A capillary electrophoresis and off-line capillary electrophoresis/electrospray ionization-quadrupole time of flight-tandem mass spectrometry approach for ganglioside analysis

A systematic study for the optimization and implementation of high-performance capillary electrophoresis (HPCE) in conjunction with negative ion electrospray ionization-quadrupole time of flight-tandem mass spectrometry (ESI-QTOF-MS/MS) for the analysis of complex glycolipids is described. The performance of the capillary electrophoresis (CE) and off-line CE/ESI-QTOF-MS approach has been explored for screening a complex ganglioside mixture from bovine brain. All instrumental and solution parameters demonstrated to require special adjustment and to have the most substantial effect on the CE separation, abundance of product ions produced in a low-energy collision-induced dissociation (CID) process and their detection by MS/MS, when attempting to identify and sequence single ganglioside molecular species from CE eluted fractions. Upon optimization of the experimental parameters, an efficient methodology emerged providing the general basic requirements for combined CE/ESI-MS analysis of this type of complex glycoconjugate.     

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.     

Sequencing of tri- and tetraantennary N-glycans containing sialic acid by negative mode ESI QTOF tandem MS

Application of the negative mode electrospray ionization-quadrupole time-of-flight mass spectrometry (ESI QTOF) tandem MS for determination of substitution patterns by sialic acid and/or fucose and extention by additional LacNAc disaccharide units in single branches of multianternary N-glycans from biological samples is described. Fragmentation patterns which can be obtained by low energy collision-induced dissociation (CID) using the QTOF instrument include cleavage ions, diagnostic for determination of antennarity and for specific structural features of single antennae. Systematic fragmentation studies in the negative ion mode were focussed toward formation of the D diagnostic ion relevant for assignment of 3- and 6-antennae in complex N-glycans carrying three and four antennae in combination with epitope-relevant B- and C-type ions. For validation of this approach ESI QTOF fragmentation of the permethylated analogues was carried out in the positive ion mode. Using this strategy, products of in vitro glycosylation reactions were investigated in order to clarify some general aspects of N-glycan acceptor specificity during biosynthesis. Alpha1-3fucosylation using GDP-fucose along with a soluble form of the recombinant human alpha1-3fucosyltransferase VI was carried out on tri- and tetraantennary precursors to test structural requirements for formation of Le(x) versus sLe(x) motifs.     

Glycoscreening by on-line sheathless capillary electrophoresis/electrospray ionization-quadrupole time of flight-tandem mass spectrometry

An analytical approach based on sheathless on-line coupling of capillary electrophoresis (CE) and electrospray ionization (ESI) quadrupole time-of-flight (QTOF) mass spectrometry (MS) has been developed for providing new insight into the characterization of carbohydrate mixtures. The home-built sheathless CE/     

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).

     

Sequential Determination of Inorganic Cations and Anions in Cerebrospinal Fluid by Microchip Electrophoresis

Analysis of inorganic ions in cerebrospinal fluid (CSF) is used mainly in the diagnostics of central nervous system diseases, such as Alzheimer’s disease or multiple sclerosis. A new analytical method for fast determination of inorganic cations (ammonium, calcium, magnesium, sodium and potassium) and anions (chloride, sulfate, nitrite and nitrate) in CSF on an electrophoretic microchip was developed in this context. Zone electrophoresis (ZE) separations were performed on the microchip with coupled channels (CC) and contact conductivity detection. Two different propionate background electrolytes were used for the sequential determination of cations at pH 3.1 and anions at pH 4.3. ZE was used for the determination of cationic constituents while ZE–ZE approach was employed for the determination of chloride in the first separation channel on the CC microchip and other anionic micro-constituents in the second channel. LOD values were in the range of 0.003–0.012 mg L⁻¹ and 0.019–0.047 mg L⁻¹ for cations and anions, respectively. Repeatability of migration time was up to 1.2 % for both cations and anions. Repeatability of peak area ranged from 0.3 to 5.6 % for cations and from 0.6 to 6.0 % for anions. Recovery of both cations and anions was in the range 90–106 %. CSF samples were only diluted appropriately without other sample pretreatment prior to analysis. Developed sequential method is suitable for fast determination of the studied cations and anions in CSF with total analysis time <15 min.

     

Evaluation of Bookkeeper mass deacidification based on historical book papers

Cellulose - Bookkeeper, the most widely used deacidification process based on MgO particles, was systematically evaluated on two sets of nonvaluable historical paper samples. Established analytical...