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A method for C-terminal sequence analysis in the proteomic era (proteins cleaved with cyanogen bromide)
Bart Samyn1, Kjell Sergeant, Jozef Van Beeumen
1Department 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
Nature Protocols
|April 5, 2007
Summary
This study introduces a novel protocol for identifying protein C-terminal sequences. The method systematically analyzes proteolytic processing events, offering a new tool for studying post-translational modifications.
Area of Science:
- Biochemistry
- Proteomics
- Molecular Biology
Background:
- Post-translational modifications (PTMs) are crucial for protein function.
- Protein truncations at N or C termini are common PTMs, yet systematic analysis methods are lacking.
- Understanding proteolytic processing is key to deciphering protein regulation.
Purpose of the Study:
- To develop and present a protocol for the systematic identification of protein C-terminal sequences.
- To provide a method for analyzing proteolytic processing events.
- To enable detailed study of C-terminal protein modifications.
Main Methods:
- Protein cleavage using cyanogen bromide (CNBr) to generate a peptide mixture.
- Enzymatic digestion with carboxypeptidase Y, which acts specifically on C-terminal fragments.
- Analysis of resulting peptide fragments using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS).
Main Results:
- A peptide ladder is generated, allowing for the identification of C-terminal sequences.
- The protocol effectively identifies the C-terminal processing of proteins.
- The entire method, including CNBr cleavage, is completed within 21 hours.
Conclusions:
- The presented protocol offers a systematic approach to identify protein C-terminal sequences.
- This method facilitates the study of proteolytic processing and C-terminal PTMs.
- The protocol is versatile and applicable to proteins purified via various methods (SDS-PAGE, 2D PAGE, solution).
