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

An emerging approach for studying protein-protein inter-action 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 chal-lenges and limitations, including the difficulty of detecting the cross-links, the potential interference from non-infor-mative “cross-linked peptides ” (dead end and intrapep-tide cross-links), and unambiguous identification of the cross-links by mass spectrometry. Thus, we have synthe-sized an isotopically coded ethylene glycol bis(succinimi-dylsuccinate) derivate (D12-EGS), which contains 12 deu-terium 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 allowin