Real-time monitoring of tryptic digests using the fluorophore epicocconone

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.