Identification of disulfide-containing chemical cross-links in proteins using MALDI-TOF/TOF-mass spectrometry

Gordon J. King, Alun Jones, Bostjan Kobe, Thomas Huber, Dmitri Mouradov, David A. Hume, Ian L. Ross

Research output: Contribution to journalArticlepeer-review

24 Citations (Scopus)


Cross-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.

Original languageEnglish
Pages (from-to)5036-5043
Number of pages8
JournalAnalytical Chemistry
Issue number13
Publication statusPublished - 1 Jul 2008
Externally publishedYes


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