A gas phase cleavage reaction of cross-linked peptides for protein complex topology studies by peptide fragment fingerprinting from large sequence database

A gas phase cleavage reaction of cross-linked peptides for protein complex topology studies by peptide fragment fingerprinting from large sequence database

Journal of Proteomics, May 2014

24840472

Hansuk Buncherd, Winfried Roseboom, Leo J. de Koning, Chris G. de Koster, Luitzen de Jong

A high molecular weight fraction of a HeLa cell nuclear extract containing nearly 1100 identified proteins was cross-linked with bis(succinimidyl)-3-azidomethyl glutarate (BAMG). The azido group in cross-linked peptides can be reduced to an amine group. Reduction enables isolation of cross-linked peptides by diagonal strong cation exchange chromatography. Collision-induced dissociation (CID) of reduced cross-linked peptides shows abundant cleavage of the cross-link amide bonds, along with the cleavage of peptide bonds of the composing peptide pair. A defined relationship exists between the sum of the masses of a pair of cleavage products and the mass of the parent compound. This relationship enables accurate mass determination of the two composing peptides. With this knowledge, the identity of the pair of peptides in a cross-link is revealed at an extremely low false discovery rate by peptide fragment fingerprinting with MS1MS2 data from the entire human sequence databases with a conventional search engine for peptide identification. Our approach resulted in identification of 229 intraprotein and 18 interprotein cross-links.