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 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 for the aspartic acid residue difficult due to interference with side-chain loss from arginine and glutamic acid residues.

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.

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• -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-/yfragments and metalated b-/y-fragments.

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 ([Mϩ2H] ϩ• ) were suppressed as the number of E and N residues increases. A strong inhibition of the backbone cleavages and H • loss from [Mϩ2H] ϩ• was found for peptides with 6E residues (or 4E ϩ 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 (ϳ80% 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 [Mϩ2H] ϩ• 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 [Mϩ2H] -4]. ECD also plays a useful role in characterization and localization of post-translational modifications (PTMs) as it preserves labile PTMs in protein [5][6][7] while cleaving the protein backbone to give series of specific fragment ions. This is in contrast with that of conventional slow-heating ion dissociation methods [8,9], such as collision induced dissociation (CID) [10 -12] and infrared multiproton dissociation (IRMPD) [13]. ECD involves the reaction between multiply-charged ions and low-energy electrons. Besides the formation of the charge-reduced precursor ions, [M ϩ nH] (n-1)ϩ• , the recombination energy released might provide enough energy to cause backbone N-C ␣ cleavages producing series of c or z • ions and to a lesser extent series of a • or y ions. Other ECD events include the elimination of (1) a H • to form [M ϩ (n-1)H] (n-1)ϩ , and (2) amino acid side chains from z • ions and/or [M ϩ nH] (n-1)ϩ• . Cleavages in ECD are nonspecific [14], and the relative propensities for dissociation of various amino acid residues were found to fall in a narrow range. Because of the ring-type structure, only fragment ions resulting from the backbone cleavages at the N-terminal side of proline (P) were suppressed [15].The importance of radical in charge-reduced precursor ion in ECD was investigated by synthetically incorporating single and multiple radical trap, spin trap, and charge tag moieties in model peptides. In radical trap experiments, Belyayev et al. [16] attached coumarin labels onto the N-terminal amino group (or/an...

Electron capture dissociation (ECD) of a series of custom-synthesized oligonucleotide pentamers was performed in a Fourier-transform mass spectrometer with a conventional filamenttype 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 ϩ 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 S tructural characterization of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) is important, as DNA and RNA play important roles in many biochemical processes. Even with the completion of the human genome project, the importance of studying nucleic acid remains. For instance, a large region of DNA requires re-sequencing [1] to allow the detection and characterization of nucleotide mutation. There is a constant demand for powerful and rapid analytical methods to determine the structure of nucleic acids and their constituents. With the development of "soft" desorption/ionization techniques, matrix-assisted laser desorption/ionization (MALDI) [2,3] and electrospray ionization (ESI) [4,5] methods, mass spectrometry has become an indispensable, quick, and reliable tool for the analysis of oligonucleotides [6]. However, simple mass measurement provides little structural information for confirmation of the sequence for unknown natural and synthetic oligonucleotides. Much research effort has recently been devoted to the development of various tandem mass spectrometry (MS/MS) methods for structural elucidation of biomolecules. The critical event in tandem mass spectrometry of biomolecules is the activation of the selected precursor ions to induce unimolecular dissociation. Common ion activation methods used for analysis of biomolecules include lowand high-energy collision-induced dissociation (CID) [7, 8], surface-induced dissociation (SID) [9], infrared multiphoton dissociation (IRMPD) [10], and blackbody infrared radiative dissociation (BIRD) [11]. However, fragment ions observed in the tandem mass spectra using these ion activation methods mostly originate from cleavages of weak bonds [12].A relatively new ion activation method, electron capture dissociation (ECD) [13] has demonstrated several interesting features. For example, for protein-type biomolecules, ECD leads predominantly to the dissociation of the N-C ␣ linkage even in the presence of labile post-translational modifications [14,15]. A combination of ECD and other dissociation methods, such as IRMPD and CID, has been shown to provide complementary information for unambiguous identification of the type and the position of several important protein posttranslational modifications [16 -18] and for de novo s...