C-terminal sequence analysis of 2DE-separated proteins.
The overall study of post-translational modifications (PTMs) of proteins is gaining strong interest. Beside phosphorylation and glycosylation, truncations of the nascent polypeptide chain at the N- or C-terminus are by far the most common types of PTMs. Nevertheless, 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 sequence of proteins separated by two-dimensional polyacrylamide gel electrophoresis (2DE). For each purified protein, a peptide mixture is generated by cleavage of the protein with cyanogen bromide. During incubation with carboxypeptidases only the original C-terminal fragment forms a ladder. Ladder readout is performed using MALDI mass spectrometry. 2DE-separated proteins from Shewanella oneidensis were chosen as a model system to investigate the effectiveness of the approach.
Samyn
Bart
B
Laboratory of Protein Biochemistry and Protein Engineering, Department of Biochemistry, Physiology and Microbiology, Ghent University, K.L. Ledeganckstraat 35, B-9000, Gent, Belgium.
Sergeant
Kjell
K
Van Beeumen
Jozef
J
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D020539:Sequence Analysis, Protein / Q000379:methods*; D020592:Shewanella / Q000737:chemistry
D001426:Bacterial Proteins