Kristina Håkansson

Low-mass ions observed in plasma desorption mass spectrometry of high explosives

The low-mass ions observed in both positive and negative plasma desorption mass spectrometry (PDMS) of the high explosives HMX, RDX, CL-20, NC, PETN and TNT are reported. Possible identities of the most abundant ions are suggested and their presence or absence in the different spectra is related to the properties of the explosives as matrices in PDMS. The detection of abundant NO+ and NO2- ions for HMX, RDX and CL-20, which are efficient matrices, indicates that explosive decomposition takes place in PDMS of these three substances and that a contribution from the corresponding chemical energy release is possible. The observation of abundant C2H4N+ and CH2N+ ions, which have high protonation properties, might also explain the higher protein charge states observed with these matrices. Also, the observation of NO2-, possibly formed by electron scavenging which increases the survival probability of positively charged protein molecular ions, completes the pattern. TNT does not give any of these ions and it is thereby possible to explain why it does not work as a PDMS matrix. For NC and PETN, decomposition does not seem to be as pronounced as for HMX, RDX and CL-20, and also no particularly abundant ions with high protonation properties are observed. The fact that NC works well as a matrix might be related to other properties of this compound, such as its high adsorption ability.

Characterization of amino acid side chain losses in electron capture dissociation

We have used electrospray ionization (ESI) Fourier-transform ion cyclotron resonance (FTICR) mass spectrometry to characterize amino acid side chain losses observed during electron capture dissociation (ECD) of ten 7- to 14-mer peptides. Side-chain cleavages were observed for arginine, histidine, asparagine or glutamine, methionine, and lysine residues. All peptides containing an arginine, histidine, asparagine or glutamine showed the losses associated with that residue. Methionine side-chain loss was observed for doubly-protonated bombesin. Lysine side-chain loss was observed for triply-protonated dynorphin A fragment 1–13 but not for the doubly-protonated ion. The proximity of arginine to a methoxy C-terminal group significantly enhances the extent of side-chain fragmentation. Fragment ions associated with side-chain losses were comparable in abundance to those resulting from backbone cleavage in all cases. In the ECD spectrum of one peptide, the major product was due to fragmentation within an arginine side chain. Our results suggest that cleavages within side chains should be taken into account in analysis of ECD mass spectral data. Losses from arginine, histidine, and asparigine/glutamine can be used to ascertain their presence, as in the analysis of unknown peptides, particularly those with non-linear structures.

Electron capture dissociation of substance P using a commercially available Fourier transform ion cyclotron resonance mass spectrometer

Electron capture dissociation of the peptide Substance P is reported for the first time, with an unmodified, commercially available Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. The fragmentation pattern is compared with that obtained with collisionally induced dissociation of the ions in the electrospray ion source, and note that electron capture dissociation gives a more easily interpreted spectrum, showing mainly C-fragments. With the exception of the proline residues, which require cleavage of two chemical bonds, we observe all C-fragmental we find the bias voltage of the electron gun not to be very critical.

Electron capture dissociation and infrared multiphoton dissociation of oligodeoxynucleotide dications

We report electron capture dissociation (ECD) and infrared multiphoton dissociation (IRMPD) of doubly protonated and protonated/alkali metal ionized oligodeoxynucleotides. Mass spectra following ECD of the homodeoxynucleotides polydC, polydG, and polydA contain w or d “sequence” ions. For polydC and polydA, the observed fragments are even-electron ions, whereas radical w/d ions are observed for polydG. Base loss is seen for polydG and polydA but is a minor fragmentation pathway in ECD of polydC. We also observe fragment ions corresponding to w/d plus water in the spectra of polydC and d(GCATGC). Although the structure of these ions is not clear, they are suggested to proceed through a pentavalent phosphorane intermediate. The major fragment in ECD of d(GCATGC) is a d ion. Radical a- or z-type fragment ions are observed in most cases. IRMPD primarily results in base loss, but backbone fragmentation is also observed. IRMPD provides more sequence information than ECD, but the spectra are more complex due to extensive base and water losses. It is proposed that the smaller degree of sequence coverage in ECD, with fragmentation mostly occurring close to the ends of the molecules, is a consequence of a mechanism in which the electron is captured at a P=O bond, resulting in a negatively charged phosphate group. Consequently, at least two protons (or alkali metal cations) must be present to observe a w or d fragmention, a requirement that is less likely for small fragments.

Secondary fragmentation of linear peptides in electron capture dissociation

Abstract Inspection of the electron capture dissociation (ECD) spectra of doubly-protonated peptides, Leu4-Sar-Leu3-Lys-OH, Leu4-Ala-Leu3-Lys-OH, Gly4-Sar-Gly3-Lys-NH2 and Gly3-Pro-Sar-Gly3-Lys-NH2, reveals extensive secondary fragmentation. In addition to w ions, entire, and in some cases multiple, cleavages of amino acid side chains from backbone fragments are observed. Extensive water loss from backbone fragments is observed for the glycine-rich peptides. For Leu4-Ala-Leu3-Lys, the preferred fragmentation channel is cleavage of the amide bond to produce b7 and b8 ions. ECD of Gly3-Pro-Sar-Gly3-Lys-NH2 results in amine bond (c/z) cleavage in the proline residue accompanied by CC (or secondary NC) cleavage in the proline side chain. That fragmentation channel has not been observed previously. The peptides were also subjected to “hot” electron capture dissociation (HECD) and the resulting spectra differed markedly from those obtained under standard ECD conditions. In contrast to HECD, secondary fragmentation observed under standard ECD conditions cannot be attributed to excess energy arising from the kinetic energy of the electrons prior to capture. The results suggest that the fragmentation channels available following electron capture depend somewhat on the individual peptide structure and have mechanistic implications.

A 9.4 T Fourier transform ion cyclotron resonance mass spectrometer: description and performance

9.4 Tesla Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers (Bruker BioAPEX-94e) have been installed at the Division of Ion Physics, Uppsala University, and at the Department of Chemistry, University of Warwick. The BioAPEX-94e FT-ICR instrument is built around a high-field, superconducting magnet and a platform with easily interchangeable ion sources [matrix-assisted laser desorption/ionisation (MALDI), secondary ion mass spectrometry (SIMS), electrospray ionisation (ESI) and electron impact/chemical ionisation (EI/CI)]. In this paper a technical description of the instrument is given. Outstanding performance characteristics are demonstrated, notably clear resolution of C59N+ and C5813C2+ (mass difference 3.65 mDa) and mass measurement accuracy at the low ppm level. A wide range of applications in Warwick and Uppsala is described, demonstrating the versatility and high performance of the instrument.

Mechanistic studies of multipole storage assisted dissociation

The degree and onset of fragmentation in multipole storage assisted dissociation (MSAD) have been investigated as functions of several hexapole parameters. Strict studies of hexapole charge density (number of ions injected) and hexapole storage time were made possible by placing a pulsed shutter in front of the entrance to the mass spectrometer. The results obtained show that the charge density is the most critical parameter, but also dependencies on storage time, radio-frequency (rf) -amplitude, and pressure are seen. From these data, and from simulations of the ion trajectories inside the hexapole, a mechanism for MSAD, similar to the ones for sustained off-resonance irradiation (SORI), and for low energy collisionally induced dissociation in the collision multipole of a triple quadrupole mass spectrometer, is proposed. It is believed that, at higher charge densities, ions are pushed to larger hexapole radii where the electric potential created by the rf field is higher, forcing the ions to oscillate radially to higher amplitudes and thereby reach higher (but still relatively low) kinetic energies. Multiple collisions with residual gas molecules at these elevated energies then heat up the molecules to their dissociation threshold. Further support for this mechanism is obtained from a comparison of MSAD and SORI spectra which are almost identical in appearance.

Combination of nozzle-skimmer fragmentation and partial acid hydrolysis in electrospray ionization time-of-flight mass spectrometry of synthetic peptides

For reliable confirmation of peptide structures, nozzle-skimmer collisionally induced dissociation was found in many cases to be insufficient, and partial acid hydrolysis was employed as a complementary technique. The utility of combining these fragmentation methods is demonstrated in two examples where the complete sequences of two synthetic peptides (peptide I, MW 2290 and peptide II, MW 1482) were unambiguously determined. In a third example, three different valine deletions in a 2 kDa synthetic peptide were identified and their positions unambiguously established. A home-built electrospray ionization time-of-flight mass spectrometer with orthogonal extraction was used for these analyses. The performance of this instrument with a resolving power of up to 7500, a mass accuracy of < or = 10 ppm, and a detection limit of 1 fmol was shown to be well suited for such studies. As a substitution to conventional external calibration, a more convenient and equally accurate internal 3-point calibration is proposed, based on the low mass ions that are present in almost all peptide spectra.

An Antibiotic Linked to Peptides and Proteins is Released by Electron Capture Dissociation Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Desfuroylceftiofur (DFC) is a bioactive β-lactam antibiotic metabolite that has a free thiol group. Previous experiments have shown release of DFC from plasma extracts after addition of a disulfide reducing agent, suggesting that DFC may be bound to plasma and tissue proteins through disulfide bonds. We have reacted DFC with [Arg8]-vasopressin (which has one disulfide bond) and bovine insulin (which has three disulfide bonds) and analyzed the reaction products by use of electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry (ECD FT-ICR MS), which has previously shown preferential cleavage of disulfide bonds. We observe cleavage of DFC from vasopressin and insulin during ECD, suggesting that DFC is indeed bound to peptides and proteins through disulfide bonds. Specifically, we observed dissociative loss of one, as well as two, DFC species during ECD of [vasopressin + 2(DFC-H)+2H]2+ from a single electron capture event. Loss of two DFCs could arise from either consecutive or simultaneous loss, but in any case implies a gas phase disulfide exchange step. ECD of [insulin + DFC + 4H]4+ shows preferential dissociative loss of DFC. Combined with HPLC, ECD FT-ICR-MS may be an efficient screening method for detection of drug-biomolecule binding.

Inter- and intra-molecular migration of peptide amide hydrogens during electrospray ionization

The isotopic exchange of amide hydrogens in proteins in solution strongly depends on the surrounding protein structure, thereby allowing structural studies of proteins by mass spectrometry. However, during electrospray ionization (ESI), gas phase processes may scramble or deplete the isotopic information. These processes have been investigated by on-line monitoring of the exchange of labile deuterium atoms in homopeptides with hydrogens from a solvent suitable for ESI. The relative contribution of intra- and inter-molecular exchange in the gas phase could be studied from their distinct influence on the well-characterized exchange processes in the spraying solution. The deuterium content of individual labile hydrogens was assessed from the isotopic patterns of two consecutive collision-induced dissociation fragments, as observed with a 9.4 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. Results demonstrate that gas phase exchange in the high-pressure region between the capillary and the skimmer cause substantial depletion of the isotopic information of penta-phenylalanine and penta-aspartic acid. For penta-alanine and hexa-tyrosine, the amide hydrogens located close to the N-terminus are depleted from deuterium during mass analysis. Amide hydrogens located close to the C-terminus still retain the information of the isotopic state in solution, but they are redistributed by intra-molecular exchange of the amide hydrogens with the C-terminal hydroxyl group.