Electron capture dissociation of gaseous multiply charged ions by Fourier-transform ion cyclotron resonance

Electron capture dissociation (ECD) has been demonstrated to be an effective fragmentation technique for characterizing the site and structure of the fatty acid modification in ghrelin, a 28-residue growth-hormone-releasing peptide that has an unusual ester-linked n-octanoyl (C8:0) modification at Ser-3. ECD cleaves 21 of 23 possible backbone amine bonds, with the product ions (c and z⅐ ions) covering a greater amino acid sequence than those obtained by collisionally activated dissociation (CAD). Consistent with the ECD nonergodic mechanism, the ester-linked octanoyl group is retained on all backbone cleavage product ions, allowing for direct localization of this labile modification. In addition, ECD also induces the ester bond cleavage to cause the loss of octanoic acid from the ghrelin molecular ion; the elimination process is initiated by the capture of an electron at the protonated ester group, which is followed by the radical-site-initiated reaction known as ␣-cleavage. emerged as a promising new tool for tandem mass spectrometry (MS/MS) of multiply charged (protonated) proteins and peptides generated by electrospray ionization (ESI) [6] using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry [7,8]. ECD typically causes extensive fragmentation of backbone amine bonds to produce c and z⅐ fragment ions, in contrast to the b and y fragment ions produced from the amide bond cleavage by conventional energetic fragmentation methods, such as collisionally activated dissociation (CAD) [9 -14], surface-induced dissociation (SID) [15], infrared multiphoton dissociation (IRMPD) [16], blackbody infrared dissociation (BIRD) [17,18]. Although c and z⅐ ions were initially observed in the 193 nm ultraviolet photodissociation (UVPD) mass spectra of multiply charged protein ions [19], they were the products of unexpected ECD processes: The low-energy electrons were ejected from metal surfaces by 193 nm photons (6.4 eV), and were captured by the protein ions trapped in a FTICR cell [1]. In addition, ECD is a nonergodic process (i.e., cleavage happens prior to energy randomization); the ECD backbone cleavages impart very little internal energy into the fragments, as a result, the labile side-chain modifications can be retained on the backbone fragment ions. This is in sharp contrast to the conventional ergodic fragmentation methods that typically eject the labile modifications before cleaving the backbone bonds. These unique attributes of ECD in providing extensive, nonergodic peptide backbone fragmentation have been demonstrated to be advantageous for localizing labile posttranslational modifications, such as ␥-carboxylation [20], sulfonation [20], glycosylation [21][22][23], and phosphorylation [24,25].In this study we examine ECD of ghrelin, a posttranslationally acylated peptide hormone that has an unusual C8:0 acylation (octanoic acid modification) at Ser-3 residue [26,27]. The ester-linked C8:0 fatty acyl moiety is essential for the activities of ghrelin, which include growth hormone secretion, feeding ...

Section: Ecd Of the Ester Bond In Ghrelinmentioning

confidence: 89%

Section: Ecd Of the Ester Bond In Ghrelinmentioning

confidence: 99%

Identification and localization of the fatty acid modification in ghrelin by electron capture dissociation

Guan

2002

“…The "hot hydrogen" model postulates that the incoming electron neutralizes the solvated proton and generates a hydrogen radical, H • . The transfer and relocation of H • to backbone carbonyl group leads to the formation of labile ketylamino radical and induces the dissociation of the adjacent N-C α bond at the C-terminal side of the carbonyl group [1,2,11,12]. Alternatively, the "superbase" model involves initial capture of electron by the π* orbital of the charge-perturbed carbonyl group.…”

Section: Introductionmentioning

confidence: 99%

Formation of Peptide Radical Cations (M^+·) in Electron Capture Dissociation of Peptides Adducted with Group IIB Metal Ions

Chen

Chan

Wong

et al. 2011

Peptides adducted with different divalent Group IIB metal ions (Zn 2+ , Cd 2+ , and Hg 2+ ) were found to give very different ECD mass spectra. ECD of Zn 2+ adducted peptides gave series of c-/z-type fragment ions with and without metal ions. ECD of Cd 2+ and Hg 2+ adducted model peptides gave mostly a-type fragment ions with M +• and fragment ions corresponding to losses of neutral side chain from M +• . No detectable a-ions could be observed in ECD spectra of Zn 2+ adducted peptides. We rationalized the present findings by invoking both proton-electron recombination and metal-ion reduction processes. . The relative population of these precursor ions depends largely on the acidity of the metal-ion peptide complexes. Peptides adducted with divalent metal-ions of small ionic radii (i.e., Zn 2+ ) would form predominantly species (b) and (c); whereas peptides adducted with metal ions of larger ionic radii (i.e., Hg 2+ ) would adopt predominantly species (a). Species (b) and (c) are believed to be essential for proton-electron recombination process to give c-/z-type fragments via the labile ketylamino radical intermediates. Species (c) is particularly important for the formation of non-metalated c-/z-type fragments. Without any mobile protons, species (a) are believed to undergo metal ion reduction and subsequently induce spontaneous electron transfer from the peptide moiety to the charge-reduced metal ions. Depending on the exothermicity of the electron transfer reaction, the peptide radical cations might be formed with substantial internal energy and might undergo further dissociation to give structural related fragment ions.

“…As a result, the techniques that rely on a common neutral loss from all phosphorylated peptides are often not reliable for tyrosine-phosphorylated species. Some of the limitations of these tandem mass spectrometry techniques for the determination of phosphorylation sites can be overcome in FTICR analyzers with the use of electron capture dissociation (ECD) [31][32][33][34][35][36][37], which allows fragmentation of peptides without the loss of the phosphate group [21, 24, 29 -31]. This technique provides valuable and extensive sequence information in the form of fragment ions complementary to those produced with CAD or IRMPD, though often at lower relative abundances.…”

mentioning

confidence: 99%

Infrared multiphoton dissociation (IRMPD) and collisionally activated dissociationof peptides in a quadrupole ion trapwith selective IRMPD of phosphopeptides

Crowe

Brodbelt

2004

112

Dissociation of protonated peptides via infrared multiphoton dissociation (IRMPD) provides more extensive sequence information than is obtained with collisionally activated dissociation (CAD) in a quadrupole ion trap due to the lack of the CAD low m/z cutoff and the ability to form secondary and higher order fragments with the non-resonant photoactivation technique. In addition, IRMPD is shown to be useful for the selective dissociation of phosphopeptides over those which are not phosphorylated because the greater photon absorption efficiency of the phosphorylated peptides leads to their more rapid dissociation. Finally, the selectivity of the IRMPD technique for phosphorylated species in complex mixtures is confirmed with the analysis of a mock peptide mixture and a tryptic digest of ␣-casein. I nvestigations into the protein complement of an organism's genome seek to identify the primary structure of the proteins produced including any modifications that take place. Following transcription, the primary sequence of a protein can undergo modifications such as N-terminal acetylation, formation of disulfide bonds, sulfation, and phosphorylation, all of which affect activity. Arguably, the most important post-translational modification of proteins is the reversible phosphorylation of serine, threonine, and tyrosine residues [1,2]. This covalent modification has regulatory influence over cellular processes such as metabolism, growth, and reproduction [3][4][5]. Because phosphorylation has such an impact on living systems, it is of great interest to be able to determine when, where, and to what extent this type of modification takes place. For this, suitable analytical techniques must be developed and applied. The relative speed and sensitivity of tandem mass spectrometry make it a powerful tool [6 -30] when compared with more conventional methods of phosphoprotein mapping, namely 32 P phosphate labeling of a protein sample followed by purification, enzymatic digestion, chromatographic separation, and Edman sequencing.Collisional activated dissociation (CAD) typically reveals sites of phosphorylation based on a mass loss associated with the cleavage of a phosphate moiety from a peptide ion (Ϫ80 (HPO 3 ) and/or Ϫ98 (H 3 PO 4 ) in positive ion MS/MS) [9,14]. Precursor ion scanning [14,19], neutral loss scanning [23], and nozzle-skimmer dissociation [9] can determine the presence, absence, and location of peptide phosphorylation based on the unique mass losses associated with phosphorylated peptides. In MALDI-TOF mass spectrometry, postsource decay (PSD) of metastable ions formed in the source region via these characteristic pathways can also be used to determine sites of phosphorylation [17]. When a peptide is phosphorylated at a tyrosine residue as opposed to a serine or threonine residue, however, the unique neutral losses are not necessarily observed upon dissociation due to the greater phosphate-tyrosine binding energy and the existence of fewer ␤-elimination pathways than are present in the cases of serine and thr...