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 crosslinking and mass spectrometric analysis of the crosslinked 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 (D 12 -EGS), which contains 12 deuterium atoms for easy detection of cross-links when applied in a 1:1 mixture with its H 12 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 H 12 form of the original crosslinked 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 H 12 form of the original crosslinked 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 crosslink 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. Large protein assemblies play a key role in many biological processes. To understand the function of these protein complexes, studies of their structural organization and the proteinprotein interfaces are of major interest to modern molecular biology. The conventional approach for studying protein-protein interactions in protein complexes is to use binary binding assays with pairs of protein subunits from the complex. However, this technique is unable to detect protein-protein interactions that are stabilized in the complex by cooperative binding and becomes laborious for multicomponent protein complexes.Current technologies for studying protein-protein interaction interfaces on a structural and molecular level, such as NMR and x-ray crystallography, are limited in application because they are restricted by considerations of protein amount, purity, concentration, size, and homogeneity (1, 2). Furthermore, these technologies are o...

Protein Cross-Linking Analysis Using Mass Spectrometry, Isotope-Coded Cross-Linkers, and Integrated Computational Data Processing

“…3,4,[7][8][9][10] The choice for cross-linking reagents (length and chemical specificity) and the respective experimental cross-linking workflow are as important as the availability of computational tools for mass spectrometry data analysis as recently presented in a review by Sinz. 11 In general, the distance range within which a bifunctional cross-linker will covalently connect two amino acid residues (distance constraint) within a protein is more rigidly defined the shorter the cross-linker spacer arm.…”

Collision-induced dissociation of lys-lys intramolecular crosslinked peptides

“…Although there are several papers in the literature reporting MS/MS spectra of crosslinked peptides [2,5,9,11,12,18,33,[41][42][43], few studies discuss the fundamental issue of how these peptides fragment in the gas phase, specifically in the case of intramolecular crosslinkers [44,45]. Schilling et al [44] proposed a nomenclature for fragments generated from dissociation of crosslinked peptides, based on the previous nomenclature for regular (linear) peptides proposed by Roepstorff and Fohlman [46] and modified by Biemann [47].…”

“…Although this seems to be a promising method, relatively little success has been reported so far. Finally, cleavable crosslinkers have proved to be an interesting alternative because, after cleaving the crosslinker, both chains behave like regular modified peptides [33,39,40]. Although these methods have proved useful, a more direct and simple method would be the analysis by tandem mass spectrometry (MS/MS), a successful approach used in the study of regular peptides.…”

“…Finally, cleavable crosslinkers have proved to be an interesting alternative because, after cleaving the crosslinker, both chains behave like regular modified peptides [33,39,40]. Although these methods have proved useful, a more direct and simple method would be the analysis by tandem mass spectrometry (MS/MS), a successful approach used in the study of regular peptides.Although there are several papers in the literature reporting MS/MS spectra of crosslinked peptides [2,5,9,11,12,18,33,[41][42][43], few studies discuss the fundamental issue of how these peptides fragment in the gas phase, specifically in the case of intramolecular crosslinkers [44,45]. Schilling et al [44] proposed a nomenclature for fragments generated from dissociation of crosslinked peptides, based on the previous nomenclature for regular (linear) peptides proposed by Roepstorff and Fohlman [46] and modified by Biemann [47].…”

Making connections for life: An in vivo map of the yeast interactome

“…As modern mass spectrometers become faster, more sensitive, and more accurate, this hurdle will eventually be overcome, allowing us to address the second challenge we face: delivering and distributing the cross-linking reagents in intact cells. Cross-linking reagents are typically too large to passively cross cell membranes (Petrotchenko et al, 2005;Tang et al, 2005). While hydrophobic reagents will be retained in the membrane, more hydrophilic ones require delivery systems that can significantly alter a cell's physiology.…”