Do b ‐ions occur from vibrational excitation upon H‐desorption in electron capture dissociation?

Unmodified and amide nitrogen methylated peptide cations were reacted with azobenzene radical anions to study the utility of electron transfer dissociation (ETD) in analyzing N-methylated peptides. We show that methylation of the amide nitrogen has no deleterious effects on the ETD process. As a result, location of alkylation on amide nitrogens should be straightforward. Such a modification might be expected to affect the ETD process if hydrogen bonding involving the amide hydrogen is important for the ETD mechanism. The partitioning of the ion/ion reaction products into all of the various reaction channels was determined and compared for modified and unmodified peptide cations. While subtle differences in the relative abundances of the various ETD channels were observed, there is no strong evidence that hydrogen bonding involving the amide nitrogen plays an important role in the ETD process. (J Am Soc

Section: Resultsmentioning

confidence: 82%

Electron transfer dissociation of amide nitrogen methylated polypeptide cations

Crizer

McLuckey

2009

“…For peptides lacking amino acids of high gas-phase basicity (arginine, lysine, histidine), such as peptides G1-G5, the preferential formation of c ions (versus z · ions) upon ECD°is°due°to°their°higher°stability° [6,°24].°Moreover,°it has been shown that this kind of peptide has often a nonclassical behavior upon ECD, a common feature being the formation of b ions. It was demonstrated by Cooper° [25]°and°confirmed°very°recently°by°Haselmann and°Schmidt° [26]°that°the°formation°of°these°ions°(which are true N-terminal b-type ions) can be explained by typical MS/MS pathways (low-energy threshold backbone dissociation) from the even-electron chargedreduced cation. These b ions were not detected in the ECD spectra of all glycopeptides under study.…”

Section: Native Glycopeptidesmentioning

confidence: 88%

The combination of electron capture dissociation and fixed charge derivatization increases sequence coverage for O-glycosylated and O-phosphorylated peptides

Chamot‐Rooke

Rest

Dalleu

et al. 2007

Electron capture dissociation (ECD) has become an alternative method to collision-activated dissociation (CAD) to avoid gas-phase cleavage of post-translational modifications carried by side chains from the peptide backbone. Nonetheless, as illustrated herein by the study of O-glycosylated and O-phosphorylated peptides, the extent of ECD fragmentations may be insufficient to cover the entire peptide sequence and to localize accurately these modifications. The present work demonstrates that the derivatization of peptides at their N-terminus by a phosphonium group improves dramatically and systematically the sequence coverage deduced from the ECD spectrum for both O-glycosylated and O-phosphorylated peptides compared with their native counterparts. The exclusive presence of N-terminal fragments (c-type ions) in the ECD spectra of doubly charged molecular cations simplifies peptide sequence interpretation. Thus, the combination of ECD and fixed charge derivatization appears as an efficient analytical tool for the extensive sequencing of peptides bearing labile groups. (J Am Soc Mass Spectrom 2007, 18, 1405-1413) © 2007 American Society for Mass Spectrometry T he field of proteomics usually aims at differential characterization of the complete pattern of protein expression, for instance between cells in a normal state and those in a pathological state. Protein identification is now routinely achieved on a large scale (tens or hundreds of proteins per day) by combining enzymatic digestion, mass spectrometric analysis of the resulting peptides, and subsequent database search. If few peptides or short peptide sequences are sufficient for protein identifications into databases, a complete determination of the primary structure is nevertheless useful to detect modifications that can be involved in the regulation of protein function. Indeed, the catalytic property, the conformation, the turnover, or the subcellular localization of proteins are largely under the control of various peptide sequence processings named posttranslational modifications. These modifications (PTM) involve the proteolytic maturation of the proteins and/or the transfer of chemical groups (phosphorylation, glycosylation, acetylation, sumoylation) on specific amino-acids (serine, threonine, or tyrosine for O-phosphorylations, serine or threonine for Oglycosylations, asparagine for N-glycosylations). The most obvious strategy for mapping the modifications along the peptide chain involves a comparison between the sequence obtained by mass spectrometric data and the one deduced from the open reading frame coded by the gene sequence. However, a drawback arises from the propensity of groups conjugated to the amino acid side chains to dissociate readily upon collisional activation. For instance, collisionally-activated dissociation (CAD) MS/MS spectra of protonated precursor ions of phosphorylated peptides exhibit intense ions originating from the elimination of metaphosphoric (HPO 3 , loss of 80 Da) or orthophosphoric acid (H 3 PO 4 , loss of 98 Da) [1]. I...

“…Indeed, electron capture is expected to yield a distonic charge-reduced species [M + 3H]• 2+ that consecutively undergo c/z • cleavage. The formation of b-type product ions in ECD experiments has been reported for peptides containing or not basic residues [34][35][36][37]. They have been proposed to result from H• loss from the charge-reduced species and subsequent dissociation of the nonradical species by cleavage of the peptide backbone.…”

Section: Fragmentation Patterns Of Mccj25 and Capistruin And Their Nomentioning

confidence: 98%

“…However, the formation of radical b' • through ECD of peptides has never been reported previously. The b'• series and bipeptic product ions permitted to propose a competitive fragmentation pattern involving two isomeric charge-reduced species [ [34][35][36][37]. In MccJ25 and MccJ25-lcm, the b-type product ions could also be generated from H• migration from the b'• series.…”

Section: Fragmentation Patterns Of Mccj25 and Capistruin And Their Nomentioning

confidence: 99%

Topoisomer Differentiation of Molecular Knots by FTICR MS: Lessons from Class II Lasso Peptides

Zirah

Afonso

Linne

et al. 2011

Lasso peptides constitute a class of bioactive peptides sharing a knotted structure where the C-terminal tail of the peptide is threaded through and trapped within an N-terminal macrolactam ring. The structural characterization of lasso structures and differentiation from their unthreaded topoisomers is not trivial and generally requires the use of complementary biochemical and spectroscopic methods. Here we investigated two antimicrobial peptides belonging to the class II lasso peptide family and their corresponding unthreaded topoisomers: microcin J25 (MccJ25), which is known to yield two-peptide product ions specific of the lasso structure under collisioninduced dissociation (CID), and capistruin, for which CID does not permit to unambiguously assign the lasso structure. The two pairs of topoisomers were analyzed by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS) upon CID, infrared multiple photon dissociation (IRMPD), and electron capture dissociation (ECD). CID and ECD spectra clearly permitted to differentiate MccJ25 from its non-lasso topoisomer MccJ25-Icm, while for capistruin, only ECD was informative and showed different extent of hydrogen migration (formation of c•/z from c/z • ) for the threaded and unthreaded topoisomers. The ECD spectra of the triply-chargedMccJ25 and MccJ25-lcm showed a series of radical b-type product ions ðb 0 I n Þ. We proposed that these ions are specific of cyclic-branched peptides and result from a dual c/z • and y/b dissociation, in the ring and in the tail, respectively. This work shows the potentiality of ECD for structural characterization of peptide topoisomers, as well as the effect of conformation on hydrogen migration subsequent to electron capture.