Presentation of a homobifunctional azo-reagent for protein structure analysis by collision-induced dissociative chemical cross-linking: Proof-of-principle

Falvo, Francesco; Fiebig, Lukas; Schäfer, Matthias

doi:10.1016/j.ijms.2013.04.012

Cited by 12 publications

(31 citation statements)

References 48 publications

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“…MS 3 ‐radical‐induced methionine side chain losses of the open‐shell peptide ion [Bz‐MRFA + H] +● at m / z 641.298 leading to the formation of the product ions at m / z 580.287 and 567.279 (Figs. and D) …”

Section: Resultsmentioning

confidence: 97%

“…These initial FRIPS studies motivated us to synthesize a homobifunctional amine‐reactive N ‐hydroxysuccinimide (NHS) active ester derivative containing an azo group that is derived from the free radical initiator 4,4′‐azobis[4‐cynopentanoic]acid. Recently, we have been able to show that the benefits of FRIPS can be utilized for an efficient cross‐linking of peptides and a facilitated identification of cross‐linked products . Based on the original FRIPS strategy, Oh et al .…”

Section: Introductionmentioning

confidence: 99%

“…and Blanksby et al . as well as by the successful application of our azo‐based cross‐linker we have synthesized a homobifunctional TEMPO‐based bis‐NHS ester (1‐[2‐(2,5‐dioxo‐pyrrolidine‐1‐yloxycarbonyl)‐benzyloxy]‐2,2,6,6‐tetramethylpiperidine‐4‐carboxylic acid 2,5‐dioxo‐pyrrolidine‐1‐yl ester), which we termed TEMPO‐Bz‐linker (Scheme ). This novel amine‐reactive FRIPS cross‐linker was reacted with all 20 proteinogenic amino acids as well as with di‐ and oligopeptides to evaluate its potential for a facilitated protein 3D‐structure analysis based on CID tandem MS experiments.…”

Section: Introductionmentioning

confidence: 99%

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Dissociation behavior of a bifunctional tempo‐active ester reagent for peptide structure analysis by free radical initiated peptide sequencing (FRIPS) mass spectrometry

et al. 2015

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We have synthesized a homobifunctional active ester cross-linking reagent containing a TEMPO (2,2,6,6-tetramethylpiperidine-1-oxy) moiety connected to a benzyl group (Bz), termed TEMPO-Bz-linker. The aim for designing this novel cross-linker was to facilitate MS analysis of cross-linked products by free radical initiated peptide sequencing (FRIPS). The TEMPO-Bz-linker was reacted with all 20 proteinogenic amino acids as well as with model peptides to gain detailed insights into its fragmentation mechanism upon collision activation. The final goal of this proof-of-principle study was to evaluate the potential of the TEMPO-Bz-linker for chemical cross-linking studies to derive 3D-structure information of proteins. Our studies were motivated by the well documented instability of the central NO-C bond of TEMPO-Bz reagents upon collision activation. The fragmentation of this specific bond was investigated in respect to charge states and amino acid composition of a large set of precursor ions resulting in the identification of two distinct fragmentation pathways. Molecular ions with highly basic residues are able to keep the charge carriers located, i.e. protons or sodium cations, and consequently decompose via a homolytic cleavage of the NO-C bond of the TEMPO-Bz-linker. This leads to the formation of complementary open-shell peptide radical cations, while precursor ions that are protonated at the TEMPO-Bz-linker itself exhibit a charge-driven formation of even-electron product ions upon collision activation. MS 3 product ion experiments provided amino acid sequence information and allowed determining the cross-linking site. Our study fully characterizes the CID behavior of the TEMPO-Bz-linker and demonstrates its potential, but also its limitations for chemical cross-linking applications utilizing the special features of open-shell peptide ions on the basis of selective tandem MS analysis.

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Section: Resultsmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Dissociation behavior of a bifunctional tempo‐active ester reagent for peptide structure analysis by free radical initiated peptide sequencing (FRIPS) mass spectrometry

et al. 2015

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“…In addition to normal CID-cleavable bonds described above, FRIPS (free radical initiated peptide sequencing)-based cross-linkers have been recently explored 78,80,112 . In comparison to TEMPO- 80 and azo-linkers 78 , ABI (azobisimidoester)-linker (Table 2) appears to be the most promising as it can induce the FRIPS process during HCD in positive ion mode 112 .…”

Section: Strategies To Overcome Inherent Challenges In Xl-ms Studiesmentioning

confidence: 99%

“…In comparison to TEMPO- 80 and azo-linkers 78 , ABI (azobisimidoester)-linker (Table 2) appears to be the most promising as it can induce the FRIPS process during HCD in positive ion mode 112 . Although interesting, their performance appears to be less favorable than normal CID-cleavable cross-linkers for automated identification of cross-linked peptides due to complex fragmentation 90 .…”

Section: Strategies To Overcome Inherent Challenges In Xl-ms Studiesmentioning

confidence: 99%

Cross-Linking Mass Spectrometry: An Emerging Technology for Interactomics and Structural Biology

2017

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Strategies for generating peptide radical cations via ion/ion reactions

Gilbert

Fisher

et al. 2015

J. Mass Spectrom.

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Several approaches for the generation of peptide radical cations using ion/ion reactions coupled with either collision induced dissociation (CID) or ultraviolet photo dissociation (UVPD) are described here. Ion/ion reactions are used to generate electrostatic or covalent complexes comprised of a peptide and a radical reagent. The radical site of the reagent can be generated multiple ways. Reagents containing a carbon-iodine (C-I) bond are subjected to UVPD with 266 nm photons, which selectively cleaves the C-I bond homolytically. Alternatively, reagents containing azo functionalities are collisionally activated to yield radical sites on either side of the azo group. Both of these methods generate an initial radical site on the reagent, which then abstracts a hydrogen from the peptide while the peptide and reagent are held together by either electrostatic interactions or a covalent linkage. These methods are demonstrated via ion/ion reactions between the model peptide RARARAA (doubly protonated) and various distonic anionic radical reagents. The radical site abstracts a hydrogen atom from the peptide while the charge site abstracts a proton. The net result is the conversion of a doubly protonated peptide to a peptide radical cation. The peptide radical cations have been fragmented via CID and the resulting product ion mass spectra are compared to the control CID spectrum of the singly protonated, even-electron species. This work is then extended to bradykinin, a more broadly studied peptide, for comparison with other radical peptide generation methods. The work presented here provides novel methods for generating peptide radical cations in the gas phase through ion/ion reaction complexes that do not require modification of the peptide in solution or generation of non-covalent complexes in the electrospray process.

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Presentation of a homobifunctional azo-reagent for protein structure analysis by collision-induced dissociative chemical cross-linking: Proof-of-principle

Cited by 12 publications

References 48 publications

Dissociation behavior of a bifunctional tempo‐active ester reagent for peptide structure analysis by free radical initiated peptide sequencing (FRIPS) mass spectrometry

Dissociation behavior of a bifunctional tempo‐active ester reagent for peptide structure analysis by free radical initiated peptide sequencing (FRIPS) mass spectrometry

Cross-Linking Mass Spectrometry: An Emerging Technology for Interactomics and Structural Biology

Strategies for generating peptide radical cations via ion/ion reactions

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