title: Isotopically coded cleavable cross-linker for studying protein-protein interaction and protein complexes.
abstract: An emerging approach for studying protein-protein interaction in complexes is the combination of chemical cross-linking and mass spectrometric analysis of the cross-linked peptides (cross-links) obtained after proteolysis of the complex. This approach, however, has several challenges and limitations, including the difficulty of detecting the cross-links, the potential interference from non-informative "cross-linked peptides" (dead end and intrapeptide cross-links), and unambiguous identification of the cross-links by mass spectrometry. Thus, we have synthesized an isotopically coded ethylene glycol bis(succinimidylsuccinate) derivate (D12-EGS), which contains 12 deuterium atoms for easy detection of cross-links when applied in a 1:1 mixture with its H12 counterpart and is also cleavable for releasing the cross-linked peptides allowing unambiguous identification by MS sequencing. Moreover, hydrolytic cleavage permits rapid distinguishing between different types of cross-links. Cleavage of a dead end cross-link produces a doublet with peaks 4.03 Da apart, with the lower peak appearing at a molecular mass 162 Da lower than the mass of the H12 form of the original cross-linked peptide. Cleavage of an intrapeptide cross-link leads to a doublet 8.05 Da apart and 62 Da lower than the molecular mass of the H12 form of the original cross-linked peptide. Cleavage of an interpeptide cross-link forms a pair of 4.03-Da doublets, with the lower mass member of each pair each shifted up from its unmodified molecular weight by 82 Da because of the attached portion of the cross-linker. All of this information has been incorporated into a software algorithm allowing automatic screening and detection of cross-links and cross-link types in matrix-assisted laser desorption/ionization mass spectra. In summary, the ease of detection of these species through the use of an isotopically coded cleavable cross-linker and our software algorithm, followed by mass spectrometric sequencing of the cross-linked peptides after cleavage, has been shown to be a powerful tool for studies of multi-component protein complexes.
authors.affiliation: Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
authors.forename: Evgeniy V
authors.initials: EV
authors.lastname: Petrotchenko
authors.forename: Vyacheslav K
authors.initials: VK
authors.lastname: Olkhovik
authors.forename: Christoph H
authors.initials: CH
authors.lastname: Borchers
mesh_terms: D000465:Algorithms; D003432:Cross-Linking Reagents / Q000138:chemical synthesis / Q000737:chemistry*; D003903:Deuterium / Q000737:chemistry; D054303:HIV Reverse Transcriptase / Q000032:analysis*; D006801:Humans; D010446:Peptide Fragments / Q000737:chemistry*; D011485:Protein Binding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013388:Succinimides / Q000138:chemical synthesis / Q000737:chemistry*
chemical_list: D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D013388:Succinimides; C028722:ethylene glycolylbis(succinimidyl succinate); D003903:Deuterium; D054303:HIV Reverse Transcriptase