Identification of disulfide-containing chemical cross-links in proteins using MALDI-TOF/TOF-mass spectrometry.
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.
Cooperative Research Centre for Chronic Inflammatory Diseases, Institute for Molecular Bioscience, University of Queensland, St. Lucia Brisbane.
Gordon J
GJ
King
Alun
A
Jones
Bostjan
B
Kobe
Thomas
T
Huber
Dmitri
D
Mouradov
David A
DA
Hume
Ian L
IL
Ross
D000818:Animals; D051356:Cortactin / Q000032:analysis / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004220:Disulfides / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D051379:Mice; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013388:Succinimides / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D051356:Cortactin; D003432:Cross-Linking Reagents; C495869:Cttn protein, mouse; D004220:Disulfides; D011506:Proteins; D013388:Succinimides; C035759:3,3'-dithiobis(sulfosuccinimidyl propionate)