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Anal Chem. 2000 Jul 15;72(14):3374-8.

A method to define the carboxyl terminal of proteins.

Author information

1
Laboratory for Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, New York 10021, USA.

Abstract

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-termini 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-HPLC 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.

PMID:
10939415
DOI:
10.1021/ac000045i
[Indexed for MEDLINE]

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