Isotope-Tagged Cross-Linking Reagents. A New Tool in Mass Spectrometric Protein Interaction Analysis

Abstract

In protein interaction analysis, one promising method to identify the involved proteins and to characterize interacting sites at the same time is the mass spectrometric analysis of enzymatic hydrolysates of covalently cross-linked complexes. While protein identification can be accomplished by the methodology developed for proteome analysis, the unequivocal detection and characterization of cross-linked sites remained involved without selection criteria for linked peptides in addition to mass. To provide such criteria, we incorporated cross-links with a distinct isotope pattern into the microtubule-destabilizing protein Op18/stathmin (Op18) and into complexes formed by Op18 with tubulin. The deuterium-labeled cross-linking reagents bis(sulfosuccinimidyl)-glutarate-d4, -pimelate-d4, and -sebacate-d4 were prepared together with their undeuterated counterparts and applied as a 1:1 mixture of the respective d0 and d4 isotopomers. The resulting d0/d4 isotope tags allowed a straightforward mass spectrometric detection of peptides carrying the linker even in complex enzymatic protein hydrolysates. In the structure elucidation of the linked peptides by MS/MS, the assignment of the linked amino acids was again greatly facilitated by the d0/d4 tag. By applying two cross-linkers with similar reactivity but different spacer length 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 complexes only a limited number of peptides carried the linker, the identification of the involved proteins per se was not impeded, thus accomplishing both protein 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 tubulin "capping" activity of the N-terminal domain of Op18.

Identification of cross-linked peptides from large sequence databases

“…To exploit this information, both peptides that are involved in a crosslink need to be identified. Improvement in mass spectrometry (MS) technology has made this conceivable, in particular high mass precision spectrometers and the development of isotopically coded cross-linkers [3][4][5] . Several studies have proven the general feasibility of this approach 3,[6][7][8][9] .…”

Thermodynamics of the Op18/Stathmin-Tubulin Interaction

“…Human Op18/stathmin (referred to as stathmin (3)) is an evolutionary well conserved 17-kDa cytoplasmic phosphoprotein. The monomeric protein consists of a N-terminal capping domain (13,14) and a C-terminal helical interaction domain (13,(15)(16)(17)(18)(19). Originally, stathmin was described as a protein that binds to tubulin dimers and increases the catastrophe frequency of MT in vitro (20).…”

“…The structure of the complex has been investigated by electron microscopy (13), by a 4-Å resolution x-ray structure of the tubulin-RB3-SLD complex (note that RB3-SLD exhibits 72% identity with stathmin (17)), and by chemical cross-linking (14). The tubulinstathmin complex consists of two head-to-tail aligned ␣/␤-tubulin heterodimers (represented as light and dark gray ribbons) that are tilted with respect to each other by ϳ25°.…”

Characterization of an Antagonist Interleukin-6 Dimer by Stable Isotope Labeling, Cross-linking, and Mass Spectrometry

“…This problem has been partially addressed by "tagging" methodologies that allow rapid visual MS location of cross-linked species within complex peptide mixtures (34,(37)(38)(39). For example, the use of a 1:1 mixture of undeuterated and deuterated (d 0 /d 4 -labeled) cross-linking reagent readily allows mass spectrometric detection of all cross-linked species by the presence of d 0 /d 4 -isotope tags (39). However, for studying interactions between proteins, even these tagging methodologies fall short in that they fail to distinguish inter-and intra-cross-linked peptides.…”