Chemical cross‐linking and mass spectrometry for mapping three‐dimensional structures of proteins and protein complexes

Sinz, Andrea

doi:10.1002/jms.559

Cited by 266 publications

(290 citation statements)

References 82 publications

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“…Cross-linking of proteins is a powerful method to investigate protein conformation 1,2 . A crosslink between two peptides is indicative for spatial proximity of the two linked amino acids at the time of cross-linking.…”

Section: Introductionmentioning

confidence: 99%

Identification of cross-linked peptides from large sequence databases

et al. 2008

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We describe a method to identify cross-linked peptides from complex samples and large protein sequence databases. The advance was achieved by combining isotopically tagged cross-linkers, chromatographic enrichment, targeted proteomics, and a novel search engine called xQuest. This software reduces the search space by an upstream candidatepeptide search before the recombination step; we show that xQuest can identify cross-linked peptides from a total E. coli lysate with an unrestricted database search.

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Section: Introductionmentioning

confidence: 99%

Identification of cross-linked peptides from large sequence databases

et al. 2008

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“…where the b 4 ions are the C-terminal cleavage products and the b 3 ions are the N-terminal fragments.…”

Section: Discussionmentioning

confidence: 99%

“…Chemical crosslinking of proteins coupled with mass spectrometric analysis has become a more widely used method for determining protein-protein interactions in recent years [4][5][6]. Protein crosslinking is a low-resolution, structural analysis technique that allows one to determine distance constraints within single proteins [7,8], or protein-ligand complexes [9 -13].…”

mentioning

confidence: 99%

Impact of proline and aspartic acid residues on the dissociation of intermolecularly crosslinked peptides

Gardner

Brodbelt

2008

J. Am. Soc. Mass Spectrom.

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The dissociation of intermolecularly crosslinked peptides was evaluated for a series of peptides with proline or aspartic acid residues positioned adjacent to the crosslinking sites (lysine residues). The peptides were crosslinked with either disuccinimidyl suberate (DSS) or disuccinimidyl L-tartrate (DST), and the influence of proline and aspartic acid residues on the fragmentation patterns were investigated for precursor ions with and without a mobile proton. Collisionally activated dissociation (CAD) spectra of aspartic acid-containing crosslinked peptide ions, doublycharged with both protons sequestered, were dominated by cleavage C-terminal to the Asp residue, similar to that of unmodified peptides. The proline-containing crosslinked peptides exhibited a high degree of internal ion formation, with the resulting product ions having an N-terminal proline residue. Upon dissociation of the doubly-charged crosslinked peptides, twenty to fifty percent of the fragment ion abundance was accounted for by multiple cleavage products. Crosslinked peptides possessing a mobile proton yielded almost a full series of b-and y-type fragment ions, with only proline-directed fragments still observed at high abundances. Interestingly, the crosslinked peptides exhibited a tendency to dissociate at the amide bond C-terminal to the crosslinked lysine residue, relative to the N-terminal side. One could envision updating computer algorithms to include these crosslinker specific product ions-particularly for precursor ions with localized protons-that provide complementary and confirmatory information, to offer more confident identification of both the crosslinked peptides and the location of the crosslink, as well as affording predictive guidelines for interpretation of the product-ion spectra of crosslinked peptides. (J

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“…Preferential biotinylation of proteins at the N-terminus maintains the integrity of the binding site, allowing the baited protein to interact with the appropriate receptor and initiate a signal throughout the cell [6]. With this approach, lysine residues containing primary amines remain available for reaction with crosslinker reagents, chemicals commonly used to study protein-protein interactions [7]. We report here a new strategy to isolate signaling complexes through combination of biotin addition and crosslinking methodologies.…”

Section: Introductionmentioning

confidence: 99%

Isolation of signal transduction complexes using biotin and crosslinking methodologies

Freed¹,

Smith²,

Li³

et al. 2007

Proteomics

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We have developed a strategy to preferentially label the N-terminal a-amino groups of intact proteins allowing the internal e-amino groups to remain free to react with chemical crosslinking reagents. The convergence of these methodologies allows biotinylated ligands to bind to their receptors within the cell membrane followed by removal of the crosslinked complex from cell lysate. This technique allows for the isolation of protein complexes in an MS-compatible system, thus providing a tool for furthering our understanding of signal transduction. Keywords:Biotin / Crosslinking / Signaling / TNF-alpha Proteomics 2007Proteomics , 7, 2371Proteomics -2374 2371The emerging field of functional proteomics has facilitated the study of protein networks, interactions within the cell due to stimulation or perturbation of the cellular environment [1]. The ability to isolate signaling complexes can not only provide information regarding the components of the complex, but also aid a more complete understanding of how these components are altered in disease states. Numerous strategies have been reported to isolate multiprotein complexes followed by identification of the components of the complex by MS methods. The majority of isolation strategies utilize affinity-based techniques that require the overexpression of a fusion protein within the cell that is later immunoprecipitated by an antibody against its epitope thus allowing for the isolation of the target protein and interacting partners [2]. Methods such as tandem affinity purification (TAP) tags [3] utilize this principle and have been successful in the identification of protein assemblies. While the isolation of macromolecular complexes provides insight into regulated cellular processes, there exists a vast interest in protein complex formation due to extracellular signals [4]. While endogenous complexes are critical for cellular signaling, quite often the need for signaling is provided by an extracellular molecule that triggers a cellular response following binding to a specific receptor within the plasma membrane.Biotin has been used extensively as an affinity fusion partner due to its remarkably strong interaction with streptavidin (K d ,10 215 7M) [5]. Appropriate addition of biotin to a recombinant protein can eliminate concerns commonly encountered with the production of fusion proteins; for example protein folding, interruption of protein activity or function, yield, and solubility. Preferential biotinylation of proteins at the N-terminus maintains the integrity of the binding site, allowing the baited protein to interact with the appropriate receptor and initiate a signal throughout the cell [6]. With this approach, lysine residues containing primary amines remain available for reaction with crosslinker reagents, chemicals commonly used to study protein-protein interactions [7]. We report here a new strategy to isolate signaling complexes through combination of biotin addition and crosslinking methodologies. In contrast to other approaches, the external signal,...

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Chemical cross‐linking and mass spectrometry for mapping three‐dimensional structures of proteins and protein complexes

Cited by 266 publications

References 82 publications

Identification of cross-linked peptides from large sequence databases

Identification of cross-linked peptides from large sequence databases

Impact of proline and aspartic acid residues on the dissociation of intermolecularly crosslinked peptides

Isolation of signal transduction complexes using biotin and crosslinking methodologies

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