King, Gordon J.Jones, AlunKobe, BostjanMouradov, DmitriHume, D ARoss, Ian R.Huber, Thomas2015-12-100003-2700http://hdl.handle.net/1885/61437Cross-linking can be used to identify spatial relationships between amino acids in proteins or protein complexes. A rapid and sensitive method for identifying the site of protein cross-linking using dithiobis(sulfosuccinimidyl propionate) (DTSSP) is presented and illustrated with experiments using murine cortactin, actin and acyl-CoA thioesterase. A characteristic 66 Da doublet, which arises from the asymmetric fragmentation of the disulfide of DTSSP-modifled peptides, is observed in the mass spectra obtained under MALDI-TOF/TOF-MS conditions and allows rapid assignment of cross-links in modified proteins. This doublet is observed not only for linear cross-linked peptides but also in the mass spectra of cyclic cross-linked peptides when simultaneous fragmentation of the disulfide and the peptide backbone occurs. We suggest a likely mechanism for this fragmentation. We use guanidinylation of the cross-linked peptides with O-methyl isourea to extend the coverage of cross-linked peptides observed in this MALDI-MS technique. The methodology we report is robust and amenable to automation, and permits the analysis of native cystines along with those introduced by disulfide-containing cross-linkers.Keywords: Amines; Amino acids; Biomolecules; Crosslinking; High performance liquid chromatography; Mass spectrometers; Mass spectrometry; Organic acids; Peptides; (I ,J) conditions; Acyl-CoA; American Chemical Society (ACS); Cross-linkers; Isourea; MALDI TOF/TOF MS200810.1021/ac702277q2015-12-10