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Analysis of global DNA methylation levels in human blood using high-performance liquid ...

DNA methylation plays an important role in both normal physiological and disease processes. In this study, a sensitive and precise liquid chromatography-electrospray ionization tandem mass spectrometry method has been optimized for quantification of global DNA �methylation levels in human peripheral blood by using multiple reaction monitoring (MRM) mode. This method was then used to analyze age-dependent association of DNA methylation. The results showed that the limit of detection was 1 pg for 5-methyl-2′-dexycytidine (5mdC) (S/N = 5.6). The linear calibration curves with correlation coefficients higher than 0.9990 were ranged 0.75–10 ppm and 0.0375–0.5 ppm for 2′-dexycytidine (dC) and 5mdC, respectively. The relative standard derivatives (RSD) of inter-day, intra-day and parallel samples were less than 5%. The analysis results of the blood samples from 60 healthy individuals showed that the child group had the highest mean 5mdC content while the adult group had the lowest values.

High-throughput analysis of gangliosides in defined regions of fetal brain by fully automated ...

Gangliosides (GGs), a large group of sialylated glycosphingolipids, are considered biomarkers of human brain development, aging and certain diseases. Determination of individual GG components in complex mixtures extracted from a human brain represents a fundamental prerequisite for correlating their specificity with the specialized function of each brain area. In the context of modern glycomics, detailed investigation of GG expression and structure in human brain requires a continuous development and application of innovative methods able to improve the quality of data and speed of analysis. In this work, for the first time, a high-throughput mapping and sequencing of gangliosides in human fetal brain was performed by a novel mass spectrometry (MS)-based approach developed recently in our laboratory. Three GG mixtures extracted and purified from different regions of the same fetal brain in the 36^th gestational week: frontal neocortex (NEO36), white matter of the frontal lobe (FL36) and white matter of the occipital lobe (OL36) were subjected to comparative high-throughput screening and multi-stage fragmentation by fully automated chip-based nanoelectrospray ionization (nanoESI) high capacity ion trap (HCT) MS. Using this method, in only a few minutes of signal acquisitions, over 100 GG and asialo-GG species were detected and identified in the three mixtures. Obtained data revealed for the first time that differences in GG expression in human fetal brain are dependent on phylogenetic development rather than topographic factors. While a significant variation of GG distribution in NEO36 vs FL36 was observed, no significant differences in GG expression in white matter of frontal vs occipital lobe were detected. Additionally, the largest number of species was identified in NEO36, which correlates with the functional complexity of neocortex as the newest brain region. In the last stage of analysis, using MS2–MS3 molecular ion fragmentation at variable amplitudes, a NEO36- associated GD1b isomer could clearly be discriminated. Present results indicate that the combination of fully automated chipESI with HCT MSⁿ is able to provide ultra-fast, sensitive and reliable analyses of complex lipid-linked carbohydrates from which the pattern of their expression and structure in a certain type of bio-matrix can be determined.

Identification of the metabolites of ecabet bismuth in rat bile by ...

In the present study, a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method was developed for the screening and the structural elucidation of the metabolites of ecabet bismuth in rat bile. Solid-phase extraction cartridges were used for sample pre-treatment and a gradient liquid chromatographic system composed of 10 mM ammonium acetate buffer and methanol was used for chromatographic separation on a Phenomenex Kromasil C18 column. The triple quadrupole mass spectrometer was employed to thoroughly detect and acquire the detailed MS/MS spectra of ecabet and its metabolites. By comparing the chromatographic retention behaviors, as well as the changes in molecular weight and full-scan MS/MS spectra of the potential metabolites with those of the parent compound, two main metabolites were identified as glucuronide conjugate of carbonylated ecabet (7-oxo-ecabet) and glucuronide conjugate of ecabet. Both two metabolites have not been reported in the literatures. The metabolic pathways of ecabet in rat were also proposed in this paper.

Determination with matrix-assisted laser desorption/ionization tandem time-of- flight mass ...

Psalmopeotoxin I (PcFK1) is a 33-residue peptide isolated from the venom of the tarantula Psalmopoeus cambridgei. This peptide specifically inhibits the intra-erythrocyte stage of Plasmodium falciparum in vitro. It contains six cysteine residues forming three disulfide bridges and belongs to the superfamily of natural peptides containing the inhibitor cystine knot (ICK) fold. We produced the wild-type and mutated forms of the recombinant peptide to examine the mechanism of action of PcFK1. The purified toxins were consistently produced as two isobaric peptides (r-PcFK1-1 and r-PcFK1- 2) with different retention properties but identical anti-plasmodial �biological activity. Comparison of ¹⁵N-NMR heteronuclear single quantum correlation spectra revealed that although rPcFK1-1 was highly structured, rPcFK1-2 does not have a stable three-dimensional structure. We used high-energy collision-induced fragmentation of the peptides with a matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometer to further investigate the structure of the native peptides in its natural form and produced in E. coli. The fragmentation spectra of the native peptides were very complex due to the occurrence in the spectrum of ions resulting from (1) cross-linking of fragments through a disulfide bridge and (2) asymmetric fragmentations of the disulfide bridges and (3) multiple neutral losses. The tandem mass spectrometry fragmentation pattern of r-PcFK1-1 was similar to that of the natural peptide isolated from crude venom, but r-PcFK1-2 had a clearly distinct fragmentation pattern, more closely resembling the fragmentation spectra of reduced and alkylated peptides. Observed ions could be attributed to specific fragments by comparing spectra between the wild-type and selected variants with point mutations (Y11W, R20T, Y26W, K28V). The disulfide connections in r-PcFK1-2 differed from those of the native peptide and showed a rare disulfide bridge between vicinal cysteine residues. The r-PcFK1_(R20T) variant showed a very limited fragmentation pattern when analyzed in positive mode but displayed much more fragmentation in negative mode pointing out the importance of the R20 residue in the fragmentation of PcFK1. Using the reductive matrix 1,5-diaminonaphtalene promoted strongly in source decay fragmentation of the peptides in MS mode. Our findings illustrated the critical role of the electronic environment around the central Cys₁₈–Cys₁₉ doublet in PcFK1 in internal fragmentation of the peptide.

Doping control analysis of metamfepramone and two major metabolites using liquid ...

The sympathomimetic agent metamfepramone (2-dimethylamino-1- phenylpropan-1-one, dimethylpropion) is widely used for the treatment of the common cold or hypotonic conditions. Due to its stimulating properties and its rapid metabolism resulting in major degradation products such as methylpseudoephedrine and methcathinone, it has been considered relevant for doping controls by the World Anti-Doping Agency (WADA). The rapid degradation of the active drug complicates the detection of metamfepramone itself but the metabolites methylpseudoephedrine and methcathinone can be monitored, and the finding of the latter in particular allows the inference of a metamfepramone administration. In order to improve sports drug testing procedures, metamfepramone, methylpseudoephedrine and methcathinone were characterized using electrospray ionization-high resolution/high accuracy mass spectrometry, and a method employing liquid chromatography/tandem mass spectrometry was established that allowed the analysis of these three analytes by direct injection of 2 µL of urine specimens. The assay was validated with regard to specificity, lower limits of detection (2–10 ng mL^–1), intraday and interday precision (3–17%) and ion suppression/enhancement effects. The developed procedure has been used to verify or falsify suspicious signals observed in routine screening procedures based on gas chromatography/mass spectrometry and yielded an adverse analytical finding concerning a metamfepramone administration in an authentic doping control sample. Although the active drug was not detected, the indicative metabolites methylpseudoephedrine and methcathinone were considered sufficient to infer the application of the prohibited drug.

Differentiation of the isomeric o-(m- and p-)nitro-(chloro- and bromo-)benzyl-2,4-(and 2,1-) ...

Electron ionisation (EI) and fast atom bombardment (FAB) mass spectral fragmentations of nine 2,4-(and 2,1-) disubstituted o-(m- and p-)nitro-(chloro- and bromo-)-2-thiocytosinium halides are investigated. Fragmentation pathways, whose elucidation is assisted by accurate mass measurements and metastable transitions [EI-mass spectrometry (MS)], as well as FAB/collision-induced dissociation (CID) mass spectra measurements are discussed. The correlations between the abundances of the (C₁₁H₁₀N₄SO₂)⁺ 1–3; (C₁₁H₁₀N₃SCl)⁺ 4–6 and (C₁₁H₁₀N₃SBr)⁺ 7–9 ions and the selected fragment ions (EI-MS), as well as (C₁₈H₁₆N₅SO₄)⁺ 1–3; (C₁₈H₁₆N₃SCl₂)⁺ 4–6 and (C₁₈H₁₆N₃SBr₂)⁺ 7–9 ions and the selected ions (C₇H₆NO₂)⁺ 1–3; (C₇H₆Cl)⁺ 4–6; (C₇H₆Br)⁺ 7–9 (FAB-MS) are discussed. The data obtained can be used for distinguishing isomers.

Mass spectrometry of metal complexes of bis-quinolizidine alkaloids: electron ionization and ...

The electron ionization (EI) and fast atom bombardment (FAB) mass spectral fragmentations of ten copper(II) dichloride (dibromide, diformate, diacetate and dithiocyanate) complexes of (–)-sparteine and (–)-α-isosparteine were investigated. Fragmentation pathways, elucidation of which was assisted by accurate mass measurements and metastable transitions (EI-MS), as well as FAB/collision-induced dissociation (CID) mass spectral measurements are discussed. The data obtained create the basis for the differentiation of the ligand (sparteine or α-isosparteine) in the investigated complexes. The comparison of the results with those obtained previously for corresponding zinc(II) complexes forms the basis for the differentiation of metals in these compounds. The results show that both EI-MS and FAB-MS are very useful tools for the differentiation of ligands, as well as metals in the series of Zn(II) and Cu(II) dichloride (dibromide, diacetate, diformate, dithioacetate) complexes of (–)-sparteine and (–)-α-isosparteine.

Formation of organometallic species, [M – H]+ ions and radical cations upon mass ...

Mass spectrometric fragmentation pathways of [M + HgClO₄]⁺ (M – crown ether molecule), determined by tandem mass spectrometry experiments, are discussed in detail. The decomposition of [M + HgClO₄]⁺ proceeds along three fragmentation pathways: formation of [M – H]⁺ ions, formation of organometallic species, namely [M – H + Hg]⁺ ions, and formation of radical cations [M]^+•. The factors influencing these processes, namely crown ether cavities and the presence of electron withdrawing/electron donor groups, have been discussed.

Collision-induced dissociation of imidazolium-based zwitterionic liquids

Fragmentation pathways of some imidazolium based zwitterionic liquids—3-(3-alkyl-1- imidazolio)-propane sulfonates and 3-(2-methyl-3-alkyl-1-imidazolio)-propane sulfonates—have been studied by tandem electrospray mass spectrometry and collision- induced dissociation. The relative abundances of the lowest energy fragment ions depend on the length of the alkyl chain at the ^IIN of the imidazolium ring and the cone voltage. The first fragment ions originate from the scission of C_{non aromatic}–N bond of compounds investigated, but with increasing collision energy, scission of C–C bonds occurs. Aggregates of the general formula [(M + H)_x + (M)_y]⁺ (x;y = 1–2) formed. Methyl substituted zwitterionic liquids show higher molecular stability than 3-(3-alkyl-1-imidazolio)-propane sulfonates.

Unimolecular and collision-induced dissociation of singly-charged mono-bromide silver clusters ...

Relative intensities of singly-charged mono-bromide silver clusters Ag_xBr⁺ formed from sputtering of a pressed pellet of silver bromide were measured by mass spectrometry. The obtained results suggest that the Ag_xBr⁺ clusters have a structural formula of the form Ag_x–1⁺(AgBr). The relative stability of Ag_x–1⁺(AgBr) was determined by the intrinsic stability of the remaining metallic portion of the cluster (Ag_x–1⁺) as predicted by the spherical jellium model (SJM). Unimolecular and high-energy collision-induced dissociation (CID) spectra of Ag_xBr⁺ (x = 2, 4, 6, 8, 10) clusters were also measured. In all of the spectra, the most intense fragment peaks were assigned to the Ag_x–1⁺ ions accompanying the loss of AgBr. The difference in the relative intensities of the Ag_x–1⁺ peaks between unimolecular dissociation and CID spectra led us to conclude that the weakest bond in the excited cluster Ag_xBr^+* is the Ag_x–1⁺–AgBr bond and the structure of Ag_xBr⁺ is a metallic Ag_x–1⁺ ion cluster adduct with AgBr. The primary fragments observed in the CID spectra were also explained by the stabilities of the generated ion products and neutral fragments, both having even delocalized valence electrons. The present results were consistently explained by SJM. The dissociation behavior of Ag₂Br⁺ can be explained on the basis of the calculated thermochemical data.

The effects of entrance fields on the performance of quadrupole mass spectrometers

The theoretical performance of quadrupole mass spectrometers has been studied by means of a new computer model, with particular reference to three-dimensional ion trajectories, both within the main filter and the entrance fields, including the effects of z-energy changes in the latter. It is shown that considerable improvements in acceptance area can be obtained by suitable modification of the entrance fields.