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Abstract of ā18O Labelling of C-Termini of Cross-Linked Peptides Using Trypsin and Glu-Cā by Pascal van Alphen The pH dependency of the carboxyl oxygen exchange reaction catalysed by Glu-C has been studied. This resulted in a protocol for efficiently labelling cross-linked proteins, digested by more than one protease, by 18O incorporation into the C-termini. Cross links between amino acid residues in close proximity can provide distance constraints in order to validate computer models of the 3D structure of proteins. An 18O labelled cross-link differs from unlabelled cross-links by 8 amu whereas surface-labels (mono-link) or looplinks shift only 4 amu. Bis(succinimidyl)-3-azidomethyl-glutarate (BAMG) was used to cross-link cytochrome c and Gas2p, respectively. BAMG is a cross-linking agent with an azido group that allows for selective and efficient purification of peptide mixtures. Here, it is shown that Glu-C is able to efficiently label peptides with 18O in conditions similar to those normally chosen for trypsin. Using this method, several cross-links have been identified in cytochrome c and Gas2p.