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A method for C-terminal sequence analysis in the proteomic era (proteins cleaved with cyanogen bromide)

Abstract

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.

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Figure 1: Schematic representation of the different steps in the C-terminal sequencing method9.
Figure 2: C-terminal sequence analysis of 2D PAGE–separated β-lactoglobulin (BLG).
Figure 3: C-terminal sequence analysis of 2D PAGE–separated proteins from S. oneidensis MR-1.

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Acknowledgements

B.S. is a Postdoctoral fellow of the Fund for Scientific Research–Flanders (FWO.-Vlaanderen), Belgium. K.S. is funded by a PhD grant of the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen).

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Correspondence to Bart Samyn.

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Supplementary information

Supplementary Fig. 1

C-terminal sequence analysis of the test peptide ACTH 18–39 Human. (PDF 4149 kb)

Supplementary Fig. 2

C-terminal sequence analysis of cytochrome c (pigeon) in solution. (PDF 5100 kb)

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Samyn, B., Sergeant, K. & Van Beeumen, J. A method for C-terminal sequence analysis in the proteomic era (proteins cleaved with cyanogen bromide). Nat Protoc 1, 318–323 (2006). https://doi.org/10.1038/nprot.2006.50

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