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

Abstract

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 ...

Characterization of an N-acylated glucagon-like peptide-1 derivative by electron capture dissociation

“…Nearly all major peaks were accompanied by satellite peaks equivalent to water losses (and in some cases, two consecutive water losses), from both band y-ions, which complicated the spectrum severely. This behavior was also observed in SORI-CAD of the O-acylated polypeptide ghrelin [18]. The base peak in the SORI-CAD spectrum was the quadruply charged nonmodified polypeptide chain, corresponding to the loss of the full modification.…”

“…This loss corresponds to the incorporation of a hydrogen atom). In an equivalent study by Guan [18] of O-acylated polypeptides, a loss of the full fatty acid chain was observed as well. The second part of the modification that linked the palmitoyl moiety to the polypeptide chain was a glutamoyl residue, which was linked to the -amine group of lysine 26 through an amide bond (see Scheme 1).…”

“…Unimolecular fragmentation in the gas phase can be performed by employing several techniques, e.g., infrared multiphoton dissociation (IRMPD, [9]), black-body infrared radiative dissociation (BIRD, [10]), sustained off-resonance irradiation collisionally activated dissociation (SORI-CAD, [11]) and electron capture dissociation (ECD, [12]). The latter technique has shown promising advantages for characterizing polypeptides with labile modifications, e.g., phosphorylation [13,14], O-glycosylation [15], Nglycosylation [16], ␥-carboxylation [17], and O-acylation [18].…”

Effects of Peptide Backbone Amide-to-Ester Bond Substitution on the Cleavage Frequency in Electron Capture Dissociation and Collision-Activated Dissociation

“…ECD of peptides with ester bonds has previously been investigated [36,37]. Marshall and co-workers have found that backbone cleavages in cyclic all-ester depsipeptides mainly occur in the O À C a bond [36], while Guan investigated fragmentation of the ester bond in a side chain [37].…”

Evaluation and optimization of electron capture dissociation efficiency in fourier transform ion cyclotron resonance mass spectrometry

“…(J Am Soc Mass Spectrom 2005, 16, 1060 -1066) © 2005 American Society for Mass Spectrometry E lectron capture dissociation (ECD) [1,2] is a relatively new MS/MS technique that has become popular because it provides better peptide and protein sequence coverage compared with other dissociation methods, and retains labile post-translational modifications (e.g., glycosylation [3,4] and phosphorylation [5][6][7][8]. Recent applications also include fatty acids [9], antibiotic/protein complexes [10], ubiquitination [11], oligonucleotides [12], and histones [13][14][15]. Despite its proven analytical utility, ECD suffers from limited conversion efficiency of precursor to product ions.…”