Real-time fluorescence monitoring of tryptic digestion in proteomics.
Ensuring that proteolytic digestions are complete before submitting samples for downstream proteomic analyses is important, as failure or partial digestion can waste valuable instrument time and make results difficult to interpret. Conversely, overdigestion can also be problematic, such as when removing affinity tags from recombinant proteins or using nonspecific proteases. The techniques of HPLC, circular dichroism, SDS-PAGE, and MS have each been used to assess protein digestion. These techniques are slow, may require expensive instrumentation, can be inaccurate, and/or are unsuitable for real-time monitoring. Epicocconone is a natural fluorophore that reacts reversibly with proteins to form a highly fluorescent adduct and has previously been used to quantify proteins in 1D and 2D gels and in solution. Here, we describe a new method for the real-time monitoring of protein digestion based on epicocconone. This unique in situ fluorescent assay can tracelessly follow proteolysis of samples, at low microgram levels, destined for proteomics analysis or purification.
Department of Chemistry & Biomolecular Sciences, Macquarie University, Sydney NSW 2109, Australia. peter.karuso@mq.edu.au
Peter
P
Karuso
Angela S
AS
Crawford
Duncan A
DA
Veal
Graham B I
GB
Scott
Hung-Yoon
HY
Choi
D001578:Benzopyrans; D005456:Fluorescent Dyes; D005470:Fluorometry; D005663:Furans; D007659:Ketones; D007700:Kinetics; D011506:Proteins / Q000032:analysis* / Q000378:metabolism*; D040901:Proteomics; D014357:Trypsin / Q000378:metabolism*
D001578:Benzopyrans; D005456:Fluorescent Dyes; D005663:Furans; D007659:Ketones; D011506:Proteins; C477464:epicocconone; D014357:Trypsin