Topographic Studies of the GroEL-GroES Chaperonin Complex by Chemical Cross-linking Using Diformyl Ethynylbenzene THE POWER OF HIGH RESOLUTION ELECTRON TRANSFER DISSOCIATION FOR DETERMINATION OF BOTH PEPTIDE SEQUENCES AND THEIR ATTACHMENT SITES

摘要: Many essential cellular processes depend upon the self-assembly of stable multiprotein entities. The architectures vast majority these protein machines remain unknown because structures are difficult to obtain by biophysical techniques alone. However, recent progress in defining architecture complexes has resulted from integrating information all available biochemical and sources generate computational models. Chemical cross-linking is a technique that holds exceptional promise toward achieving this goal providing distance constraints reflect topography complexes. Combined with structural data, can yield three-dimensional models higher order molecular machines. thus far utility been thwarted insufficient yields cross-linked products tandem mass spectrometry methods unable unambiguously establish identity covalently labeled peptides their sites modification. We report amino moieties 1,3-diformyl-5-ethynylbenzene (DEB) analysis high resolution electron transfer dissociation. This new reagent coupled energy deposition addresses obstacles generating containing two additional protonation relative conventional reagents. In addition excellent coverage sequence ions dissociation, DEB produces gas-phase precursor 4+, 5+, or 6+ charge states readily segregated unmodified dead-end modified using charge-dependent selection only quadruply state ions. Furthermore, induces dissociation DEB-peptide bonds diagnostic ion signals reveal "molecular ions" peptides. demonstrate power strategy 21-protein, ADP-bound GroEL-GroES chaperonin complex. Twenty-five unique were determined.