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At least 1,693 records · Page 94Linked to original sources
PubMed2001

A strategy for quantitative bioanalysis of non-polar neutral compounds by liquid chromatography/electrospray ionization tandem mass spectrometry: determination of TS-962, a novel acyl-CoA:cholesterol acyltransferase inhibitor, in rabbit aorta and liver tissues.

A strategy for the sensitive and reliable quantitative determination of non-polar neutral compounds in biological matrices by liquid chromatography/electrospray ionization tandem mass spectrometry is described in the context of assay development for TS-962, a novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, in rabbit aorta and liver tissues. The electrospray ionization (ESI) mass spectrum of this compound with a mobile phase of water/acetonitrile did not give abundant [M + H]+ ions, but did give alkali metal cation adducts such as [M + Na]+, [M + CH3CN + Na]+ and [M + K]+ ions. The cationized species are acknowledged as unsuitable precursor ions for selected reaction monitoring (SRM) for various reasons, such as difficulty in obtaining characteristic product ions in low-energy collision-induced dissociation, and irreproducibility of the adduct-ion intensities. To overcome this problem, a solution of 3.4 mM trifluoroacetic acid in 2-propanol was added to the mobile phase as a postcolumn additive, resulting in a decrease of the undesirable adduct formation and significant enhancement of [M + H]+ ion intensity. An attempt was then made to prevent the matrix effect by employing a column-switching system, which allowed direct injection of a large volume of 2-propanolic tissue homogenate (950 microL) followed by sufficient clean-up, separation, and ESI-SRM on-line. This enabled development of a sensitive and reliable assay method for TS-962 in rabbit aorta and liver tissues in the concentration range of 5-500 ng/g wet tissue using a 25-mg aliquot of tissue sample. Application of this method to the determination of aortic TS-962 levels at 24 h after repeated oral administration of this compound (3 mg/kg) once a day for 12 weeks to 1% cholesterol-fed rabbits is also presented. Results showed that TS-962 is well distributed to both the thoracic and abdominal aorta tissues, at levels higher than the 50% inhibitory concentration value of this compound for microsomal ACAT activity from rabbit aorta.

Acetamides↗
PubMed2007

Rapid identification of C21 steroidal saponins in Cynanchum versicolor Bunge by electrospray ionization multi-stage tandem mass spectrometry and liquid chromatography/tandem mass spectrometry.

Electrospray ionization multi-stage tandem mass spectrometry (ESI-MSn) and liquid chromatography coupled with on-line electrospray ionization tandem mass spectrometry (LC/ESI-MSn) were performed to elucidate the clearage rule of nine investigated C21 steroidal saponins and identify them in the saponin fraction of 90% ethanolic extracts from the root and rhizome of Cynanchum versicolor Bunge. The fragments of C21 steroidal saponins in positive and negative ESI-MSn were used to deduce their mass spectral fragmentation mechanisms, and their structures were further confirmed by ESI-MSn in positive mode. The MSn spectra of the [M+Na]+ ions for saponins provided a wealth of structural information on glycosidic bond cleavage, which allowed a straightforward interpretation of spectra, with respect to the identifications of features such as the sequences of sugars attached to saponins and sugar type. By using LC/ESI-MSn, nine C21 steroidal saponins were detected in the saponin fraction of C. versicolor, and an isomer of atratoglaucoside A was elucidated simultaneously. All nine compounds showed an abundant ion for the loss of 46 Da (HCOOH) from [M+Na]+. The losses of monosaccharide sequences and aglycone as neutral fragmentation from [M+Na-HCOOH]+ were also acquired as the characteristic ions of these C21 steroidal saponins. It provided important information on monosaccharide sequences and in particular on sugar types and could be used to identify and elucidate other C21 steroidal saponins. These studies allowed us to rapidly identify C21 steroidal saponins from Radix cynanchi atrati. It is indicated that the described method had wide applicability to rapidly screen and provide structural confirmation on C21 steroidal saponins in crude materials.

Chromatography, High Pressure Liquid↗
PubMed2001

Automated molecular weight assignment of electrospray ionization mass spectra.

Process improvements in the synthesis of therapeutic agents and their intermediates are often facilitated by identification of reaction by-products. Analysis by liquid chromatography/mass spectrometry (LC/MS) with electrospray ionization is a powerful approach for obtaining molecular weight information for these compounds. Such analyses are well suited for 'open-access' mass spectrometry using generic chromatographic conditions, provided spectral interpretation for unknown compounds is facile. We have developed a software application (MassAssign) that facilitates automated data processing and molecular weight assignment for chromatographic peaks detected by any standard ultraviolet-visible wavelength detector. The program assigns [M + H](+) ions (and thus molecular weight) in the mass spectra using predetermined criteria. This evaluation process differentiates [M + H](+) ions from other signals in a complex mass spectrum such as those resulting from chromatographic coelution or the presence of multiple species (i.e., fragment ions, singly charged ions, doubly charged ions, adduct ions, proton-bound dimers, etc.). Once the program has evaluated all ions in a mass spectrum that exceed a preset abundance threshold, MassAssign reports either a numeric value-indicating the chromatographic peak consists of a single component having the displayed molecular weight, 'MC'-indicating the peak consisted of multiple components, or 'ND'-that a molecular weight could not be determined unequivocally. The performance of the program was evaluated by comparing mass assignments made by MassAssign against manual interpretation for 55 samples analyzed by positive electrospray ionization using a generic HPLC method. Correct molecular weight assignments were obtained in 90% of the cases.

Algorithms↗
PubMed2002

Determination of selected sulfonamide antibiotics and trimethoprim in manure by electrospray and atmospheric pressure chemical ionization tandem mass spectrometry.

A method for the determination of sulfonamides and trimethoprim in the complex matrix liquid manure has been developed using reversed-phase liquid chromatography and atmospheric pressure chemical ionization (APCI) tandem mass spectrometry. A comparison was made between electrospray and atmospheric pressure chemical ionization. APCI proved to be more robust and less sensitive to matrix effects. High-performance liquid chromatographic (HPLC) separation of the analytes was achieved in less than 7 min. The compounds were extracted with ethyl acetate and the extracts were cleaned up by solid-phase extraction on an aminopropyl column. Recoveries were not dependent on the concentration level. The mean recoveries were as follows: trimethoprim 79.0%, sulfadiazine 80.5%, N(4)-acetylsulfadiazine 91.0%, sulfamerazine 78.6%, sulfadimidine 77.2% and sulfamethoxazole 82.8%. Linearity was established over a concentration range of 5 to 5000 microg/kg with correlation coefficients greater than 0.99. The method had a limit of quantitation (LOQ) of 5 microg/kg manure.

Animals↗
PubMed2002

Electrospray ionization mass spectrometric studies of phosphorus oxychloride directed synthesis of homo-oligopeptide ester libraries.

With the assistance of phosphorus oxychloride, alpha-amino acids were assembled into homo-peptides, which were analyzed by electrospray ionization mass spectrometry (ESI-MS) and multistage electrospray ionization mass spectrometry (ESI-MS/MS). On quenching with water or various alcohols, the reaction mixtures yielded the corresponding peptides or peptide esters, respectively. This paper reports a simple method to synthesize the homo-oligo-peptide-ester conjugated library by phosphorus oxychloride activation.

Amino Acids↗
PubMed2002

New example of proline-induced fragmentation in electrospray ionization mass spectrometry of peptides.

The positive ion electrospray ionization (ESI+) mass spectra of peptides usually display only protonated molecules provided that soft ionization conditions are applied (low cone voltage to prevent in-source dissociations). Such ions can be multiply charged depending on the molecular weight of the studied compounds. We have experienced an unexpected behavior during the ESI analysis of a modified peptide of relatively high mass (3079 Da). A specific fragmentation occurred even under soft energetic conditions, leading to a mass spectrum containing multiply charged molecular and fragment ions. The selective rupture involved the amide bond between the glutamic acid and proline residues (E-P sequence). The successive replacement of each amino acid by an alanine residue (positional scanning study) was undertaken to assess which part of the sequence induced such selective and abundant fragmentation on multiply charged species. The succession P-P was evidenced as the minimum unit giving rise to the first peptide bond rupture in the sequence X-P-P. Any acidic amino acid at the X position (X = D, E) favored the fragmentation by an intramolecular interaction. Such proline-induced fragmentation occurring readily in the source differed from the literature data on the specific behavior of proline-containing peptides where bond ruptures occur solely in dissociation conditions.

Amino Acids↗
PubMed2003

Analysis of ceramides in cosmetics by reversed-phase liquid chromatography/electrospray ionization mass spectrometry with collision-induced dissociation.

A rapid, sensitive and selective method involving reversed-phase liquid chromatography (LC) with electrospray ionization (ESI) mass spectrometry (MS) was employed for determination of commercial ceramides in cosmetics for quality control of the product formulation. Using this LC/ESI-MS technique, simultaneous separation and characterization of ceramides and an impurity substance were possible. Informative fragmentation patterns were obtained by employing LC/ESI-MS in both positive and negative ionization modes to identify the structures of both sphingoid base and N-acyl chains of ceramides, and also of an impurity. The combination of positive and negative mass spectra can be used for unambiguous confirmation of ceramides and for characterization of unknown species. In-source collision-induced fragmentation resulted in characteristic product anions for the ceramides containing a phytosphingosine moiety at m/z 267, 255 and 225, and for those with a sphingosine moiety at m/z 263 and 237, regardless of the length of the fatty acyl chains. The detection limit was about 0.5 pmol in selected-ion monitoring mode. Quantification using internal standards showed good linearity and a relative standard deviation of 4%. These ceramides were more sensitively detected in positive than in negative ion mode.

Calibration↗
PubMed2003

Heuristic charge assignment for deconvolution of electrospray ionization mass spectra.

We propose a new algorithm for deconvolution of electrospray ionization mass spectra based on direct assignment of charge to the measured signal at each mass-to-charge ratio (m/z). We investigate two heuristics for charge assignment: the entropy-based heuristic is adapted from a deconvolution algorithm by Reinhold and Reinhold;10 the multiplicative-correlation heuristic is adapted from the multiplicative-correlation deconvolution algorithm of Hagen and Monnig.6 The entropy-based heuristic is insensitive to overestimates of z(max), the maximum ion charge. We test the deconvolution algorithm on two single-component samples: the measured spectrum of human beta-endorphin has two prominent and one very weak line whereas myoglobin has a well-developed quasi-gaussian envelope of 17 peaks. In both cases, the deconvolution algorithm gives a clean deconvoluted spectrum with one dominant peak and very few artefacts. The relative heights of the peaks due to the parent molecules in the deconvoluted spectrum of a mixture of two peptides, which are expected to ionize with equal efficiency, give an accurate measure of their relative concentration in the sample.

Algorithms↗
PubMed2003

A novel nanoflow interface for atmospheric pressure ionization mass spectrometry.

A novel spray-ionization technique for nanoflow liquid chromatography/mass spectrometry (nLC/MS) has been developed by modifying the sonic spray ionization (SSI) technique. A solution from a tapered fused-silica capillary is sprayed by a gas flow coaxial to the capillary, and ions produced are analyzed with an ion-trap mass spectrometer. The ion intensity is shown to have a steep threshold at a low gas velocity and to be much less dependent on the gas velocity than that of conventional SSI, in which the ion intensity is strongly dependent on the gas velocity and reaches its maximum at sonic velocity. Thus, we conclude that the concentration of charge in the solution at the tapered capillary tip with an inner diameter of 15 microm is almost at saturation so that charged droplets are produced from the solution by electrical force, rather than by sheer stress due to the gas flow. The ions are readily produced from these charged droplets. Preliminary results are compared with results obtained with a miniaturized electrospray unit.

Bradykinin↗
PubMed1997

Damage-sensing mechanisms in human cells after ionizing radiation.

Human cells have evolved several mechanisms for responding to damage created by ionizing radiation. Some of these responses involve the activation or suppression of the transcriptional machinery. Other responses involve the downregulation of enzymes, such as topoisomerase I, which appear to be necessary for DNA repair or apoptosis. Over the past five years, many studies have established links between DNA damage, activation of transcription factors that are coupled to DNA repair mechanisms, increased gene transcription and altered cell cycle regulation to allow for repair or cell death via apoptosis or necrosis. Together these factors determine whether a cell will survive with or without carcinogenic consequences. The immediate responses of human cells to ionizing radiation, in terms of sensing and responding to damage, are therefore, critical determinants of cell survival and carcinogenesis.

Animals↗
PubMed1996

Analysis of carotenoids and carotenol fatty acid esters by matrix-assisted laser desorption ionization (MALDI) and MALDI-post-source-decay mass spectrometry.

Matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry and MALDI post-source-decay (PSD) fragment ion mass analysis were employed to examine various model carotenoids and some of their fatty acid esters. It was demonstrated that the use of MALDI helps overcome problems resulting from the inherent thermal instability and lack of solubility which render this group of compounds rather difficult for conventional ionization techniques. Detection limits were in the subpicomolar range. Rather abundant metastable fragmentation occurred under conditions of prompt extraction, but was significantly restricted under conditions of delayed extraction (DE). In DE the quasimolecular ion signals (mainly as odd electron radical ions) became the base peak even in spectra of the most delicate fatty acid esters. PSD fragment ion patterns were similar to those recorded under linked B/E scanning in conventional fast atom bombardment-MS and contained information on structural end group substituents such as hydroxyl, epoxide, or carbonyl functions and on the extent of double bond conjugations. The mono- and bis-fatty acid esters fragmented mainly by simple fatty acid cleavage but also furnished some of the end group-specific fragments.

Carotenoids↗
PubMed1998

Determination of diethylpyrocarbonate-modified amino acid residues in alpha 1-acid glycoprotein by high-performance liquid chromatography electrospray ionization-mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry.

The chemical modification reagent diethylpyrocarbonate (DEPC) was used to modify alpha 1-acid glycoprotein (orosomucoid, OMD) under various conditions. The extents of DEPC modification of the histidine and tyrosine residues were followed by UV spectrophotometry. The resulting modified OMD was analyzed using enzyme digestion, reverse-phase HPLC, electrospray ionization-mass spectrometry (ESI/MS), and matrix-assisted laser desorption ionization time-of-flight-mass spectrometry (MALDI-TOF/MS). The inherent problem of instability of DEPC-modified histidine residues was overcome by adjusting the time scale of the postreaction processing of modified OMD. There were observed differences in reactivity of histidine 97 and histidine 100 that were consistent throughout the pH range 6-8. Furthermore, several lysine residues were modified and the amount of modification increased over the pH range 6-8. These experiments show that HPLC-ESI/MS and MALDI-TOF/MS analysis coupled with enzyme digestion provide the necessary information to describe the reaction of DEPC with OMD. In addition, the results provide the carbethoxy-histidine stability and histidine reactivity information of DEPC-modified OMD necessary for the design of experiments to characterize the drug binding properties of OMD.

Amino Acid Sequence↗
PubMed1998

Nonradioactive phosphopeptide assay by matrix-assisted laser desorption ionization time-of-flight mass spectrometry: application to calcium/calmodulin-dependent protein kinase II.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) was used to quantify the phosphopeptide produced by calcium/calmodulin-dependent protein kinase II (CaMK II). MALDI-TOF measurements were performed in a linear and positive ion mode with delayed extraction excited at various laser powers and at different sampling positions, i.e., different loci of laser illumination. We find that the ratio of the peak area of the substrate (S) to that of its monophosphorylated form (SP) for a given mixture is constant, independent of the laser powers and/or of the sample loci illuminated by the laser. We also find that the fraction of phosphorylation determined by MALDI-TOF, or fMALDI-TOF, is proportionally smaller than that determined by HPLC, or fHPLC; the ratio fMALDI-TOF/fHPLC was 0.797 +/- 0.0229 (99% confidence limit, n = 7) for a 30-mer peptide substrate used in this study. A low mass gate, which turns off the detector temporarily, improved the ratio fMALDI-TOF/fHPLC to 0.917 +/- 0.0184 (99% confidence limit, n = 7). Our interpretation of this result is that the reduction of the phosphopeptide peak in the MALDI-TOF measurement is likely to be caused by a temporal loss of detector function rather than by a lower efficiency of ionization for the phosphopeptide compared with its parent species. In these measurements the experimental errors, up to the 50% phosphorylation state, were less than 5%. After an adjustment made based on the fMALDI-TOF/fHPLC ratio of 0.917, MALDI-TOF gave an accurate measurement for the kinetics of the CaMK II phosphorylation reaction. Since only a small volume of the reaction mixture, typically containing 3 to 50 pmol of substrate, is required for the MALDI-TOF measurement, this method can be adapted to a nonradioactive microscale assay for CaMK II and also for other protein kinases.

Amino Acid Sequence↗
PubMed2001

Homogeneous preparations of 3'-phosphoglycolate-terminated oligodeoxynucleotides from bleomycin-treated DNA as verified by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

Single- and double-strand breaks bearing 3'-phosphoglycolate termini are among the most frequent lesions formed in DNA by ionizing radiation and other oxidative mutagens. In order to obtain homogeneous preparations of defined 3'-phosphoglycolate substrates for repair studies, 5'-(32)P-end-labeled partial duplex DNAs were treated with bleomycin, and individual cleavage products were isolated from polyacrylamide gels. The fragments were then treated with alkaline phosphatase and further purified by reverse-phase HPLC. Electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry of the purified oligomers produced molecular ions of the expected masses, with no detectable contaminants. Gas-phase sequencing by tandem mass spectrometry of these single species yielded the expected sequence ions and confirmed the presence of phosphoglycolate on the 3'-terminal fragments only. The fragments could be relabeled with polynucleotide kinase to yield highly purified, high-specific-activity substrates for repair studies.

Alkaline Phosphatase↗
PubMed2001

Quantitative determination of saccharide surfactants in protein samples by liquid chromatography coupled to electrospray ionization mass spectrometry.

A direct and highly selective method, combining liquid chromatography (LC) with electrospray ionization mass spectrometry (ESI-MS), has been developed for quantifying saccharide surfactants. Saccharide surfactants, such as n-octyl-beta-d-glucopyranoside (NOG), are widely used to solubilize or refold membrane-bound or lipophilic proteins. In the present study, we have developed an LC-MS method to quantify NOG in protein samples. Protein-bound NOG was completely dissociated from proteins by reversed-phase LC, allowing the total amount of saccharide surfactant in protein samples to be quantified by MS. A chemical analog of NOG was used as an internal standard for improving the reproducibility of the method. Linearity was found in the range of 10 microg/mL-1.0 mg/mL NOG concentrations. Seven major surfactant oligomeric ions were detected under the ionization conditions applied and their relative abundance was essentially unchanged over the range of 0.05-1.0 mg/mL NOG concentrations. Consequently, ions with characteristic mass-to-charge ratios could be used for quantification of NOG. Analytical accuracy of the method was examined by determining the amounts of NOG recovered from apolipoprotein A-I and myoglobin samples spiked with NOG.

Animals↗
PubMed2001

Liquid chromatographic-electrospray ionization-mass spectrometric analysis of cytochrome P450 metabolites of arachidonic acid.

Arachidonic acid (AA) can be metabolized by cytochrome P450 (CYP) enzymes to many biologically active compounds including 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs), their corresponding dihydroxyeicosatrienoic acids (DHETs), and 20-hydroxyeicosatetraenoic acid (20-HETE). These eicosanoids are potent regulators of vascular tone. We developed a liquid chromatography-electrospray ionization-mass spectrometry method to simultaneously determine 5,6-, 8,9-, 11,12-, and 14,15-EETs; 5,6-, 8,9-, 11,12-, and 14,15-DHETs; and 20-HETE. [2H8]EETs, [2H8]DHETs, and [2H2]20-HETE were used as internal standards. These compounds are readily separated on a C18 reverse-phase column using water:acetonitrile with 0.005% acetic acid as a mobile phase. The internal standards, [2H8]EETs, [2H8]DHETs, and [2H2]20-HETE, eluted slightly faster than the natural eicosanoids. The samples were ionized by electrospray with fragmentor voltage of 120 V and detected in a negative mode. The negative ion detection gave a lower background than the positive ion detection for these compounds. These eicosanoids exhibited high abundance of the ions corresponding to [M - 1]-. The m/z = 319, 337, and 319 ions were used for quantitation of EETs, DHETs, and 20-HETE, respectively. The detection limits using selected ion monitoring of these compounds are about 1 pg per injection. The position of functional groups and water content of mobile phase had a significant effect on the sensitivity of detection. Water content of 40% was found to give maximal sensitivity. The method was used to determine EETs, DHETs, and 20-HETE in bovine coronary artery endothelial cells, dog plasma, rat astrocytes, and rat kidney microsome samples.

Animals↗
PubMed1997

The proto-oncogene c-fos mediates apoptosis in murine T-lymphocytes induced by ionizing radiation and dexamethasone.

Expression of the immediate early response gene c-fos is induced by several cellular and extracellular stress factors including ionizing radiation. We examined the role of c-fos in mediating stress-induced apoptosis of isogenic CD4+ and CD8+ mouse T-lymphocytes differing only in their c-fos status after treatment with ionizing radiation and the synthetic glucocorticoid dexamethasone. The amount of radiation-induced apoptosis was decreased (up to 37%) in the T-lymphocyte population derived from the knockout mice lacking endogenous c-fos compared to the wildtype T-lymphocyte population. The difference in apoptosis induction in T-lymphocytes from wildtype and c-fos knockout mice was even more prominent (up to 55%) after dexamethasone treatment. Comparative experiments were performed with T-lymphocytes from isogenic mouse littermates differing only in the status of the tumor-suppressor gene p53. Whereas p53 plays a primary role in radiation-induced apoptosis, our results suggest that c-fos enhances both p53-dependent radiation- and p53-independent steroid-induced apoptosis in T-lymphocytes.

Animals↗
PubMed2001

Identification of respiratory complexes I and III as mitochondrial sites of damage following exposure to ionizing radiation and nitric oxide.

In 32D cl 3 hematopoietic progenitor cells, the overexpression of manganese superoxide dismutase (MnSOD, SOD2), the enzyme normally found in mitochondria, protects against the damaging effects of ionizing radiation. In the presence of a nitric oxide donor, which exacerbates the damage, inhibition of mitochondrial function can be demonstrated to be associated with respiratory complexes I (NADH dehydrogenase) and III (cytochrome c reductase), but not II (succinate dehydrogenase), IV (cytochrome c oxidase), or V (ATP synthase). The same pattern of inhibition is observed in the case of isolated bovine heart mitochondria exposed to ionizing radiation and the nitric oxide donor. The addition of authentic peroxynitrite (ONO2(-)) to isolated mitochondria also results in damage to complexes I and III (but not II, IV, and V), as shown by assays of electron-transfer activities and electron paramagnetic resonance (EPR) spectroscopic measurements, suggesting ONO2(-) to be responsible for most of the observed radiation damage in both the cultured cell lines and isolated mitochondria. It is argued that, in general, production of ONO2(-) is an important contributor to radiation damage in biological systems and the implications of these findings in relation to possible mechanisms of oxidant-linked apoptosis are briefly considered.

Adenosine Diphosphate↗