Tandem mass spectrometry acquisition approaches to enhance identification of protein‐protein interactions using low‐energy collision‐induced dissociative chemical crosslinking reagents

Soderblom, Erik J.; Bobay, Benjamin G.; Cavanagh, John; Goshe, Michael B.

doi:10.1002/rcm.3213

Cited by 48 publications

(70 citation statements)

References 24 publications

(32 reference statements)

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“…3B), presumably because the energy used to break the Rink labile bond remains trapped in the peptide ion. Similar behavior was also observed for loop-links derived from other cross-linkers containing a single labile bond, such as the D-P cross-linker (33). The fragmentation behavior of BDRG loop-links permits their identification as doubly modified peptides by database searching of their MS2 spectra (Fig.…”

Section: Fig 2 Identification Of Bdrg Cross-linked Peptidessupporting

confidence: 62%

“…The reduced complexity of the MS3 spectra facilitates confident peptide identification. Previously described MS labile cross-linkers have exploited the labile properties of the aspartyl-prolyl bond (D-P) (32,33), various forms of a carbon-sulfur bond (13,15), a urea moiety (14), and a Rink moiety (11,23). Except for the D-P-based cross-linker, all of these reagents can produce multiple fragments (four or more) during CID of interpeptide cross-links because of the presence of two labile bonds.…”

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confidence: 99%

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An Integrated Chemical Cross-linking and Mass Spectrometry Approach to Study Protein Complex Architecture and Function

Luo

Fishburn

Hahn

et al. 2012

Molecular & Cellular Proteomics

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Knowledge of protein structures and protein-protein interactions is essential for understanding biological processes. Chemical cross-linking combined with mass spectrometry is an attractive approach for studying protein-protein interactions and protein structure, but to date its use has been limited largely by low yields of informative cross-links (because of inefficient crosslinking reactions) and by the difficulty of confidently identifying the sequences of cross-linked peptide pairs from their fragmentation spectra. Here we present an approach based on a new MS labile cross-linking reagent, BDRG (biotin-aspartate-Rink-glycine), which addresses these issues. BDRG incorporates a biotin handle (for enrichment of cross-linked peptides prior to MS analysis), two pentafluorophenyl ester groups that react with peptide amines, and a labile Rink-based bond between the pentafluorophenyl groups that allows crosslinked peptides to be separated during MS and confidently identified by database searching of their fragmentation spectra. We developed a protocol for the identification of BDRG cross-linked peptides derived from purified or partially purified protein complexes, including software to aid in the identification of different classes of cross-linker-modified peptides. Importantly, our approach permits the use of high accuracy precursor mass measurements to verify the database search results. We demonstrate the utility of the approach by applying it to purified yeast TFIIE, a heterodimeric transcription factor complex, and to a single-step affinitypurified preparation of the 12-subunit RNA polymerase II complex. The results show that the method is effective at identifying cross-linked peptides derived from purified and partially purified protein complexes and provides complementary information to that from other structural approaches. As such, it is an attractive approach to study the topology of protein complexes. Molecular & Cellular Proteomics 11: 10.1074/mcp.M111.008318, 1-16, 2012.Most cellular processes are carried out by macromolecular complexes, and knowledge of the structure of these complexes is an essential step toward understanding how they function to control diverse cellular functions (1). Unfortunately, our ability to decipher the structure of many complexes has been hampered by the lack of robust technologies that can efficiently accomplish this goal. Although high resolution structures have been determined for many proteins and some protein complexes by x-ray crystallography, its ability to generate high resolution structures of large complexes is often limited by difficulties obtaining sufficient quantities of purified complexes, insolubility of complexes during crystallization trials, or difficulties obtaining diffraction quality crystals. When structures are obtained they often comprise only parts of the proteins because difficult areas have been removed to improve solubility or crystallization properties. Furthermore, protein crystallization typically occurs under conditions which are very differe...

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Section: Fig 2 Identification Of Bdrg Cross-linked Peptidessupporting

confidence: 62%

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confidence: 99%

An Integrated Chemical Cross-linking and Mass Spectrometry Approach to Study Protein Complex Architecture and Function

Luo

Fishburn

Hahn

et al. 2012

Molecular & Cellular Proteomics

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“…MS-cleavable cross-linkers have received considerable attention because the resulting cross-linked products can be identified based on their characteristic fragmentation behavior observed during MS analysis. Gas-phase cleavage sites result in the detection of a "reporter" ion (26), single peptide chain fragment ions (35)(36)(37)(38), or both reporter and fragment ions (16,34). In each case, further structural characterization of the peptide product ions generated during the cleavage reaction can be accomplished by subsequent MS n1 analysis.…”

Section: Dsso Contains Two Symmetric Collision-induced Dissociation (mentioning

confidence: 99%

Development of a Novel Cross-linking Strategy for Fast and Accurate Identification of Cross-linked Peptides of Protein Complexes

Kao¹,

Chiu²,

Vellucci³

et al. 2011

Molecular & Cellular Proteomics

349

570

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Knowledge of elaborate structures of protein complexes is fundamental for understanding their functions and regulations. Although cross-linking coupled with mass spectrometry (MS) has been presented as a feasible strategy for structural elucidation of large multisubunit protein complexes, this method has proven challenging because of technical difficulties in unambiguous identification of cross-linked peptides and determination of cross-linked sites by MS analysis. In this work, we developed a novel cross-linking strategy using a newly designed MS-cleavable cross-linker, disuccinimidyl sulfoxide (DSSO). DSSO contains two symmetric collision-induced dissociation (CID)-cleavable sites that allow effective identification of DSSO-cross-linked peptides based on their distinct fragmentation patterns unique to cross-linking types (i.e. interlink, intralink, and dead end). The CID-induced separation of interlinked peptides in MS/MS permits MS 3 analysis of single peptide chain fragment ions with defined modifications (due to DSSO remnants) for easy interpretation and unambiguous identification using existing database searching tools. Integration of data analyses from three generated data sets (MS, MS/MS, and MS Proteins form stable and dynamic multisubunit complexes under different physiological conditions to maintain cell viability and normal cell homeostasis. Detailed knowledge of protein interactions and protein complex structures is fundamental to understanding how individual proteins function within a complex and how the complex functions as a whole. However, structural elucidation of large multisubunit protein complexes has been difficult because of a lack of technologies that can effectively handle their dynamic and heterogeneous nature. Traditional methods such as nuclear magnetic resonance (NMR) analysis and x-ray crystallography can yield detailed information on protein structures; however, NMR spectroscopy requires large quantities of pure protein in a specific solvent, whereas x-ray crystallography is often limited by the crystallization process.In recent years, chemical cross-linking coupled with mass spectrometry (MS) has become a powerful method for studying protein interactions (1-3). Chemical cross-linking stabilizes protein interactions through the formation of covalent bonds and allows the detection of stable, weak, and/or transient protein-protein interactions in native cells or tissues (4 -9). In addition to capturing protein interacting partners, many studies have shown that chemical cross-linking can yield low resolution structural information about the constraints within a molecule (2, 3, 10) or protein complex (11-13). The application of chemical cross-linking, enzymatic digestion, and subsequent mass spectrometric and computational analyses for the elucidation of three-dimensional protein structures offers distinct advantages over traditional methods because of its speed, sensitivity, and versatility. Identification of cross-linked peptides provides distance constraints that aid in constructing...

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“…Several cleavable cross-linkers have been developed to assist in the identification process, including collision induced dissociation (CID) cleavable reagents [12,[20][21][22][23][24][25], as well as others that are chemically cleaved prior to MS analysis [11,16]. CID cleavable crosslinkers may afford products corresponding to a reporter ion of known m/z [12,22], or cleavage of the cross-linker at a defined position, giving fragment ions corresponding to each of the two linked peptides [21,23,24,26,27]. In certain designs, both are incorporated [12,28].…”

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confidence: 99%

A Negative Ion Mass Spectrometry Approach to Identify Cross-Linked Peptides Utilizing Characteristic Disulfide Fragmentations

Calabrese

Good

Wang

et al. 2012

J. Am. Soc. Mass Spectrom.

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Chemical cross-linking combined with mass spectrometry (MS) is an analytical tool used to elucidate the topologies of proteins and protein complexes. However, identification of the low abundance cross-linked peptides and modification sites amongst a large quantity of proteolytic fragments remains challenging. In this work, we present a strategy to identify cross-linked peptides by negative ion MS for the first time. This approach is based around the facile cleavages of disulfide bonds in the negative mode, and allows identification of cross-linked products based on their characteristic fragmentations. MS 3 analysis of the cross-linked peptides allows for their sequencing and identification, with residue specific location of cross-linking sites. We demonstrate the applicability of the commercially available cystine based cross-linking reagent dithiobis(succinimidyl) propionate (DSP) and identify cross-linked peptides from ubiquitin. In each instance, the characteristic fragmentation behavior of the cross-linked species is described. The data presented here indicate that this negative ion approach may be a useful tool to characterize the structures of proteins and protein complexes, and provides the basis for the development of high throughput negative ion MS chemical cross-linking strategies.

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Tandem mass spectrometry acquisition approaches to enhance identification of protein‐protein interactions using low‐energy collision‐induced dissociative chemical crosslinking reagents

Cited by 48 publications

References 24 publications

An Integrated Chemical Cross-linking and Mass Spectrometry Approach to Study Protein Complex Architecture and Function

An Integrated Chemical Cross-linking and Mass Spectrometry Approach to Study Protein Complex Architecture and Function

Development of a Novel Cross-linking Strategy for Fast and Accurate Identification of Cross-linked Peptides of Protein Complexes

A Negative Ion Mass Spectrometry Approach to Identify Cross-Linked Peptides Utilizing Characteristic Disulfide Fragmentations

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