Abstract
We describe the use of the 13C-labeled artificial amino acid p-benzoyl-L-phenylalanine (Bpa) to improve the reliability of cross-linked product identification. Our strategy is exemplified for two protein–peptide complexes. These studies indicate that in many cases the identification of a cross-link without additional stable isotope labeling would result in an ambiguous assignment of cross-linked products. The use of a 13C-labeled photoreactive amino acid is considered to be preferred over the use of deuterated cross-linkers as retention time shifts in reversed phase chromatography can be ruled out. The observation of characteristic fragment ions additionally increases the reliability of cross-linked product assignment. Bpa possesses a broad reactivity towards different amino acids and the derived distance information allows mapping of spatially close amino acids and thus provides more solid structural information of proteins and protein complexes compared to the longer deuterated amine-reactive cross-linkers, which are commonly used for protein 3D-structure analysis and protein–protein interaction studies.
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Acknowledgments
This work was funded by the Deutsche Forschungsgemeinschaft (DFG, project Si 867/13-1) and the region of Sachsen-Anhalt. Dr. Thomas Schröder is acknowledged for providing GCAP-2
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Supporting information is available for this manuscript on synthesis of Fmoc-13C6-Bpa, chromatograms of peptide separation, protein and peptide sequences, SDS-PAGE, MS and MS/MS data of cross-linked products, and details of identified cross-linked products.
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Pettelkau, J., Ihling, C.H., Frohberg, P. et al. Reliable Identification of Cross-Linked Products in Protein Interaction Studies by 13C-Labeled p-Benzoylphenylalanine. J. Am. Soc. Mass Spectrom. 25, 1628–1641 (2014). https://doi.org/10.1007/s13361-014-0944-6
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DOI: https://doi.org/10.1007/s13361-014-0944-6