Mass Spectrometry Identifiable Cross-Linking Strategy for Studying Protein−Protein Interactions

Tang, Xiaoting; Munske, Gerhard R.; Siems, William F.; Bruce, James E.

doi:10.1021/ac0488762

Cited by 201 publications

(269 citation statements)

References 27 publications

(32 reference statements)

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“…Steen et al exploited this low-energy fragmentation characteristic of mono-oxidized thio-ethers to profile phosphorylation by ␤-elimination and Michael addition chemical strategy [7]. It was reported that this characteristic neutral loss was generated by the formation of dehydroalanine in a cysteinyl residue after releasing a sulfenic acid fragment (RSOH, where R stands for added tag mass from the blocking reagent) from the modified peptide in a low-energy MS/MS experiment.This study was carried out to investigate new features for novel low-energy mass spectrometry cleavable cross linkers where bonds can be selectively dissociate with low-energy MS/MS in the gas-phase environment of a mass spectrometer [8,9]. Low-energy fragmentation characteristics of oxidized thio-ether bonds have been reported [6,7].…”

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confidence: 99%

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Evaluation of low energy CID and ECD fragmentation behavior of mono-oxidized thio-ether bonds in peptides

Chowdhury

Munske

Ronald

et al. 2007

J. Am. Soc. Mass Spectrom.

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Thio-ether bonds in the cysteinyl side chain of peptides, formed with the most commonly used cysteine blocking reagent iodoacetamide, after conversion to sulfoxide, releases a neutral fragment mass in a low-energy MS/MS experiment in the gas phase of the mass spectrometer [6]. In this study, we show that the neutral loss fragments produced from the mono-oxidized thio-ether bonds (sulfoxide) in peptides, formed by alkyl halide or double-bond containing cysteine blocking reagents are different under low-energy MS/MS conditions. We have evaluated the low-energy fragmentation patterns of mono-oxidized modified peptides with different cysteine blocking reagents, such as iodoacetamide, 3-maleimidopropionic acid, and 4-vinylpyridine using FTICR-MS. We propose that the mechanisms of gas-phase fragmentation of mono-oxidized thio-ether bonds in the side chain of peptides, formed by iodoacetamide and double-bond containing cysteine blocking reagents, maleimide and vinylpyridine, are different because of the availability of acidic ␤-hydrogens in these compounds. Moreover, we investigated the fragmentation characteristics of mono-oxidized thio-ether bonds within the peptide sequence to develop novel mass-spectrometry identifiable chemical cross-linkers. This methionine type of oxidized thio-ether bond within the peptide sequence did not show anticipated low-energy fragmentation. Electron capture dissociation (ECD) of the side chain thio-ether bond containing oxidized peptides was also studied. ECD spectra of the oxidized peptides showed a greater extent of peptide backbone cleavage, compared with CID spectra. This fragmentation information is critical to researchers for accurate data analysis of this undesired modification in proteomics research, as well as other methods that may utilize sulfoxide derivatives. . Before digestion, the disulfide bonds in the proteins are first cleaved with either DTT or ␤-mercaptoethanol, and their reformation must be blocked using different cysteine blocking reagents. Several cysteine-blocking reagents exist; the most common are iodoacetamide, N-ethylmaleimide, acrylamide, and 4-vinylpyridine [2][3][4]. These methods that use iodoacetamide rely on an alkyl halide to react with the sulfhydryl group of cysteine. The other three blocking reagents, maleimide, acrylamide, and vinylpyridine utilize double bonds to react with the sulfhydryl group of cysteine by the Michael addition type mechanism [5]. All of these blocking reagents form thio-ether bonds in the cysteinyl side chain of proteins. Steen et al. reported that these thio-ether bonds can be oxidized in current protein profiling techniques, such as tryptic digestion and gel electrophoresis [6]. The oxidized thio-ether bond formed with iodoacetamide in the cysteinyl side chain of the peptide loses a neutral fragment mass in the gas phase under low-energy MS/MS conditions. Steen et al. exploited this low-energy fragmentation characteristic of mono-oxidized thio-ethers to profile phosphorylation by ␤-elimination and Michael addition chemical st...

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confidence: 99%

“…This study was carried out to investigate new features for novel low-energy mass spectrometry cleavable cross linkers where bonds can be selectively dissociate with low-energy MS/MS in the gas-phase environment of a mass spectrometer [8,9]. Low-energy fragmentation characteristics of oxidized thio-ether bonds have been reported [6,7].…”

mentioning

confidence: 99%

Evaluation of low energy CID and ECD fragmentation behavior of mono-oxidized thio-ether bonds in peptides

Chowdhury

Munske

Ronald

et al. 2007

J. Am. Soc. Mass Spectrom.

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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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“…High  (Gruhler & Kratchmarova, 2008;Mann, 2006) 15 N-labeling High  Nelson et al, 2007) Chemical crosslinking-MS using protein interaction reporter (PIRs) Anderson et al, 2007;Tang et al, 2005) Protein microarrays High  (Angenendt et al, 2006;Popescu et al, 2007;Ramachandran et al, 2004) …”

Section: Stable-isotope Labeling Of Amino Acids In Cell Culture (Silac)mentioning

confidence: 99%

The Prediction and Analysis of Inter- and Intra-Species Protein-Protein Interaction

Tsao¹,

Chan²,

Huang³

et al. 2011

Systems and Computational Biology - Molecular and Cellular Experimental Systems

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Mass Spectrometry Identifiable Cross-Linking Strategy for Studying Protein−Protein Interactions

Cited by 201 publications

References 27 publications

Evaluation of low energy CID and ECD fragmentation behavior of mono-oxidized thio-ether bonds in peptides

Evaluation of low energy CID and ECD fragmentation behavior of mono-oxidized thio-ether bonds in peptides

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

The Prediction and Analysis of Inter- and Intra-Species Protein-Protein Interaction

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