A new chemical approach to differentiate carboxy terminal peptide fragments in cyanogen bromide digests of proteins.
We present a novel approach to perform C-terminal sequence analysis by discriminating the C-terminal peptide in a mass spectral analysis of a CNBr digest. During CNBr cleavage, all Met-Xxx peptide bonds are cleaved and the generated internal peptides all end with a homoserine lactone (hsl)-derivative. The partial opening of the hsl-derivatives, by using a slightly basic buffer solution, results in the formation of m/z doublets (Deltam=18 Da) for all internal peptides and allows to identify the C-terminal peptide which appears as a singlet in the mass spectra. Using two model proteins we demonstrate that this approach can be applied to study proteins purified in gel or in solution. The chemical opening of the hsl-derivative does not require any sample clean-up and therefore, the sensitivity of the C-terminal sequencing approach is increased significantly. Finally, the new protocol was applied to characterize the C-terminal sequence of two recombinant proteins. Tandem mass spectrometry by MALDI-TOF/TOF allowed to identify the sequence of the C-terminal peptides. This novel approach will allow to perform a proteome-wide study of C-terminal proteolytic processing events in a high-throughput fashion.
Ghent University, Department Biochemistry and Microbiology, Ghent, Belgium.
P P
PP
Moerman
K
K
Sergeant
G
G
Debyser
B
B
Devreese
B
B
Samyn
D015107:4-Butyrolactone / Q000031:analogs & derivatives / Q000737:chemistry; D000595:Amino Acid Sequence; D003488:Cyanogen Bromide; D010446:Peptide Fragments / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D020539:Sequence Analysis, Protein / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D010446:Peptide Fragments; D011506:Proteins; C088386:homoserine lactone; D015107:4-Butyrolactone; D003488:Cyanogen Bromide