Novel Amidinating Cross-Linker for Facilitating Analyses of Protein Structures and Interactions

A novel bifunctional thioimidate cross-linking reagent (diethyl suberthioimidate) that modifies amines without sacrificing their native basicity is developed. Intermolecular cross-linking of neurotensin and intramolecular cross-linking of cytochrome c under physiological conditions is investigated with this reagent. Because it does not perturb the electrostatic properties of a protein, it is unlikely to lead to artifactual conclusions about native protein structure. The interpeptide cross-links formed with this reagent are easily separated from other tryptic fragments using strong cation exchange chromatography, and they have a readily identified mass spectrometric signature. The use of this novel amidinating protein cross-linking reagent holds great promise for efficient, large-scale structural analysis of complex systems.