Protein C-Terminal Labeling and Biotinylation Using Synthetic Peptide and Split-Intein
Figure 4
Detection of possible N-cleavage as a side reaction.
A. Illustration of the detection strategy. The predicted N-cleavage product MC is compared with the trans-splicing (C-terminally labeled) product ML. Site-specific cleavage of MC and ML with protease Factor Xa releases the C-terminal peptide pepXa and pepXa-L, respectively. B. RP-HPLC analysis. The left chromatogram shows a synthetic pepXa used as a standard. The middle and right chromatograms show the sample peptide detected at 210 nm (middle) and 497 nm (right). The increasing baseline absorbance at 497 nm over time was most likely due to the absorbance of the increasing concentration of the mobile phase B solution. C. MS-coupled HPLC analysis. The pepXa-L was much more abundant than pepXa, and both peptides were identified by their corresponding molecular weights in mass spectrometry analysis (data not shown).