Applications of electron-ion dissociation reactions for analysis of polycationic chitooligosaccharides in Fourier transform mass spectrometry.

Singly protonated, doubly protonated, and sodiated pentaglucosamide (GlcNAc)(5), oligoglucosamines (GlcN)(m)(), and (GlcN)(3)GlcN(3OH14:0) were analyzed in an FTICR mass spectrometer by electron-ion dissociation reactions and compared to collision activation. The general fragmentation mode was found as the asymmetrical sequence fragments (B(n)() and minor C(n)() ion series) with full sequence coverage. Molecular mass information of each glucosamide or glucosamine residue can be readily obtained from the ion series. Fragmentation by electron capture dissociation revealed additional fragmentation of the N-acetyl moiety compared to sustained off-resonance irradiation collision-activated dissociation (SORI-CAD) and electron-induced dissociation (EID). Sodiated GlcNAc(5) molecular adduct ions were analyzed by EID and compared to CAD. Both techniques provided full sequence coverage. EID was more effective, but CAD resulted in the cross-ring ion products (0,2)A(n)() and (2,4)A(n)() for all relevant glucosamide residues.

Towards the standard-module approach to disulfide-linked polypeptide nanostructures. I. Methodological prerequisites and mass spectrometric characterization of the test two-loop structure.

Potentially biologically-active nanostructures can be created from single chains of unmodified peptides by cross-linking different regions of the chain by disulfide bonds and cleaving the chain at specified sites to obtain the final configuration. The availability of techniques for assembly and characterization of such structures was tested on a two-loop structure created from a 21-residue linear peptide. Directed intra-molecular disulfide bond formation was performed by inserting partial sequences favoring intra-molecular SS bond formation ("loops") separated by partial sequences disfavoring such a process ("spacers") into the precursor sequence. Peptide bond cleavage by partial acid hydrolysis at specific sites (GG, NP/DP) inside the loops opened them; the same process in the spacer separated the loops. Synthesis, oxidation and bond cleavage were monitored by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI ToF MS). The hydrolysis fragments of the produced nanostructures were characterized by tandem electrospray ionization Fourier transform mass spectrometry (ESI FT-MS) with collisional and electron capture dissociations. The latter technique was especially useful as it cleaves SS bonds preferentially. The feasibility of the proposed synthesis approach and the adequacy of the analysis techniques for the test structure were demonstrated.

Improved low-energy electron injection systems for high rate electron capture dissociation in Fourier transform ion cyclotron resonance mass spectrometry.

New low-energy electron injection systems based on indirectly heated dispenser cathodes facilitate electron capture dissociation (ECD) in Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. In this joint report, details are presented of the design and performance of these systems on two commercial FTICR instruments, 9.4 T Bruker BioAPEX in Uppsala and 4.7 T IonSpec Ultima in Odense. New results include obtaining meaningful one-scan MS/MS data for isolated precursor ions with millisecond irradiation times. The ECD rate improvement is not only due to the larger total electron current, but the larger emitting area as well.

Advantages of external accumulation for electron capture dissociation in Fourier transform mass spectrometry.

A combination of external accumulation (XA) with electron capture dissociation (ECD) improves the electron capture efficiency, shortens the analysis time, and allows for rapid integration of multiple scans in Fourier transform mass spectrometry. This improves the signal-to-noise ratio and increases the number of detected products, including structurally important MS3 fragments. With XA-ECD, the range of the labile species amenable to ECD is significantly extended. Examples include the first-time determination of the positions of six GalNAc groups in a 60-residue peptide, five sialic acid and six O-linked GalNAc groups in a 25-residue peptide, and the sulfate group position in a 11-residue peptide. Even weakly bound supramolecular aggregates, including nonspecific peptide complexes, can be analyzed with XA-ECD. Preliminary results are reported on high-rate XA-ECD that uses an indirectly heated dispenser cathode as an electron source. This shortens the irradiation time to > or = 1 ms and increases the acquisition rate to 3 scans/s, an improvement by a factor of 10-100.

Comparison of electron capture dissociation and collisionally activated dissociation of polycations of peptide nucleic acids.

Electron capture dissociation (ECD) in Fourier transform ion cyclotron resonance mass spectrometry coupled with electrospray ionization enhances the sequence elucidation of peptide nucleic acids compared with conventional low-energy collisionally activated dissociation (CAD). Examples are shown where ECD produced complete or extensive sequence coverage in PNAs six to ten nucleobases long. However, facile base losses from the reduced species and low abundances of backbone ECD fragments presented a significant problem. This was rationalized through the lower degree of charge solvation on the backbone compared to polypeptides. Combination of both CAD and ECD data is advantageous, as these techniques produce cleavages at different sites.

Electron capture dissociation of b (2+) peptide fragments reveals the presence of the acylium ion structure.

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 identical with 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.

Formation of chloroform in spruce forest soil--results from laboratory incubation studies.

The release of chloroform, 1,1,1-trichloroethane, tetrachloromethane, trichloroethene and tetrachloroethene from an organic rich spruce forest soil was studied in laboratory incubation experiments by dynamic headspace analysis, thermodesorption and gas chromatography. Performance parameters are presented for the dynamic headspace system. For spruce forest soil, the results showed a significant increase in chloroform concentration in the headspace under aerobic conditions over a period of seven days, whereas the concentration of the other compounds remained fairly constant. A biogenic formation of chloroform is suggested, whereas for the other compounds anthropogenic sources are assumed. The addition of trichloroacetic acid to the soil increased the release of chloroform from the soil. It is, therefore, suggested that trichloroacetic acid also contributed to the formation of chloroform. Under the experimental conditions, the spruce forest soil released chloroform concentrations corresponding to a rate of 12 microg m(-2) day(-1). Data on chloroform production rates are presented and compared with literature results, and possible formation mechanisms for chloroform are discussed.

Electron capture dissociation of singly and multiply phosphorylated peptides.

Analysis of phosphotyrosine and phosphoserine containing peptides by nano-electrospray Fourier transform ion cyclotron resonance (FTICR) mass spectrometry established electron capture dissociation (ECD) as a viable method for phosphopeptide sequencing. In general, ECD spectra of synthetic and native phosphopeptides appeared less complex than conventional collision activated dissociation (CAD) mass spectra of these species. ECD of multiply protonated phosphopeptide ions generated mainly c- and z(.)-type peptide fragment ion series. No loss of water, phosphate groups or phosphoric acid from intact phosphopeptide ions nor from the c and z(.) fragment ion products was observed in the ECD spectra. ECD enabled complete or near-complete amino acid sequencing of phosphopeptides for the assignment of up to four phosphorylation sites in peptides in the mass range 1400 to 3500 Da. Nano-scale Fe(III)-affinity chromatography combined with nano-electrospray FTMS/ECD facilitated phosphopeptide analysis and amino acid sequencing from crude proteolytic peptide mixtures.