Abstract
Proteome-wide analysis of protein C-termini has long been inaccessible, but is now enabled by a newly developed negative selection strategy we term C-terminomics. In this procedure, amine- and carboxyl groups of full-length proteins are chemically protected. After trypsin digestion, N-terminal and internal tryptic peptides – but not C-terminal peptides – posses newly formed, unprotected C-termini that are removed by coupling to the high-molecular-weight polymer poly-allylamine. Ultrafiltration separates the uncoupled, blocked C-terminal peptides that are subsequently analyzed by liquid chromatography-tandem mass spectrometry. On a proteome-wide scale, this strategy profiles native protein C-termini together with neo C-termini generated by endoproteolytic cleavage or processive C-terminal truncations (“ragging”). In bacterial proteomes, hundreds of protein C-termini were identified. Stable isotope labeling enables quantitative comparison of protein C-termini and C-terminal processing in different samples. Using formaldehyde-based chemical labeling, this quantitative approach termed “carboxy-terminal amine-based isotope labeling of substrates (C-TAILS)” identified >100 cleavage sites of exogenously applied GluC protease in an Escherichia coli proteome. C-TAILS complements recently developed N-terminomic techniques for endoprotease substrate discovery and is essential for the characterization of carboxyprotease processing.
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Acknowledgments
O.S. acknowledges support from the Deutsche Forschungsgemeinschaft (DFG, SCHI 871/2-1). P.F.H. is supported by Postdoctoral Fellowships of the German Academic Exchange Service (DAAD) and the Michael Smith Foundation for Health Research (MSFHR). O.B. acknowledges support from the Swiss National Science Foundation and Canadian Institutes of Health Research (CIHR). C.M.O is supported by a Canada Research Chair in Metalloproteinase Proteomics and Systems Biology. This work was supported by a grant from the CIHR and from a program project grant in Breast Cancer Metastases from the Canadian Breast Cancer Research Alliance (CBCRA) with funds from the Canadian Breast Cancer Foundation and the Cancer Research Society as well as with an Infrastructure Grant from MSHFR.
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Schilling, O., Huesgen, P.F., Barré, O., Overall, C.M. (2011). Identification and Relative Quantification of Native and Proteolytically Generated Protein C-Termini from Complex Proteomes: C-Terminome Analysis. In: Cagney, G., Emili, A. (eds) Network Biology. Methods in Molecular Biology, vol 781. Humana Press. https://doi.org/10.1007/978-1-61779-276-2_4
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