Download Abstract of “18O Labelling of C-Termini of Cross

Abstract of “18O Labelling of C-Termini of Cross-Linked Peptides Using Trypsin
and Glu-C” 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.