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A new method for C-terminal sequence analysis in the proteomic era

Nature Methods volume 2, pages 193–200 (2005)Cite this article

Abstract

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 amino or carboxy terminus are by far the most common types of PTMs in proteins. In contrast to the analysis of phosphorylation and glycosylation sites, relatively little attention has been paid to the development of approaches for the systematic analysis of proteolytic processing events. Here we present a new mass spectrometry (MS)-based strategy that allows the identification of the C-terminal sequence of proteins. The method can be directly applied to proteins cleaved with cyanogen bromide (CNBr) and purified either by SDS-PAGE, by two-dimensional (2D) PAGE or in solution, and it therefore eliminates the need for specific isolation of the C-terminal peptide. Using Shewanella oneidensis as a model system, we have demonstrated that this approach can be used for C-terminal sequence analysis at a proteomic scale. We also applied the method to study the C-terminal proteolytic processing of procardosin A.

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Figure 1: Schematic representation of the steps in the C-terminal sequencing method.
Figure 2: C-terminal sequence analysis of pigeon cytochrome c.
Figure 3: C-terminal sequence analysis of 2D PAGE–separated proteins from S. oneidensis MR-1.
Figure 4: Analysis of recombinant procardosin A.

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Acknowledgements

B.S. is a postdoctoral fellow of the Fund for Scientific Research-Flanders (F.W.O.-Vlaanderen, Belgium). K.S. is funded by a Ph.D. grant of the Institute for the Promotion of Innovation through Science and Technology in Flanders (I.W.T.-Vlaanderen). P.C. is the recipient of a Ph.D. grant of the Portuguese government (PRAXIS XXI Programme–Fundação para a Ciência e a Tecnologia). This work was supported by Research Grant G.0132.02 (F.W.O.-Vlaanderen, Belgium) to J.V.B. and by the PRAI programme of the Centro Region of Portugal (X-PROT project) (C.F.). The authors thank G. Debyser for preparing the figures, F. Vanrobaeys for statistical analysis and E. Lecocq for excellent technical assistance.

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Authors and Affiliations

  1. Department of Biochemistry, Physiology and Microbiology, Laboratory of Protein Biochemistry and Protein Engineering, Ghent University, K.L. Ledeganckstraat 35, Ghent, B-9000, Belgium

    Bart Samyn, Kjell Sergeant & Jozef Van Beeumen

  2. Department of Biochemistry and Center of Neuroscience and Cellular Biology, University of Coimbra, Apt. 3126, Coimbra, 3000, Portugal

    Pedro Castanheira & Carlos Faro

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  1. Bart Samyn
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  2. Kjell Sergeant
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Corresponding authors

Correspondence to Bart Samyn or Jozef Van Beeumen.

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Supplementary information

Supplementary Fig. 1

2D-PAGE analysis of Shewanella oneidensis proteins. (PDF 2174 kb)

Supplementary Fig. 2

SDS-PAGE of the spiked Shewanella oneidensis protein extract. (PDF 1978 kb)

Supplementary Fig. 3

C-terminal sequence analysis of the Shewanella extract spiked with the intact procardosin protein. (PDF 477 kb)

Supplementary Table 1

C-terminal sequence analysis of standard proteins. (PDF 50 kb)

Supplementary Table 2

Proteins of Shewanella oneidensis identified by PMF-analysis of CNBr fragments without characterization of the C termini. (PDF 56 kb)

Supplementary Methods (PDF 85 kb)

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Samyn, B., Sergeant, K., Castanheira, P. et al. A new method for C-terminal sequence analysis in the proteomic era. Nat Methods 2, 193–200 (2005). https://doi.org/10.1038/nmeth738

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