Our R&D team has developed a new workflow that utilizes MT-Trypsin to facilitate rapid in-gel digestion of proteins followed by trypsin removal from peptide samples.
In-gel digestion, the enzymatic digestion of proteins in bands or plugs excised from one- or two-dimensional polyacrylamide gels, is a commonly used method of preparing peptides for mass spectrometry (MS) analysis in bottom-up proteomics workflows.
Utilizing click chemistry technology, we developed an in-gel digestion protocol that enables rapid, two-hour enzymatic digestion of proteins. This workflow includes aspects of a canonical method of in-gel digestion combined with the utilization of methyltetrazine (MT) modified trypsin (MT-Trypsin) that covalently binds to trans-cyclooctene (TCO) resin, allowing its removal from the digestion reaction. We also introduced homogenization steps using a pestle to crush the gel band into small fragments that facilitate MT-Trypsin migration into the gel matrix.
Using Cy3-labeled proteins of various molecular weights, we demonstrate the efficacy of this in-gel digestion procedure and show that this method enables detection of proteins present in gel samples at femtomolar quantities.