Characterization of an Antagonist Interleukin-6 Dimer by Stable Isotope Labeling, Cross-linking, and Mass Spectrometry

Taverner, Thomas; Hall, Nathan E.; O’Hair, Richard A. J.; Simpson, Richard J.

doi:10.1074/jbc.m207370200

Cited by 110 publications

(105 citation statements)

References 61 publications

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“…One major shortcoming of chemical cross-linking approaches, however, is the tremendous sample complexity attributable to the wide variety of cross-linked products that can be created during the cross-linking reaction. To circumvent this disadvantage, a number of different strategies have been developed that have in common to facilitate identification of cross-linker containing species, e.g., by employing isotope-labeled cross-linkers [6,7], isotope-labeled proteins [8], cleavable cross-linkers [9], fluorogenic crosslinkers [10], or cross-linkers creating a characteristic marker ion during MS/MS analysis [11]. Another strategy to selectively enrich cross-linker containing species by using trifunctional cross-linkers containing a biotin moiety has been described recently [12][13][14].…”

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confidence: 99%

Isotope-labeled cross-linkers and fourier transform ion cyclotron resonance mass spectrometry for structural analysis of a protein/peptide complex

Ihling

Schmidt

Kalkhof

et al. 2006

J. Am. Soc. Mass Spectrom.

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For structural studies of proteins and their complexes, chemical cross-linking combined with mass spectrometry presents a promising strategy to obtain structural data of protein interfaces from low quantities of proteins within a short time. We explore the use of isotope-labeled cross-linkers in combination with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry for a more efficient identification of cross-linker containing species. For our studies, we chose the calcium-independent complex between calmodulin and a 25-amino acid peptide from the C-terminal region of adenylyl cyclase 8 containing an "IQ-like motif." Cross-linking reactions between calmodulin and the peptide were performed in the absence of calcium using the amine-reactive, isotope-labeled (d 0 and d 4 ) cross-linkers BS 3 (bis[sulfosuccinimidyl]suberate) and BS 2 G (bis[sulfosuccinimidyl]glutarate). Tryptic in-gel digestion of excised gel bands from covalently cross-linked complexes resulted in complicated peptide mixtures, which were analyzed by nano-HPLC/nano-ESI-FTICR mass spectrometry. In cases where more than one reactive functional group, e.g., amine groups of lysine residues, is present in a sequence stretch, MS/MS analysis is a prerequisite for unambiguously identifying the modified residues. MS/MS experiments revealed two lysine residues in the central ␣-helix of calmodulin as well as three lysine residues both in the C-terminal and N-terminal lobes of calmodulin to be cross-linked with one single lysine residue of the adenylyl cyclase 8 peptide. Further cross-linking studies will have to be conducted to propose a structural model for the calmodulin/peptide complex, which is formed in the absence of calcium. The combination of using isotope-labeled cross-linkers, determining the accurate mass of intact cross-linked products, and verifying the amino acid sequences of cross-linked species by MS/MS presents a convenient approach that offers the perspective to obtain structural data of protein assemblies within a few

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confidence: 99%

Isotope-labeled cross-linkers and fourier transform ion cyclotron resonance mass spectrometry for structural analysis of a protein/peptide complex

Ihling

Schmidt

Kalkhof

et al. 2006

J. Am. Soc. Mass Spectrom.

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“…Despite these advantages, however, identification and correct assignment of the cross-linking products can be hampered by the complexity of the peptide mixtures derived from intermolecular crosslinking of two proteins. Tagging cross-linking reagents or proteins with isotope (46,47) or fluorogenic (48) labels has been used in attempts to facilitate identification of the crosslinking products.…”

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confidence: 99%

Mapping the Topology and Determination of a Low-Resolution Three-Dimensional Structure of the Calmodulin−Melittin Complex by Chemical Cross-Linking and High-Resolution FTICRMS: Direct Demonstration of Multiple Binding Modes

et al. 2004

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Calmodulin serves as a calcium-dependent regulator in many metabolic pathways and is known to bind with high affinity to various target proteins and peptides. One such target is the small peptide melittin, the principal component of honeybee venom. The calmodulin-melittin system was used as a model system to gain further insight into target recognition of calmodulin. Using chemical cross-linking in combination with high-resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS), we have determined the interacting regions within the calcium-dependent calmodulin-melittin complex and thus the orientation of bound melittin. Using ambiguous distance restraints derived from the chemical cross-linking data in combination with recently developed computational methods of conjoined rigid body/torsion angle simulated annealing, we were able to generate low-resolution three-dimensional structure models of the calmodulin-melittin complex, for which no high-resolution structure exists to date. Our data provide evidence for the first time that calmodulin can recognize target peptides in two opposing orientations simultaneously. The general procedure for mapping interacting regions within the complex involves conjugation of calmodulin and melittin with several cross-linking reagents possessing different specificities and spacer lengths, followed by enzymatic proteolysis of the cross-linked complex. The highly complex peptide mixtures were subsequently analyzed by nano-HPLC, which was online coupled to a FTICR mass spectrometer equipped with a nano-electrospray ionization source. The mass spectra obtained in this manner were screened for possible cross-linking products using customized software programs. This integrated approach, exemplified for mapping the topology of the calmodulin-melittin complex, is likely to have wide-ranging implications for structural studies on protein-protein interactions.Calmodulin (CaM) 1 is a small (148 amino acids) acidic protein belonging to the class of EF-hand proteins, which is found ubiquitously in animals, plants, fungi, and protozoa (1). CaM serves as a calcium-dependent regulator in many metabolic pathways (2). Upon calcium binding, CaM adopts a dumbbell structure (3, 4) consisting of two lobes connected by a flexible central helix (5, 6). CaM is known to bind with high affinity to various target proteins and peptides, with a dissociation constant in the low nanomolar range (7). One such target is the small, 26-residue peptide, melittin, the principal component of honeybee (Apis mellifera) venom. Concomitant with complex formation, melittin adopts an R-helical conformation (8) consisting of two R-helical segments interrupted by proline, with a bent rod-shaped appearance (9, 10). Our aim was to gain further insight into the determinants of target recognition by CaM, as exemplified by the CaM-melittin system. Melittin has been widely used as a tool for studying protein-protein interactions and is known to interact with a number of proteins, including CaM (11), ...

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“…Observed trends in the dissociation patterns obtained for these species will allow for improvements to software used in the automated interpretation of crosslinked peptide MS/MS data. hemical crosslinking has been demonstrated to be an important tool for probing protein structure, particularly where a crystal structure is not yet available [1][2][3][4][5][6][7]. Crosslinking chemistry also is used in the analysis of protein complexes, where it has been applied to identify binding partners [8 -15], characterize the interface of interacting subunits [16 -20], and stabilize transient interactions [21,22].…”

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confidence: 99%

Influence of crosslinker identity and position on gas-phase dissociation of lys-lys crosslinked peptides

Gaucher

Hadi

Young

2006

J. Am. Soc. Mass Spectrom.

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A systematic study of the dissociation patterns of crosslinked peptides analyzed by tandem mass spectrometry is reported. A series of 11-mer peptides was designed around either a polyalanine or polyglycine scaffold with arginine at the C terminus. One or two lysine residues were included at various locations within the peptides to effect inter-or intra-molecular crosslinking, respectively. Crosslinked species were generated with four commonly used amine-specific chemical crosslinking reagents: disuccinimidyl suberate (DSS), disuccinimidyl tartarate (DST), dithiobis(succinimidylpropionate) (DSP), and disuccinimidyl glutarate (DSG). The influence of precursor charge state, location of crosslink, and specific crosslinking reagent on the MS/MS dissociation pattern was examined. Observed trends in the dissociation patterns obtained for these species will allow for improvements to software used in the automated interpretation of crosslinked peptide MS/MS data. hemical crosslinking has been demonstrated to be an important tool for probing protein structure, particularly where a crystal structure is not yet available [1][2][3][4][5][6][7]. Crosslinking chemistry also is used in the analysis of protein complexes, where it has been applied to identify binding partners [8 -15], characterize the interface of interacting subunits [16 -20], and stabilize transient interactions [21,22]. There are several possible experimental approaches, many of which rely on specifically identifying and characterizing the crosslinked species by mass spectrometry. Both LCMS and LCMS/MS have been used. A current challenge for MS/MS studies is that little data exists to aid interpretation of crosslinked peptide MS/MS, unlike the wealth of data available for model peptide MS/MS.To improve the methodology, work has been done to more closely scrutinize the specificity of crosslinker reactivity [23,24], develop software for data analysis [25][26][27][28], and develop and test crosslinkers with certain properties such as affinity tags for product purification or stable isotope labels [29 -34]. Interpretation of the peptide sequences from a crosslinked species still remains a challenge, however. Much "unexpected reactivity" has been found, thus MS/MS confirmation can be important.Although much fundamental research has been done on gas-phase dissociation of protonated and cationized peptides [35][36][37][38][39][40], only limited MS/MS studies have been performed on crosslinked peptide species [25,41]. Given that the ability to determine crosslinked peptide sequence is a key component of many experimental approaches incorporating chemical crosslinking, it would be of great advantage to know more about how the crosslinked peptides dissociate in MS/MS so that database search methods and de novo analysis can be optimized for these spectra.In this manuscript we report initial findings of ongoing work to systematically study the dissociation patterns of crosslinked peptides. In the current study we report results on the influence of precursor charge state, ...

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Characterization of an Antagonist Interleukin-6 Dimer by Stable Isotope Labeling, Cross-linking, and Mass Spectrometry

Cited by 110 publications

References 61 publications

Isotope-labeled cross-linkers and fourier transform ion cyclotron resonance mass spectrometry for structural analysis of a protein/peptide complex

Isotope-labeled cross-linkers and fourier transform ion cyclotron resonance mass spectrometry for structural analysis of a protein/peptide complex

Mapping the Topology and Determination of a Low-Resolution Three-Dimensional Structure of the Calmodulin−Melittin Complex by Chemical Cross-Linking and High-Resolution FTICRMS: Direct Demonstration of Multiple Binding Modes

Influence of crosslinker identity and position on gas-phase dissociation of lys-lys crosslinked peptides

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