Isotope tagged cross linking reagents. A new tool in mass spectrometric protein interaction analysis

In protein interaction analysis, one promising method to identify the invol ved proteins and to characterize interacting sites at the same time is the mass spectrometric analysis of enzymatic hydrolysates of covalently crossli nked complexes. While protein identification can be accomplished by the met hodology developed for proteome analysis, the unequivocal detection and cha racterization of cross-linked sites remained involved without selection cri teria for linked peptides in addition to mass, To provide such criteria, we incorporated cross-links with a distinct isotope pattern into the microtub ule-destabilizing protein Op18/stathmin (Op13) and into complexes formed by Op18 with tubulin. The deuterium-labeled cross-linking reagents bis(sulfos uccinimidyl)-glutarate-d(4), -pimelate-d(4), and -sebacate-d(4) were prepar ed together with their undeuterated counterparts and applied as a 1:1 mixtu re of the respective do and dr isotopomers. The resulting d(0)/ dq isotope tags allowed a straightforward mass spectrometric detection of peptides car rying the linker even in complex enzymatic protein hydrolysates. In the str ucture elucidation of the linked peptides by MS/MS, the assignment of the l inked amino acids was again greatly facilitated by the d(0)/d(4) tag. By ap plying two cross-linkers with similar reactivity but different spacer lengt h in parallel, even doublets with very low intensity could be assigned with high confidence in MS and MS/MS spectra. Since in the Op18-tubulin complex es only a limited number of peptides carried the linker, the identification of the involved proteins per se was not impeded, thus accomplishing both p rotein identification and characterization of interacting sites in the same experiment, This novel methodology allowed us to significantly refine the current view of the complex between Op18 and tubulin corroborating the tubu lin "capping" activity of the N-tenninal domain of Op18.