Journal
ANALYTICAL CHEMISTRY
Volume 72, Issue 14, Pages 3374-3378Publisher
AMER CHEMICAL SOC
DOI: 10.1021/ac000045i
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- NCRR NIH HHS [RR00862] Funding Source: Medline
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Accurate definition of the carboxyl terminal of proteins is necessary for elucidating posttranslational processing at the C-terminal and more generally for characterizing protein primary structures. Here, we describe a strategy for isolating and characterizing the C-terminal peptide of a protein after proteolysis with endoprotease Lys-C, Isolation is achieved using anhydrotrypsin, a catalytically inert derivative of trypsin that binds peptides containing lysine or arginine residues at their C-terminl without cleaving them. Rapid, accurate characterization of the isolated C-terminal peptide is achieved by mass spectrometry. Initial identification of the C-terminal peptide is obtained by comparing matrix-assisted laser desorption/ionization time-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 isolated peptide using a quadrupole ion trap mass spectrometer in the selective reaction monitoring mode. This strategy was successfully applied to the characterization of the C-terminal of proteins with molecular masses ranging up to 56 kDa.
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