Identification of Disulfide-Containing Chemical Cross-Links in Proteins Using MALDI-TOF/TOF-Mass Spectrometry

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• 作者：Gordon J. King ; Alun Jones ; Bostjan Kobe ; Thomas Huber ; Dmitri Mouradov ; David A. Hume ; Ian L. Ross
• 刊名：Analytical Chemistry
• 出版年：2008
• 出版时间：July 1, 2008
• 年：2008
• 卷：80
• 期：13
• 页码：5036 - 5043
• 全文大小：152K
• 年卷期：v.80,no.13(July 1, 2008)
• ISSN：1520-6882

文摘

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-modified 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.