De novo sequencing of antimicrobial peptides isolated from the venom glands of the wolf spider Lycosa singoriensis.

Antimicrobial peptides (AMPs), named lycocitin 1, 2 and 3, and a peptide with a monoisotopic molecular mass of 3038.70 Da were detected in the venom glands of the wolf spider Lycosa singoriensis. Two of the peptides, lycocitin 1 and 2, are new AMPs whereas lycocitin 3 is highly homologous to lycotoxin II isolated from the venom of spider Lycosa carolinensis. In addition, two other peptides with monoisotopic masses of 2034.20 and 2340.28 Da showing the motif typical for antimicrobial peptides were also identified. These peptides and lycocitin 1, 2 and 3 were de novo sequenced using electron capture dissociation and low-energy collisional tandem mass spectrometry. The amino acid sequence of lycocitin 1 was determined as GKLQAFLAKMKEIAAQTL-NH(2). Lycocitin 2 differs from lycocitin 1 by a replacement of a lysine residue for an arginine residue at the second position. Lycocitin 3 differs from the known lycotoxin II consisting of 27 amino acid residues by a deletion of Gly-26. Both lycocitin 1 and 2 inhibit growth of Gram-positive (Staphylococcus aureus, Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria and fungi (Candida albicans, Pseudomonas aeruginosa) at micromolar concentrations.

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.

Leishmanicidal, antiplasmodial, and cytotoxic activity of novel diterpenoid 1,2-quinones from Perovskia abrotanoides: new source of tanshinones.

Cryptotanshinone (1), a quinoid diterpene with a nor-abietane skeleton, and three new natural products, 1beta-hydroxycryptotanshinone (2), 1-oxocryptotanshinone (3), and 1-oxomiltirone (4), were isolated from roots of the Iranian medicinal plant Perovskia abrotanoides. Their structures were established using homo- and heteronuclear two-dimensional NMR experiments, supported by HRMS. The total amount of tanshinones isolated from dry roots of Perovskia abrotanoides was about 1.5%. The compounds exhibited leishmanicidal activity in vitro (IC(50) values in the range 18-47 microM). These findings provide a rationale for traditional use of the roots in Iran as a constituent of poultices for treatment of cutaneous leishmaniasis. The isolated tanshinones also inhibited growth of cultured malaria parasites (3D7 strain of Plasmodium falciparum), drug-sensitive KB-3-1 human carcinoma cell line, multidrug-resistant KB-V1 cell line, and human lymphocytes activated with phytohaemagglutinin A (IC(50) values in the range 5-45 microM). The toxicity of tanshinones toward the drug-sensitive KB-3-1 and the multidrug-resistant KB-V1 cells was the same, indicating that the compounds are not substrates for the P-glycoprotein drug efflux pump.

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.

Benefits of 2.94 micron infrared matrix-assisted laser desorption/ionization for analysis of labile molecules by Fourier transform mass spectrometry.

A 2.94 microm Er:YAG laser was used together with a commercial Fourier transform mass spectrometer to study labile biomolecules. The combination has shown superior performance over conventional 337 nm ultraviolet matrix-assisted laser desorption/ionization (UV-MALDI) Fourier transform mass spectrometry (FTMS), especially for the analysis of peptides with post-translational modifications. With succinic acid as a matrix, the sensitivity of the single-shot analysis was increased by an order of magnitude to the low femtomole level, with significantly less fragmentation observed. Intact molecular ions of a range of O-glycosylated and sulfated peptides were detected. Urea was found to induce even less fragmentation, although at the expense of the total ion yield. Molecular ions of a noncovalent complex (vancomycin + diacetyl-L-Lys-D-Ala-D-Ala) have been observed for the first time in MALDI-FTMS. 2.94 microm infrared (IR) MALDI also produced abundant molecular ions of a range of nonbiological samples, including C60 and C70 fullerenes as well as dimetal coordination complexes.