Direct observation of covalent adducts with Cys34 of human serum albumin using mass spectrometry

The interactions of the unpaired thiol residue (Cys34) of human serum albumin (HSA) with low-molecular-weight thiols and an Au(I)-based antiarthritic drug have been examined using electrospray ionization mass spectrometry. Early measurements of the amount of HSA containing Cys34 as the free thiol suggested that up to 30% of circulating HSA bound cysteine as a mixed disulfide. It has also been suggested that reaction of HSA with cysteine, occurs only on handling and storage of plasma. In our experiments, there were three components of HSA in freshly collected plasma from normal volunteers, HSA, HSA + cysteine, and HSA + glucose in the ratio similar to50:25:25. We addressed this controversy by using iodoacetamide to block the free thiol of HSA in fresh plasma, preventing its reaction with plasma cysteine. When iodoacetamide was injected into a vacutaner tube as blood was collected, the HSA was modified by iodoacetamide, with 20-30% present as the mixed disulfide with cysteine (HSA + cys). These data provide strong evidence that 20-30% of HSA in normal plasma contains one bound cysteine. Reaction of HSA with [Au(S2O3)(2)](3-) resulted in formation of the adducts HSA + Au(S2O3) and HSA + Au. Reaction of HSA with iodoacetamide prior to treatment with [Au(S2O3)(2)](3-) blocked the formation of gold adducts. (C) 2003 Elsevier Inc. All rights reserved.

Formation of mononuclear and chloro-bridged binuclear copper(II) complexes of patellamide D, a naturally occurring cyclic peptide: influence of anion and solvent

Patellamide D (patH(4)) is a cyclic octapeptide isolated from the ascidian Lissoclinum patella. The peptide possesses a 24-azacrown-8 macrocyclic structure containing two oxazoline and two thiazole rings, each separated by an amino acid. The present spectrophotometric, electron paramagnetic resonance (EPR) and mass spectral studies show that patellamide D reacts with CuCl, and triethylamine in acetonitrile to form mononuclear and binuclear copper(II) complexes containing chloride. Molecular modelling and EPR studies suggest that the chloride anion bridges the copper(II) ions in the binuclear complex [Cu-2(patH(2))(mu-Cl)](+). These results contrast with a previous study employing both base and methanol, the latter substituting for chloride in the copper(II) complexes en route to the stable mu-carbonato binuclear copper(II) complex [Cu-2 (patH(2))(mu-CO3)]. Solvent clearly plays an important role in both stabilising these metal ion complexes and influencing their chemical reactivities. (C) 2004 Elsevier Inc. All rights reserved.

Enhancing the ratio of molecular ions to non-covalent compounds in the electrospray interface of LC-MS in quantitative analysis

A common problem encountered during the development of MS methods for the quantitation of small organic molecules by LGMS is the formation of non-covalently bound species or adducts in the electrospray interface. Often the population of the molecular ion is insignificant compared to those of all other forms of the analyte produced in the electrospray, making it difficult to obtain the sensitivity required for accurate quantitation. We have investigated the effects of the following variables: orifice potential, nebulizer gas flow, temperature, solvent composition and the sample pH on the relative distributions of ions of the types MH+, MNa+, MNH+, and 2MNa(+), where M represents a 4 small organic molecule: BAY 11-7082 ((E)-3-[4-methylphenylsulfonyl]-2-propenenitrile). Orifice potential, solvent composition and the sample pH had the greatest influence on the relative distributions of these ions, making these parameters the most useful for optimizing methods for the quantitation of small molecules.

A recombinant diheme SoxAX cytochrome - Implications for the relationship between EPR signals and modified heme-ligands

The multiheme SoxAX proteins are notable for their unusual heme ligation (His/Cys-persulfide in the SoxA subunit) and the complexity of their EPR spectra. The diheme SoxAX protein from Starkeya novella has been expressed using Rhodobacter capsulatus as a host expression system. rSoxAX was correctly formed in the periplasm of the host and contained heme c in similar amounts as the native SoxAX. ESI-MS showed that the full length rSoxA, in spite of never having undergone catalytic turnover, existed in several forms, with the two major forms having masses of 28 687 +/- 4 and 28 718 +/- 4 Da. The latter form exceeds the expected mass of rSoxA by 31 4 Da, a mass close to that of a sulfur atom and indicating that a fraction of the recombinant protein contains a cysteine persulfide modification. EPR spectra of rSoxAX contained all four heme-dependent EPR signals (LS1a, LS1b, LS2, LS3) found in the native SoxAX proteins isolated from bacteria grown under sulfur chemolithotrophic conditions. Exposure of the recombinant SoxAX to different sulfur compounds lead to changes in the SoxA mass profile as determined by ESI while maintaining a fully oxidized SoxAX visible spectrum. Thiosulfate, the proposed SoxAX substrate, did not cause any mass changes while after exposure to dimethylsulfoxide a + 112 +/- 4 Da form of SoxA became dominant in the mass spectrum. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Late pleistocene to holocene composite speleothem O-18 and C-13 chronologies from south island, new Zealand-did a global younger dryas really exist?

Oxygen and carbon data from eight stalagmites from northwest South Island are combined to produce composite records of delta(18)O and delta(13)C from 23.4 ka to the present. The chronology is anchored by 43 thermal ionization mass spectrometry (TIMS) uranium series ages. Delta O-18 values are interpreted as having a first order positive relationship to temperature, but also to be influenced by precipitation in a complex manner. Delta C-13 is interpreted as responding negatively to increases in atmospheric CO, concentration, biological activity and precipitation amount. Six climatic phases are recognized. After adjustment of 1.2parts per thousand for the ice volume effect, the delta(18)O record between 23 and 18 ka varies around -3.72parts per thousand compared to the Holocene average of -3.17parts per thousand. Late-glacial warming commenced between 18.2 and 17.8 ka and accelerated after 16.7 ka, culminating in a positive excursion between 14.70 and 13.53 ka. This was followed by a significant negative excursion between 13.53 and 11.14 ka of up to 0.55parts per thousand depth that overlapped the Antarctic Cold Reversal (ACR) and spanned the Younger Dryas (YD). Positive delta(18)O excursions at 11.14 ka and 6.91-6.47 ka represent the warmest parts of the Holocene. The mid-Holocene from 6 to 2 ka was marked by negative excursions that coincide with increased glacial activity in the South Island. A short positive excursion from 0.71 to 0.57 ka was slightly later than the Medieval Warm Period of Europe. Delta C-13 values were high until 17.79 ka after which there was an abrupt decrease to 17.19 ka followed by a steady decline to a minimum at 10.97 ka. Then followed a general increase, suggesting a drying trend, to 3.23 ka followed by a further general decline. The abrupt decrease in delta-values after 17.79 ka probably corresponds to an increase in atmospheric CO2 concentration, biological activity and wetness at the end of the Last Glaciation, but the reversal identified in the delta(18)O record from 13.53 to 11.14 ka was not reflected in delta(13)C changes. The lowest delta(13)C values coincided with the early Holocene climatic suboptimum when conditions were relatively wet as well as mild. Major trends in the delta(18)O(c) record are similar to the Northern Hemisphere, but second order detail is often distinctly different. Consequently, at the millennial scale, a more convincing case can be made for asymmetric climatic response between the two hemispheres rather than synchronicity. (C) 2004 Elsevier B.V. All rights reserved.

Taxonomy of Australian Funnel-web spiders using rp-HPLC/ESI-MS profiling techniques

The complex nature of venom from spider species offers a unique natural source of potential pharmacological tools and therapeutic leads. The increased interest in spider venom molecules requires reproducible and precise identification methods. The current taxonomy of the Australian Funnel-web spiders is incomplete, and therefore, accurate identification of these spiders is difficult. Here, we present a study of venom from numerous morphologically similar specimens of the Hadronyche infensa species group collected from a variety of geographic locations in southeast Queensland. Analysis of the crude venoms using online reversed-phase high performance liquid chromatography/electrospray ionisation mass spectrometry (rp-HPLC/ESI-MS) revealed that the venom profiles provide a useful means of specimen identification, from the species level to species variants. Tables defining the descriptor molecules for each group of specimens were constructed and provided a quick reference of the relationship between one specimen and another. The study revealed that the morphologically similar specimens from the southeast Queensland region are a number of different species/species variants. Furthermore, the study supports aspects of the current taxonomy with respect to the H. infensa species group. Analysis of Australian Funnel-web spider venom by rp-HPLC/ESI-MS provides a rapid and accurate method of species/species variant identification. (c) 2006 Elsevier Ltd. All rights reserved.

Mechanism and kinetics of dithiobenzoate-mediated RAFT polymerization. I. The current situation

Investigations into the kinetics and mechanism of dithiobenzoate-mediated Reversible Addition-Fragmentation Chain Transfer (RAFT) polymerizations, which exhibit nonideal kinetic behavior, such as induction periods and rate retardation, are comprehensively reviewed. The appreciable uncertainty in the rate coefficients associated with the RAFT equilibrium is discussed and methods for obtaining RAFT-specific rate coefficients are detailed. In addition, mechanistic studies are presented, which target the elucidation of the fundamental cause of rate retarding effects. The experimental and theoretical data existing in the literature are critically evaluated and apparent discrepancies between the results of different studies into the kinetics of RAFT polymerizations are discussed. Finally, recommendations for further work are given. (c) 2006 Wiley Periodicals, Inc.

Simultaneous determination of morphine, oxycodone, morphine-3-glucuronide, and noroxycodone concentrations in rat serum by high performance liquid chromatography-electrospray ionization-tandem mass spectrometry

An assay using high performance liquid chromatography (HPLC)-electrospray ionization-tandem mass spectrometry (ESI-MS-MS) was developed for simultaneously determining concentrations of morphine, oxycodone, morphine-3-glucuronide, and noroxycodone, in 50 mul samples of rat serum. Deuterated (d(3)) analogues of each compound were used as internal standards. Samples were treated with acetonitrile to precipitate plasma proteins: acetonitrile was removed from the supernatant by centrifugal evaporation before analysis. Limits of quantitation (ng/ml) and their between-day accuracy and precision (%deviation and %CV) were-morphine, 3.8 (4.3% and 7.6%); morphine-3-glucuronide, 5.0 (4.5% and 2.9%); oxycodone, 4.5 (0.4% and 9.3%); noroxycodone, 5.0 (8.5% and 4.6%). (C) 2004 Elsevier B.V. All rights reserved.

Measurement and stability of FTY720 in human whole blood by high-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry

We report here a validated method for the quantification of a new immunosuppressant drug FTY720, using HPLC-tandem mass spectrometry. Whole blood samples (500 mu l) were subjected to liquid-liquid extraction, in the presence of an internal standard (Y-32919). Mass spectrometric detection was by selected reaction monitoring with an atmospheric pressure chemical ionization source in positive ionization mode (FTY720: m/z 308.3 -> 255.3). The assay was linear from 0.2 to 25 mu g/l (r(2) > 0.997, n = 5). The inter- and intra-day analytical recovery and imprecision for quality control samples (0.5, 7 and 15 mu g/l) were 95.8-103.2 and < 5.5%, respectively. At the lower limit of quantification (0.2 mu g/l) the interand intra-day analytical recovery was 99.0-102.8% with imprecision of < 7.6% (n = 5). The assay had a mean relative recovery of 100.5 +/- 5.8% (n = 15). Extracted samples were stable for 16 h. IFTY720 quality control samples were stable at room temperature for 16 h at 4 degrees C for at least 8 days and when taken through at least three freeze-thaw cycles. In conclusion, the method described displays analytical performance characteristics that are suitable for pharmacokinetic studies in humans. (c) 2006 Elsevier B.V. All rights reserved.

A rapid HPLC-mass spectrometry cyclosporin method suitable for current monitoring practices

Objectives: Cyclosporin is an immunosuppressant drug with a narrow therapeutic window. Trough and 2-h post-dose blood samples are currently used for therapeutic drug monitoring in solid organ transplant recipients. The aim of the current study was to develop a rapid HPLC-tandem mass spectrometry (HPLC-MS) method for the measurement of cyclosporin in whole blood that was not only suitable for the clinical setting but also considered a reference method. Methods: Blood samples (50 mu L) were prepared by protein precipitation followed by C-18 solid-phase extraction while using d(12) cyclosporin as the internal standard. Mass spectrometric detection was by selected reaction monitoring with an electrospray interface in positive ionization mode. Results: The assay was linear from 10 to 2000 mu g/L (r(2) > 0.996, n = 9). Inter-day,analytical recovery and imprecision using whole blood quality control samples at 10, 30, 400, 1500, and 2000 mu g/L were 94.9-103.5% and

Matrix effects: The Achilles heel of quantitative high-performance liquid chromatography-electrospray-tandem mass spectrometry

High-performance liquid chromatography coupled by an electrospray ion source to a tandem mass spectrometer (HPLC-EST-MS/ MS) is the current analytical method of choice for quantitation of analytes in biological matrices. With HPLC-ESI-MS/MS having the characteristics of high selectivity, sensitivity, and throughput, this technology is being increasingly used in the clinical laboratory. An important issue to be addressed in method development, validation, and routine use of HPLC-ESI-MS/MS is matrix effects. Matrix effects are the alteration of ionization efficiency by the presence of coeluting substances. These effects are unseen in the chromatograrn but have deleterious impact on methods accuracy and sensitivity. The two common ways to assess matrix effects are either by the postextraction addition method or the postcolumn infusion method. To remove or minimize matrix effects, modification to the sample extraction methodology and improved chromatographic separation must be performed. These two parameters are linked together and form the basis of developing a successful and robust quantitative HPLC-EST-MS/MS method. Due to the heterogenous nature of the population being studied, the variability of a method must be assessed in samples taken from a variety of subjects. In this paper, the major aspects of matrix effects are discussed with an approach to address matrix effects during method validation proposed. (c) 2004 The Canadian Society of Clinical Chemists. All rights reserved.

The survey for ionization in neutral gas galaxies. I. Description and initial results

We introduce the Survey for Ionization in Neutral Gas Galaxies (SINGG), a census of star formation in H I selected galaxies. The survey consists of H alpha and R-band imaging of a sample of 468 galaxies selected from the H I Parkes All Sky Survey (HIPASS). The sample spans three decades in H I mass and is free of many of the biases that affect other star-forming galaxy samples. We present the criteria for sample selection, list the entire sample, discuss our observational techniques, and describe the data reduction and calibration methods. This paper focuses on 93 SINGG targets whose observations have been fully reduced and analyzed to date. The majority of these show a single emission line galaxy (ELG). We see multiple ELGs in 13 fields, with up to four ELGs in a single field. All of the targets in this sample are detected in H alpha, indicating that dormant (non-star-forming) galaxies with M-H I greater than or similar to 3x10(7) M-circle dot are very rare. A database of the measured global properties of the ELGs is presented. The ELG sample spans 4 orders of magnitude in luminosity (H alpha and R band), and H alpha surface brightness, nearly 3 orders of magnitude in R surface brightness and nearly 2 orders of magnitude in H alpha equivalent width (EW). The surface brightness distribution of our sample is broader than that of the Sloan Digital Sky Survey (SDSS) spectroscopic sample, the EW distribution is broader than prism-selected samples, and the morphologies found include all common types of star-forming galaxies (e.g., irregular, spiral, blue compact dwarf, starbursts, merging and colliding systems, and even residual star formation in S0 and Sa spirals). Thus, SINGG presents a superior census of star formation in the local universe suitable for further studies ranging from the analysis of H II regions to determination of the local cosmic star formation rate density.

The survey for ionization in neutral gas galaxies. II. The star formation rate density of the local universe

We derive observed H alpha and R-band luminosity densities of an H I-selected sample of nearby galaxies using the SINGG sample to be l'(H alpha) = (9.4 +/- 1.8) x 10(38) h(70) ergs s(-1) Mpc(-3) for H alpha and l'(R) = (4.4 +/- 9.7) x 10(37) h(70) ergs s(-1) angstrom(-1) Mpc(-3) in the R band. This R-band luminosity density is approximately 70% of that found by the Sloan Digital Sky Survey. This leads to a local star formation rate density of log ((rho)over dot(SFR) [M-circle dot yr(-1) Mpc(-3)]) = -1.80(-0.07)(+0.13)(random) +/- 0.03(systematic) + log (h(70)) after applying a mean internal extinction correction of 0.82 mag. The gas cycling time of this sample is found to be t(gas) = 7.5(-2.1)(+1.3) Gyr, and the volume-averaged equivalent width of the SINGG galaxies is EW(H alpha) = 28.8(-4.7)(+7.2) angstrom (21.2-3.5+4.2 angstrom without internal dust correction). As with similar surveys, these results imply that (rho)over dot(SFR)(z) decreases drastically from z similar to 1.5 to the present. A comparison of the dynamical masses of the SINGG galaxies evaluated at their optical limits with their stellar and H I masses shows significant evidence of downsizing: the most massive galaxies have a larger fraction of their mass locked up in stars compared with H I, while the opposite is true for less massive galaxies. We show that the application of the Kennicutt star formation law to a galaxy having the median orbital time at the optical limit of this sample results in a star formation rate decay with cosmic time similar to that given by the. (rho)over dot(SFR)(z) evolution. This implies that the (rho)over dot(SFR)(z) evolution is primarily due to the secular evolution of galaxies, rather than interactions or mergers. This is consistent with the morphologies predominantly seen in the SINGG sample.