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Molecular & Cellular Proteomics 4:1167-1179, 2005.
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

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Technology

Isotopically Coded Cleavable Cross-linker for Studying Protein-Protein Interaction and Protein Complexes^*

Evgeniy V. Petrotchenko, Vyacheslav K. Olkhovik and Christoph H. Borchers^,¶

From the Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599; The Institute of Chemistry of New Materials, The National Academy of Sciences of Belarus, Minsk 220141, Belarus

An emerging approach for studying protein-protein interaction in complexes is the combination of chemical cross-linking and mass spectrometric analysis of the cross-linked peptides (cross-links) obtained after proteolysis of the complex. This approach, however, has several challenges and limitations, including the difficulty of detecting the cross-links, the potential interference from non-informative "cross-linked peptides" (dead end and intrapeptide cross-links), and unambiguous identification of the cross-links by mass spectrometry. Thus, we have synthesized an isotopically coded ethylene glycol bis(succinimidylsuccinate) derivate (D₁₂-EGS), which contains 12 deuterium atoms for easy detection of cross-links when applied in a 1:1 mixture with its H₁₂ counterpart and is also cleavable for releasing the cross-linked peptides allowing unambiguous identification by MS sequencing. Moreover, hydrolytic cleavage permits rapid distinguishing between different types of cross-links. Cleavage of a dead end cross-link produces a doublet with peaks 4.03 Da apart, with the lower peak appearing at a molecular mass 162 Da lower than the mass of the H₁₂ form of the original cross-linked peptide. Cleavage of an intrapeptide cross-link leads to a doublet 8.05 Da apart and 62 Da lower than the molecular mass of the H₁₂ form of the original cross-linked peptide. Cleavage of an interpeptide cross-link forms a pair of 4.03-Da doublets, with the lower mass member of each pair each shifted up from its unmodified molecular weight by 82 Da because of the attached portion of the cross-linker. All of this information has been incorporated into a software algorithm allowing automatic screening and detection of cross-links and cross-link types in matrix-assisted laser desorption/ionization mass spectra. In summary, the ease of detection of these species through the use of an isotopically coded cleavable cross-linker and our software algorithm, followed by mass spectrometric sequencing of the cross-linked peptides after cleavage, has been shown to be a powerful tool for studies of multi-component protein complexes.

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