A method to define the carboxyl terminal of proteins

Accurate definition of the carboxyl terminal of proteins is necessary for e lucidating posttranslational processing at the C-terminal and more generall y for characterizing protein primary structures. Here, we describe a strate gy for isolating and characterizing the C-terminal peptide of a protein aft er proteolysis with endoprotease Lys-C, Isolation is achieved using anhydro trypsin, a catalytically inert derivative of trypsin that binds peptides co ntaining lysine or arginine residues at their C-terminl without cleaving th em. Rapid, accurate characterization of the isolated C-terminal peptide is achieved by mass spectrometry. Initial identification of the C-terminal pep tide is obtained by comparing matrix-assisted laser desorption/ionization t ime-of-flight mass spectra of the digest prior to and after incubation with anhydrotrypsin, Characterization of the C-terminal sequence is achieved by capillary-HPU: electrospray ionization tandem mass spectrometry of the iso lated peptide using a quadrupole ion trap mass spectrometer in the selectiv e reaction monitoring mode. This strategy was successfully applied to the c haracterization of the C-terminal of proteins with molecular masses ranging up to 56 kDa.