A method for C-terminal sequence analysis in the proteomic era (proteins cleaved with cyanogen bromide).
There is growing interest in the overall study of post-translational modifications (PTMs) of proteins. Beside phosphorylation and glycosylation, truncations of the nascent polypeptide chain at the N or C termini are by far the most common types of PTMs found in proteins. However, little attention has been paid to the development of approaches that allow a systematic analysis of these proteolytic processing events. Here we present a protocol that allows the identification of the C-terminal sequences of proteins. A peptide mixture is generated by cleavage of the protein with cyanogen bromide and is incubated with carboxypeptidase Y. The enzyme is only able to act on the C-terminal fragment, because this is the only peptide without a homoserine lactone residue at its C terminus. The resulting fragments, forming a peptide ladder, are analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The entire protocol, including the CNBr cleavage, takes 21 h and can be applied to proteins purified either by SDS-PAGE or by 2D PAGE or in solution.
Samyn
Bart
B
Department of Biochemistry, Physiology and Microbiology, Laboratory of Protein Biochemistry and Protein Engineering, Ghent University, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium. bart.samyn@UGent.be
Sergeant
Kjell
K
Van Beeumen
Jozef
J
D043402:Cathepsin A; D003488:Cyanogen Bromide; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D020539:Sequence Analysis, Protein / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D011506:Proteins; D043402:Cathepsin A; D003488:Cyanogen Bromide