Fragmentation features of intermolecular cross-linked peptides using N-hydroxy- succinimide esters by MALDI- and ESI-MS/MS for use in structural proteomics.

The use of mass spectrometry coupled with chemical cross-linking of proteins has become one of the most useful tools for proteins structure and interactions studies. One of the challenges in these studies is the identification of the cross-linked peptides. The interpretation of the MS/MS data generated in cross-linking experiments using N-hydroxy succinimide esters is not trivial once a new amide bond is formed allowing new fragmentation pathways, unlike linear peptides. Intermolecular cross-linked peptides occur when two different peptides are connected by the cross-linker and they yield information on the spatial proximity of different domains (within a protein) or proteins (within a complex). In this article, we report a detailed fragmentation study of intermolecular cross-linked peptides, generated from a set of synthetic peptides, using both ESI and MALDI to generate the precursor ions. The fragmentation features observed here can be helpful in the interpretation and identification of cross-linked peptides present in cross-linking experiments and be further implemented in search engine's algorithms.