RESUMEN
Electron capture dissociation (ECD) of model peptides adducted with first row divalent transition metal ions, including Mn(2+), Fe(2+), Co(2+), Ni(2+), Cu(2+), and Zn(2+), were investigated. Model peptides with general sequence of ZGGGXGGGZ were used as probes to unveil the ECD mechanism of metalated peptides, where X is either V or W; and Z is either R or N. Peptides metalated with different divalent transition metal ions were found to generate different ECD tandem mass spectra. ECD spectra of peptides metalated by Mn(2+) and Zn(2+) were similar to those generated by ECD of peptides adducted with alkaline earth metal ions. Series of c-/z-type fragment ions with and without metal ions were observed. ECD of Fe(2+), Co(2+), and Ni(2+) adducted peptides yielded abundant metalated a-/y-type fragment ions; whereas ECD of Cu(2+) adducted peptides generated predominantly metalated b-/y-type fragment ions. From the present experimental results, it was postulated that electronic configuration of metal ions is an important factor in determining the ECD behavior of the metalated peptides. Due presumably to the stability of the electronic configuration, metal ions with fully-filled (i.e., Zn(2+)) and half filled (i.e., Mn(2+)) d-orbitals might not capture the incoming electron. Dissociation of the metal ions adducted peptides would proceed through the usual ECD channel(s) via "hot-hydrogen" or "superbase" intermediates, to form series of c-/z(â¢)- fragments. For other transition metal ions studied, reduction of the metal ions might occur preferentially. The energy liberated by the metal ion reduction would provide enough internal energy to generate the "slow-heating" type of fragment ions, i.e., metalated a-/y- fragments and metalated b-/y- fragments.
Asunto(s)
Espectrometría de Masas/métodos , Péptidos/química , Elementos de Transición/química , Secuencia de Aminoácidos , Bradiquinina/química , Cationes/química , Electrones , Datos de Secuencia MolecularRESUMEN
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(+â¢) and fragment ions corresponding to losses of neutral side chain from M(+â¢). 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. As previously postulated, divalent metal-ions adducted peptides could adopt several forms, including (a) [M + Cat](2+), (b) [(M + Cat - H) + H](2+), and (c) [(M + Cat - 2H) + 2H](2+). 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.
Asunto(s)
Metales Pesados/química , Péptidos/química , Secuencia de Aminoácidos , Aminoácidos/química , Cationes/química , Radicales Libres/química , Espectrometría de MasasRESUMEN
Series of doubly and triply protonated diarginated peptide molecules with different number of glutamic acid (E) and asparagine (N) residues were analyzed under ECD conditions. ECD spectra of doubly-protonated peptides show a strong dependence on the number of E and N residues. Both the backbone cleavages and hydrogen radical (H*) loss from the charge-reduced precursor ions ([M+2H](+*)) were suppressed as the number of E and N residues increases. A strong inhibition of the backbone cleavages and H* loss from [M+2H](+*) was found for peptides with 6E residues (or 4E + 2N residues). The results obtained using these model peptides were re-confirmed by analyzing N-arginated Fibrinopeptide-B (i.e., REGVNDNEEGFFSAR). In contrast to the N-arginated peptide, ECD of the doubly-protonated Fibrinopeptide-B and its analogues show extensive backbone cleavages leading to series of c- and z-ions ( approximately 80% sequence coverage). Based on these results, it is believed that peptide ions with all surplus protons sequestered in arginine-residues would show enhanced stability under ECD conditions as the number of acid-residue increases. The suppression of backbone cleavages and H* loss from [M+2H](+*) are presumably attributed to the low reactivity of the charge-reduced precursor ions. One of the possible hypothesis is that diarginated E-rich peptides may contain hydrogen bonds between carbonyl oxygen of E side chains and backbone amide hydrogen. These hydrogen bonds would provide extra stabilization for [M+2H](+*). This is the first demonstration of natural structural motifs in peptides that would inhibit the backbone fragmentation of the charge-reduced peptide ions under ECD conditions.
Asunto(s)
Secuencias de Aminoácidos , Iones/química , Espectrometría de Masas/métodos , Péptidos/química , Secuencia de Aminoácidos , Fibrinopéptido B/química , Datos de Secuencia Molecular , ProtonesRESUMEN
Electron-transfer dissociation (ETD) with supplemental activation of the doubly charged deamidated tryptic digested peptide ions allows differentiation of isoaspartic acid and aspartic acid residues using the c + 57 or z*-57 peaks. The diagnostic peak clearly localizes and characterizes the isoaspartic acid residue. Supplemental activation in ETD of the doubly charged peptide ions involves resonant excitation of the charge reduced precursor radical cations and leads to further dissociation, including extra backbone cleavages and secondary fragmentation. Supplemental activation is essential to obtain a high quality ETD spectrum (especially for doubly charged peptide ions) with sequence information. Unfortunately, the low-resolution of the ion trap mass spectrometer makes detection of the diagnostic peak, [M-60], for the aspartic acid residue difficult due to interference with side-chain loss from arginine and glutamic acid residues.
Asunto(s)
Ácido Aspártico/química , Ácido Isoaspártico/química , Espectrometría de Masas/métodos , Fragmentos de Péptidos/química , Secuencia de Aminoácidos , Cationes/química , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismoRESUMEN
Electron capture dissociation (ECD) of a series of custom-synthesized oligonucleotide pentamers was performed in a Fourier-transform mass spectrometer with a conventional filament-type electron gun. Dissociation of oligonucleotide ions by electron capture generates primarily w/d-type and z/a-type ions with and without the loss of a nucleobase fragment ions. Minor yields of radical [z/a + H]. fragment ions were also observed in many cases. It is interesting to note that some nucleoside-like fragment ions and protonated nucleobase ions (except thymine-related nucleobases and nucleoside-like fragments) were observed in most ECD spectra. The formation of these low-mass fragment ions was tentatively attributed to the secondary fragmentation of the radical [z + H]. fragment ions. From the ECD tandem mass spectra of a series of C/T based binary oligonucleotide ions, including d(CTCTC), d(CTTTC), d(TCCCT), d(CCCCT), and d(TCCCC), it was clearly demonstrated that the formation of many sequence ions was sensitive to the position of cytosine (or the position of charge carrier). The findings of this work support a notion that the ECD of protonated oligonucleotide molecules is charge-directed with the electron being captured by the protonated nucleobase.