Duration: 11 Feb 200614 Feb 2006


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