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

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. BIOLOGICAL SIGNIFICANCE: Mapping protein-protein interactions in complex samples like digests of in vitro cross-linked extracts, by interrogation of entire species specific sequence database with tandem mass spectrometric data may yield repositories of cross-linked peptides. Results will reveal interactions between proteins, the identity of which may lead to new hypotheses about molecular mechanisms and regulations of biological function, or new targets for drug development. In this paper we describe a new analytical strategy that improves existing approaches of cross-link mapping in complex samples. The cross-linker that we have designed and synthesized for our approach is membrane permeable. This opens avenues for in vivo cross-linking for better understanding of dynamic protein complex topologies involved in many biological processes.