Electron capture dissociation of b 2 peptide fragments reveals the presence of the acylium ion structure Kim F. Haselmann, Bogdan A. Budnik and Roman A. Zubarev* Department of Chemistry, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark Electron capture dissociation (ECD) of peptides and their fragments has now been extended to b n 2 ions, where it also produced far more structural information than collisional activation. Interestingly, b n 2 ions revealed abundant loss of CO from radical monocations and the presence of c (n  1) . fragments. The CO loss from peptide radical cations is unusual and was attributed to neutralization of the CO  group in the acylium ion structure, supported by the observation of c (n  1) . ions that can only be formed from an open- chain ion. These characteristic features were most prominent for b 12 2 ions of renin substrate and least prominent for b n 2 ions of substance P (n = 9,10). Totally, out of seven b n 2 ions studied, CO loss above 3% level was present in all spectra as well as c (n  1) . fragments of three species, suggesting that the acylium ion structure plays a significant role for at least some b 2 ions. This is an unexpected result in view of the literature data for small, singly charged b ions, for which the protonated oxazolone structure is favoured in ab initio calculations. Apparently, more studies are required before extrapolating the small molecule results to large species. The CO loss in ECD can be used for distinguishing between b and y ions in the MS/MS spectrum of larger molecules. Copyright # 2000 John Wiley & Sons, Ltd. Received 10 August 2000; Revised 4 October 2000; Accepted 9 October 2000 Vibrational (collisional or infrared) activation of protonated polypeptides produces b and y fragments upon cleavage of the peptide bond. 1 While it is generally accepted that y ions are simply truncated peptides, there is a great deal of disagreement in the literature about the structure of the complementary and less stable b ions. Four possible structures have been suggested: the open chain acylium ion, the protonated diketopiperazine ion, the protonated oxazolone structure and the immonium ion structure (Fig. 1). Historically, the acylium ion structure was proposed first; this structure is known from the classical EI spectra. 2 But because b 1 ions of simple amino acids and short peptides have not been observed, 3 peptide acylium ions have been ruled out by many. 3–5 Furthermore, calculations have indicated that the loss of CO to form the a 1 ions is an exothermic reaction, and thus the acylium-type b 1 ions are thermodynamically unstable. 6 A cyclic dipeptide with the diketopiperazine structure has been found to produce different CAD spectra than the equivalent b ions derived by collisions from a linear peptide chain. 4,7 This has left the protonated oxazolone ion as the generally recognised structure of b ions. Extensive ab initio calculations also seem to favour this structure. 4,8 However, these calculations concerned only small ions (b 1 and b 2 , and, at a lower level of theory, up to b 4 ), and the experimental evidence presented so far has mostly been circumstantial. Recently, Eckert et al. 9 have proposed the immonium ion structure based almost solely on ab initio calculations. Harrison et al. 10 have, however, pointed out that the protonated oxazolone structure can also account for the observations and therefore no immonium ion structure is necessary. Since the results obtained for small, singly charged ions (the biggest species studied experimentally was b 5  ) 3 may not necessarily be applicable to large, multiply charged species, we employed a novel experimental technique to derive structural information on doubly charged b ions 9–13 residues long. The experimental techniques used so far for studying b ion structures included metastable ion decay in sector instruments equipped with fast atom bombardment (FAB) 6,9,10 and low energy collision activated dissociation (CAD) in quadrupole, 9,11,12 hybrid 4,5 or quadrupolar ion trap 3 instruments with electrospray interfaces. Only a few studies have used other experimental techniques, i.e. ion- molecular H/D exchange reactions 3 and neutralization- reionization mass spectrometry (NRMS). 6 Here we report the use of electron capture dissociation (ECD) 13 to probe the structure of b 2 ions. Electron capture dissociation fragments through a different mechanism than vibrational excitation techniques, and leads to c, z . instead of b, y products. 13,14 An important feature of ECD is the preference for cleavage of just one bond near the site of charge neutralization, 13 with S–S bonds being a possible exception. 15 As a result, cyclic structures in the absence of disulphide bonds show in ECD spectra predominantly side-chain losses but not backbone cleavages (internal cleavage does not change the m/z value of the ions). The small side-chain losses in ECD 13 also result from single bond cleavages, in contrast to vibrational excitation, where the loss of small molecules such as H 2 O occurs 16 through a low-activation-energy rearrangement reaction. Furthermore, the non-ergodic cleavage in ECD is accompanied by a rather small increment in the internal *Correspondence to: R. A. Zubarev, Department of Chemistry, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark. Contract/grant sponsor: The Danish National Research Foundation; Contract/grant number: 9801448. Copyright # 2000 John Wiley & Sons, Ltd. RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 14, 2242–2246 (2000)