Improvement of electron capture efficiency by resonant excitation

A novel pulse sequence improving the efficiency for electron capture dissociation (ECD) of an unmodified Fourier transform ion cyclotron resonance (FTICR) mass spectrometer by more than an order of magnitude is presented. Commercially available FTICR instruments are usually equipped with a filament-based electron source producing an electron beam that has a rather small cross section. An ideal overlap between the rotating ion cloud and the electron beam appears to be a prerequisite for a high ECD efficiency. A reduced interception of the ion cloud and the electron beam is probably due to the contribution of the magnetron motion to the trajectory of the ions, resulting in a precession about the z-axis of the instrument. By increasing the kinetic energy and therefore increasing the cyclotron radii of the precursor ions by resonant excitation, the overlap of the rotating ion cloud with the electron beam is improved. By use of this protocol the efficiency of electron capture is substantially increased and consequently the acquisition time of ECD spectra is reduced significantly. The capability of resonant excitation of the precursor ions during the irradiation with electrons is demonstrated for standard peptides. This approach is particularly valuable for analysis and characterization of O-glycosylated peptides. In addition to amino acid sequence information, the attachment site of the labile glycan moiety is determined, and also radical-site-induced fragmentations of the glycosidic bonds are observed.     

Structural characterization of chondroitin/dermatan sulfate oligosaccharides from bovine aorta by capillary electrophoresis and electrospray ionization quadrupole time-of-flight tandem mass spectrometry

An analytical approach based on high-performance capillary electrophoresis (CE) in conjunction with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-QTOF-MS/MS) has been developed for providing the basis to obtain new insights into the domain structure of the glycosaminoglycan (GAG) moiety of proteoglycans. The feasibility and performance of the off-line CE/ESI-QTOF-MS approach in GAG oligosaccharide analysis were assessed by screening a chondroitin/dermatan sulfate (DS) oligosaccharide mixture obtained from bovine aorta by enzymatic depolymerization by chondroitin B lyase. The CS/DS mixture was analyzed by CE using 50 mM ammonium acetate, pH 12.0, dissolved in aqueous methanol (2:3; v/v), as a CE carrier. Structural identification of the GAG components was achieved using off-line CE/nanoESI-QTOF-MS and-MS/MS experiments. ESI-QTOF instrumental parameters were found to play an important role in the MS screening of the CE-separated GAG species. By optimizing the ESI conditions, oligosaccharides differing in chain length and degree of sulfation could be detected. The building block composition, the size of the carbohydrate chain, as well as structural features of the repeating HexA-GalNAc, HexA-GalNAc(S) units, have been determined using MS/MS by applying collision-induced dissociation at low energies. Cleavage of GAG chains by chondroitin B lyase occurs with formation of structural markers useful for identification of IdoA-containing domains.     

Umwandlung von 6-Methylen-tricyclo[3.2.1.02,7]oct-3-en-8-onen in Polymethyltropyliumsalze

When dissolved in trifluoroacetic or fluorosulfonic acid, 6-methylene-tricyclo[3.2.1.0^2,7]oct-3-ene-8-one derivatives of type 2 (;scheme 1); give polymethyltropylium salts in moderate to good yields by CO-extrusion. These tropylium salts can be isolated pure as hexachloroplatinates. Thus the tricyclic compound 6 gives 1,2,4-trimethyltropylium trifluoroacetate 19 in trifluoroacetic acid (;scheme 3);. This salt in CDCl₃ is in equilibrium with its covalent cycloheptatriene (;tropilidene); form 20 , the ratio of the two forms being 1.5–2.1/1. The tropylium salt 19 is reduced by lithium aluminium hydride to a mixture of 1,2,4-trimethylcycloheptatrienes, isomeric with respect to the double bonds, which on hydride abstraction with trityl-tetrafluoroborate gives again the 1,2,4-trimethyltropylium salt 19 (;scheme 3);. From the trimethyl-substituted tricyclic compounds 7 and 8 , in trifluoroacetic acid, are obtained respectively the 1,2,4,6- and 1,2,3,4-tetramethyltropylium ions (; 22 and 24 ); (;schemes 4 and 5);. In this way the 1,2,3,5,6-pentamethyl-tropylium ion (; 26 ); was obtained from 9 (;scheme 6);. With the higher substituted tropylium trifluoroacetates in CDCl₃ the equilibrium tropylium trifluoroacetate ? trifluoroacetoxycycloheptatrienes lies well to the left. The hexamethylated tricyclic compound 10 gives a small quantity of heptamethyltropylium trifluoroacetate (; 27 ); and as the main product the C(;3);-protonated species 28 (;scheme 7);, which when treated with aqueous sodium hydrogencarbonate yielded unchanged educt 10 . - The heptamethyltropylium ion (; 27 ); was, apart from polymeric species, the only product from the treatment of starting material 10 with fluorosulfonic acid (;50%);; its salts have as yet not been isolated in their pure form, however. The mechanism for the rearrangement of the tricyclic compounds of type 2 into tropylium salts is presented for compound 6 in scheme 8: The first step is the protonation at C(;9);. Ring opening of the cyclopropane of the tertiary carbenium ion 29 gives the allylic ion 30 , which then yields the aromatic tropylium salt 19 by carbon monoxide extrusion in a linear cheletropic reaction. The smooth conversion with strong acids of the easily accessible tricyclic compounds of type 2 to the corresponding polymethylated tropylium salts, presents a new and useful method for the synthesis of the latter compounds.     

Thin chip microsprayer system coupled to quadrupole time-of-flight mass spectrometer for glycoconjugate analysis

A thin chip polymer-based microsprayer has been coupled to a hybrid quadrupole time-of-flight mass spectrometer (QTOF MS) and introduced in carbohydrate research. The feasibility of the approach is demonstrated for mapping, sequencing and structural elucidation of glycoconjugates originating from human body fluids and tissues such as a glycopeptide mixture from normal human urine and an isolated and purified GT1 ganglioside fraction from normal adult human brain. The optimization procedure required by each glycoconjugate category is described and the advantages of the system in terms of flexibility and adaptability to QTOF MS, stability of the ESI MS signal, carbohydrate ionization and sequencing, sensitivity, speed of analysis and sample consumption are discussed.     

β-Tricarbonyl compounds. I. 2,4-Disubstituted 1,3-cyclopentanediones

Summary Stable enolic isomers of 2-aroyl-4-aracyl-1,3-cyclopentanediones such as3 and4 were prepared by condensation of aryl methyl ketones and diethyl maleate using an excess of sodium ethoxide (Aryl=C₆H₅, 4-C₆H₄CH₃, 4-C₆H₄Br and 4-C₆H₄Cl).

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-Tricarbonyl Verbindungen. I. 2,4-Disubstituierte 1,3-Cyclopentandione

Zusammenfassung Stabile Enol-Isomere von 2-Aroyl-4-aracyl-1,3-cyclopentandionen wie3 und4 wurden durch Kondensation von Arylmethylketonen und Diethylmaleat mit einem Überschuß von Natriumethoxid dargestellt (Aryl=C₆H₅, 4-C₆H₄CH₃, 4-C₆H₄Br und 4-C₆H₄Cl).

     

Supercritical CO2 Plant Extracts Show Antifungal Activities against Crop-Borne Fungi

Fungal infections of cultivated food crops result in extensive losses of crops at the global level, while resistance to antifungal agents continues to grow. Supercritical fluid extraction using CO₂ (SFE-CO₂) has gained attention as an environmentally well-accepted extraction method, as CO₂ is a non-toxic, inert and available solvent, and the extracts obtained are, chemically, of greater or different complexities compared to those of conventional extracts. The SFE-CO₂ extracts of Achillea millefolium, Calendula officinalis, Chamomilla recutita, Helichrysum arenarium, Humulus lupulus, Taraxacum officinale, Juniperus communis, Hypericum perforatum, Nepeta cataria, Crataegus sp. and Sambucus nigra were studied in terms of their compositions and antifungal activities against the wheat- and buckwheat-borne fungi Alternaria alternata, Epicoccum nigrum, Botrytis cinerea, Fusarium oxysporum and Fusarium poae. The C. recutita and H. arenarium extracts were the most efficacious, and these inhibited the growth of most of the fungi by 80% to 100%. Among the fungal species, B. cinerea was the most susceptible to the treatments with the SFE-CO₂ extracts, while Fusarium spp. were the least. This study shows that some of these SFE-CO₂ extracts have promising potential for use as antifungal agents for selected crop-borne fungi.     

Fragmentation of intra-peptide and inter-peptide disulfide bonds of proteolytic peptides by nanoESI collision-induced dissociation

Characterisation and identification of disulfide bridges is an important aspect of structural elucidation of proteins. Covalent cysteine-cysteine contacts within the protein give rise to stabilisation of the native tertiary structure of the molecules. Bottom-up identification and sequencing of proteins by mass spectrometry most frequently involves reductive cleavage and alkylation of disulfide links followed by enzymatic digestion. However, when using this approach, information on cysteine-cysteine contacts within the protein is lost. Mass spectrometric characterisation of peptides containing intra-chain disulfides is a challenging analytical task, because peptide bonds within the disulfide loop are believed to be resistant to fragmentation. In this contribution we show recent results on the fragmentation of intra and inter-peptide disulfide bonds of proteolytic peptides by nano electrospray ionisation collision-induced dissociation (nanoESI CID). Disulfide bridge-containing peptides obtained from proteolytic digests were submitted to low-energy nanoESI CID using a quadrupole time-of-flight (Q-TOF) instrument as a mass analyser. Fragmentation of the gaseous peptide ions gave rise to a set of b and y-type fragment ions which enabled derivation of the sequence of the amino acids located outside the disulfide loop. Surprisingly, careful examination of the fragment-ion spectra of peptide ions comprising an intramolecular disulfide bridge revealed the presence of low-abundance fragment ions formed by the cleavage of peptide bonds within the disulfide loop. These fragmentations are preceded by proton-induced asymmetric cleavage of the disulfide bridge giving rise to a modified cysteine containing a disulfohydryl substituent and a dehydroalanine residue on the C-S cleavage site.     

Domoic acid--a new toxin in the Croatian Adriatic shellfish toxin profile

This is the first study that presents concentrations of domoic acid detected in the whole shellfish tissue from breeding and harvesting areas along the Croatian coast of the Adriatic Sea during the period 2006 to 2008. Shellfish sample analyses after SAX cleaning procedures, using a UV-DAD-HPLC system, showed the presence of domoic acid in four species. The most prevalent of those species were the blue mussel (Mytilus galloprovincialis), followed by European flat oyster (Ostrea edulis), Mediterranean scallop (Pecten jacobaeus) and proteus scallop (Flexopecten proteus). Domoic acid, a potentially lethal phycotoxin that causes amnesic shellfish poisoning (ASP), was detected for the first time in January 2006 with the highest value of 6.5486 μg g?1 in whole shellfish tissue. Pseudo-nitzschia spp. bloom events preceded these high domoic acid concentrations. According to this study, retention of domoic acid in the blue mussel M. galloprovincialis is more than 42 days. This investigation indicates the first presence of domoic acid in Croatian shellfish, but in concentrations under the regulatory limit (20 μg g?1), therefore shellfish consumption was not found to endanger human health.     

Lipophilic toxin profile in Mytilus galloprovincialis during episodes of diarrhetic shellfish poisoning (DSP) in the N.E. Adriatic Sea in 2006

Dinophysis spp. blooms and related shellfish toxicity events of diarrhetic shellfish poisoning (DSP) have been the most reported toxicity event through the Croatian National monitoring program. With the aim to characterize the DSP toxin profile in shellfish farmed in Croatia, for the first time a complete analysis of the toxin profile of Croatian mussels has been carried out using the LC-MS/MS technique. The obtained results showed okadaic acid (OA) as the main toxin contaminating Croatian mussels at that time. The maximum concentration of OA in shellfish tissue was recorded 12 days after the Dinophysis fortii bloom, thus suggesting that rapid growth of the toxin level in the shellfish occurred in the first week after the bloom while it was slower in the second week. Furthermore, the presence of only OA at concentrations which could endanger human health suggests D. fortii as the main organism responsible for the toxic event that occurred in Lim Bay. The presence of gymnodimine and spirolides in Croatian mussel has been detected for the first time, while the presence of yessotoxin and pectenotoxin-2 is confirmed.     

Use of nonspecific cleavage products for protein sequence analysis as shown on calcyclin isolated from human granulocytes

In this paper, analysis strategies developed for a sequencing problem concerning the identification of an S100 protein isolated from human granulocytes are discussed. The analysis of a trypsinized lyophilized sample suggested the presence of a number of peptides which are non-tryptic in origin. During purification of proteins from cell lysates nonspecific cleavage can be observed which may reflect biological processes and can become an unavoidable analytical problem. Current mass spectrometric software is evaluated for the analysis of nonspecific digests in this context. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), high-performance liquid chromatography (HPLC)-MS/MS, and selected ion monitoring (SIM)-MS/MS have been used for peptide analysis and in addition HPLC-MS was carried out for protein analysis leading to the detection of an N-terminal modification of the protein. The success of the study is mainly due to the careful investigation of nonspecific cleavage products. Data obtained from the routine mass spectrometric analysis of an in-gel-digest allowed the identification of this protein as S100 calcium-binding protein A6-calcyclin whose expression in granulocytes has not been described so far.