| Abstract: | Combining the properties of a zero‐length cross‐linker with cleavability by tandem mass spectrometry (MS/MS) poses great advantages for protein structure analysis using the cross‐linking/MS approach. These include a reliable, automated data analysis and the possibility to obtain short‐distance information of protein 3D‐structures. We introduce 1,1′‐carbonyldiimidazole (CDI) as an easy‐to‐use and commercially available, low‐cost reagent that ideally fulfils these features. CDI bridges primary amines and hydroxy groups in proteins with the lowest possible spacer length of one carbonyl unit (ca. 2.6 Å). The cross‐linking reaction can be conducted under physiological conditions in the pH range between 7.2 and 8. Urea and carbamate cross‐linked products are cleaved upon collisional activation during MS/MS experiments generating characteristic product ions, greatly improving the unambiguous identification of cross‐links. Our innovative analytical concept is exemplified and applied for bovine serum albumin (BSA), wild‐type tumor suppressor p53, an intrinsically disordered protein, and retinal guanylyl cyclase activating protein‐2 (GCAP‐2). |
