Bogdan A. Budnik

Towards an understanding of the mechanism of electron-capture dissociation: a historical perspective and modern ideas

Electron-capture dissociation (ECD) is a new fragmentation technique that utilizes ion–electron recombination reactions. The latter have parallels in other research fields; revealing these parallels helps to understand the ECD mechanism. An overview is given of ECD-related phenomena and of the history of ECD discovery and development. Current views on the ECD mechanism are discussed using both published and new examples.

Electron detachment dissociation of peptide di-anions: an electron–hole recombination phenomenon

A novel electron–ion reaction mimicking positron capture is reported for gas-phase polypeptide di-anions. Bombardment of the latter by >10 eV electrons produced electron detachment followed by backbone dissociation, noticeably cleavage of the N–Cα bond. On the other hand, reaction with hydrogen cations resulted in losses of neutral groups but not in backbone cleavage. It is proposed that the N–Cα dissociation is due to recombination along the peptide chain of a positive radical charge (hole) with a negative charge. The new reaction is likely to find application in mass spectrometry for primary structure determination of acidic polypeptides.

Dissociative capture of hot (3–13 eV) electrons by polypeptide polycations: an efficient process accompanied by secondary fragmentation

Dissociative capture of hot (3-13 eV) electrons by polypeptide polycations: an efficient process accompanied by secondary fragmentation

Can the (M - X) region in electron capture dissociation provide reliable information on amino acid composition of polypeptides?

It has been suggested that small losses from reduced peptide molecular species in electron capture dissociation (ECD) could indicate the presence of certain amino acids [H.J. Cooper, R.R. Hudgins, K. Håkansson and A.G. Marshall, J. Am Soc. Mass Spectrom 13, 241 (2002)], similarly to immonium ions in high-energy collision-activated dissociation. The diagnostic value in ECD of the (M•–X) region (1 Da ≤ X ≤ 130 Da) was tested on several synthetic peptides. The insufficiency of the existing knowledge for making correct conclusions on the amino acid composition is demonstrated and new suggestions of the origin of losses are presented based on the “hot hydrogen atom” ECD mechanism. Generally, it is shown that not only protonation but also charge solvation is responsible for the small losses. The origin of 17 Da and 59 Da losses is revisited and a new mechanism for the 18 Da loss is suggested. The loss of a side chain plus a hydrogen atom is found to be a rather reliable indicator of the presence of histidine, tryptophan, tyrosine and, to a lesser degree, threonine. The overall conclusion is that the (M• - X) region does contain information on the amino acid composition, but extraction of this information requires additional studies.

Intramolecular hydrogen atom transfer in hydrogen-deficient polypeptide radical cations

Irradiation of protonated polypeptides NH2‐RH a ‐COOH by >10 eV electrons leads to further ionization and fast intramolecular charge transfer to the free N-terminus. The resulting species may undergo further hydrogen atom rearrangement to form distonic ions N a H3‐RH a ‐COOAE. Such transfer is exothermic but can involve an appreciable barrier, e.g., 2:3 0:5 eV for MH 2aAE ions of the peptide ACTH 1‐10. Radical polypeptide dications can, therefore, be viewed as hydrogen atom wires. Subsequent capture of low energy electrons results in fragmentation. The pattern of this electronic excitation dissociation (EED) is consistent with hydrogen transfer prior to electron capture. ” 2000 Elsevier Science B.V. All rights reserved.

MH2+⋅ ion production from protonated polypeptides by electron impact: observation and determination of ionization energies and a cross-section

Irradiation of gas-phase MH+ ions of polypeptides up to MW 3493 by 11 to 70 eV electrons produced further ionization: MH++e−→MH2+·+2 e−, with ionization thresholds of 11.4±0.5 eV for [Arg-8]-vasopressin (MW 1084), 10.7±0.5 eV for substance P (MW 1347), 11.4±0.6 eV for renin substrate (MW 1759) and 10.6±0.4 eV for melittin (MW 2846). The ionization cross-section of vasopressin MH+ ions was found to be (1.3±0.4)·10−15 cm2 for 20 eV electrons. The gas-phase MH2+⋅ ions are rather stable at room temperature, with lifetimes of hundreds of seconds at 10−10 Torr.

Electronic excitation gives informative fragmentation of polypeptide cations and anions

A Fourier transform mass spectrometer is a versatile instrument with a range of available fragmentation techniques. Comparison of polypeptide fragmentation patterns revealed that the techniques involving electronic excitation, such as hot-electron-capture dissociation (HECD) and electron-detachment dissociation (EDD), are even more informative than vibrational excitation (VE) techniques such as collisional activation. For dications of the peptide KIMHASELMANN, 11 eV HECD cleaved all inter-residue links in at least two places, with up to five fragments characterizing each link. For dianions of the same molecule, VE produced only one backbone cleavage whereas EDD gave ten, including five internal cleavage fragments. This is consistent with the general postulate that homogeneous electronic excitation yields more types of cleavage than near-equilibrium processes such as VE.

Can relative cleavage frequencies in peptides provide additional sequence information

The ambiguity of attributing N- vs. C-terminal origin to a fragment ion can pose a problem in mass spectrometric de novo peptide sequencing even when all inter-residue bonds are cleaved. This makes additional sequence information highly desirable. The question is investigated whether relative abundances of fragment ions in MS/MS are capable of providing this information. Statistical analysis of data on peptides 10–24 residues long reveled that the frequencies of N–C bond cleavage in electron capture dissociation (ECD) are less dependent upon the total number of basic sites, the charge states of the parent ions and the nature of the terminal group than the peptide bond fragmentation in collisionally activated dissociation (CAD). The consensus sequences in peptides of different lengths also showed similar fragmentation patterns, with the typical correlation factor of 0.7. The conclusion is that frequencies of ECD cleavages are promising as a source of additional sequence information provided that strong intra-molecular bonds are absent or broken prior to MS/MS analysis. (Int J Mass Spectrom 219 (2002) 283–294)

Cyanohydrin glycosides of Passiflora: distribution pattern, a saturated cyclopentane derivative from P. guatemalensis, and formation of pseudocyanogenic α-hydroxyamides as isolation artefacts

Nineteen species of Passiflora (Passifloraceae) were examined for the presence of cyanogenic glycosides. Passibiflorin, a bisglycoside containing the 6-deoxy-beta-D-gulopyranosyl residue, was isolated from P. apetala, P. biflora, P. cuneata, P. indecora, P. murucuja and P. perfoliata. In some cases this glycoside co-occurs with simple beta-D-glucopyranosides: tetraphyllin A, deidaclin, tetraphyllin B, volkenin, epivolkenin and taraktophyllin. P. citrina contains passicapsin, a rare glycoside with the 2,6-dideoxy-beta-D-xylo-hexopyranosyl moiety, while P. herbertiana contains tetraphyllin A, deidaclin, epivolkenin and taraktophyllin, P. discophora tetraphyllin B and volkenin, and P. x violacea tetraphyllin B sulfate. The remaining species were noncyanogenic. The glycosides were identified by 1H and 13C NMR spectroscopy following isolation by reversed-phase preparative HPLC. From P. guatemalensis, a new glucoside named passiguatemalin was isolated and identified as a 1-(beta-D-glucopyranosyloxy)-2,3-dihydroxycyclopentane-1-carbonitrile. An isomeric glycoside was prepared by catalytic hydrogenation of gynocardin. alpha-Hydroxyamides corresponding to the cyanogenic glycosides were isolated from several Passiflora species. These alpha-hydroxyamides, presumably formed during processing of the plant material, behave as cyanogenic compounds when treated with commercial Helix pomatia crude enzyme preparation. Thus, the enzyme preparation appears to contain an amide dehydratase, which converts alpha-hydroxyamides to cyanohydrins that liberate cyanide; this finding is of interest in connection with analysis of plant tissues and extracts using Helix pomatia enzymes.

High-performance liquid chromatography–mass spectrometry and electron-capture dissociation tandem mass spectrometry of osteocalcin: Determination of γ-carboxyglutamic acid residues

Two mass spectrometry methods, high-performance liquid chromatography combined on-line with electrospray ionization mass spectrometry (HPLC-ESI-MS) and electron-capture (EC) dissociation tandem mass spectrometry (MS-MS), were applied for structural analysis of bovine and human osteocalcins. Osteocalcin contains gamma-carboxyglutamic acid (Gla) residues, which bind metal ions, among its amino acids. Ethylenediaminetetraacetic acid (EDTA) was added to all samples in order to chelate bound metal ions. After elimination of interfering metal ions MS spectra became uncomplicated to interpret. EDTA is incompatible with ESI and it was removed from samples using either on-line HPLC or micropurification method. The number of Gla residues varies in osteocalcin. These subforms, which contain different amounts of Gla residues, were separated using the HPLC-ESI-MS method. In order to determine locations of Gla residues in human osteocalcin, which contained two Gla residues, dissociation MS-MS method was successfully applied.