Automatic internal calibration in liquid chromatography/Fourier transform ion cyclotron resonance mass spectrometry of protein digests

Accurately measured peptide masses can be used for large-scale protein identification from bacterial whole-cell digests as an alternative to tandem mass spectrometry (MS/MS) provided mass measurement errors of a few parts-per-million (ppm) are obtained. Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) routinely achieves such mass accuracy either with internal calibration or by regulating the charge in the analyzer cell. We have developed a novel and automated method for internal calibration of liquid chromatography (LC)/FTICR data from whole-cell digests using peptides in the sample identified by concurrent MS/MS together with ambient polydimethyl-cyclosiloxanes as internal calibrants in the mass spectra. The method reduced mass measurement error from 4.3 +/- 3.7 ppm to 0.3 +/- 2.3 ppm in an E. coli LC/FTICR dataset of 1000 MS and MS/MS spectra and is applicable to all analyses of complex protein digests by FTICRMS. Copyright (c) 2006 John Wiley & Sons, Ltd.

Pressure-jump X-ray studies of liquid crystal transitions in lipids

In this paper, we give an overview of our studies by static and time-resolved X-ray diffraction of inverse cubic phases and phase transitions in lipids. In 1, we briefly discuss the lyotropic phase behaviour of lipids, focusing attention on non-lamellar structures, and their geometric/topological relationship to fusion processes in lipid membranes. Possible pathways for transitions between different cubic phases are also outlined. In 2, we discuss the effects of hydrostatic pressure on lipid membranes and lipid phase transitions, and describe how the parameters required to predict the pressure dependence of lipid phase transition temperatures can be conveniently measured. We review some earlier results of inverse bicontinuous cubic phases from our laboratory, showing effects such as pressure-induced formation and swelling. In 3, we describe the technique of pressure-jump synchrotron X-ray diffraction. We present results that have been obtained from the lipid system 1:2 dilauroylphosphatidylcholine/lauric acid for cubic-inverse hexagonal, cubic-cubic and lamellar-cubic transitions. The rate of transition was found to increase with the amplitude of the pressure-jump and with increasing temperature. Evidence for intermediate structures occurring transiently during the transitions was also obtained. In 4, we describe an IDL-based 'AXCESS' software package being developed in our laboratory to permit batch processing and analysis of the large X-ray datasets produced by pressure-jump synchrotron experiments. In 5, we present some recent results on the fluid lamellar-Pn3m cubic phase transition of the single-chain lipid 1-monoelaidin, which we have studied both by pressure-jump and temperature-jump X-ray diffraction. Finally, in 6, we give a few indicators of future directions of this research. We anticipate that the most useful technical advance will be the development of pressure-jump apparatus on the microsecond time-scale, which will involve the use of a stack of piezoelectric pressure actuators. The pressure-jump technique is not restricted to lipid phase transitions, but can be used to study a wide range of soft matter transitions, ranging from protein unfolding and DNA unwinding and transitions, to phase transitions in thermotropic liquid crystals, surfactants and block copolymers.

Liquid matrices for analyses by UV-MALDI mass spectrometry

Data are presented for a pH-adjustable liquid UV-matrix-assisted laser desorption ionization (MALDI) matrix for mass spectrometry analysis. The liquid matrix system possesses high analytical sensitivity within the same order of magnitude as that achievable by the commonly used solid UV-MALDI matrices such as 2,5-dihydroxybenzoic acid but with improved spot homogeneity and reproducibility. The pH of the matrix has been adjusted by the addition of up to 0.35% trifluoroacetic acid and up to 200 mM ammonium bicarbonate, achieving an on-target pH range of 3.5-8.6. Alteration of the pH does not seem to affect the overall sample signal intensity or signal-to-noise ratio achievable, nor does it affect the individual peptide ion signals from a mixture of peptides with varying isoelectric points (p1). In addition, the pH adjustment has allowed for the performance of a tryptic digest within the diluted pH-optimized liquid matrix.

Analysis of 'APN' naphthenic acids by high temperature gas chromatoagraphy and high performance liquid chromatography

Examination by high temperature GC (HTGC) of the methyl esters of the so-called 'ARN' naphthenic acids from crude oils of North Sea UK, Norwegian Sea and West African oilfields revealed the distributions of resolved 4-8 ring C-80 tetra acids and trace amounts of other acids. Whilst all three oils contained apparently the same the proportions of each differed, possibly reflecting the growth tempe acids, ratures of the archaebacteria from which the acids are assumed to have originated. The structures of the 4, 5, 7 and 8 ring acids are tentatively assigned by comparison with the known 6 ring acid and related natural products and an HPLC method for the isolation of the individual acids is described. ESI-MS of individual acids isolated by preparative HPLC established the elution order of the 4-8 ring acids on the HPLC and HTGC systems and revealed the presence of previously unreported acids tentatively identified as C-81 and C-82 7 and 8 ring analogues.

Development of a liquid chromatographic time-of-flight mass spectrometric method for the determination of unlabelled and deuterium-labelled alpha-tocopherol in blood components

A method is described for the analysis of deuterated and undeuterated alpha-tocopherol in blood components using liquid chromatography coupled to an orthogonal acceleration time-of-flight (TOF) mass spectrometer. Optimal ionisation conditions for undeuterated (d0) and tri- and hexadeuterated (d3 or d6) alpha-tocopherol standards were found with negative ion mode electrospray ionisation. Each species produced an isotopically resolved single ion of exact mass. Calibration curves of pure standards were linear in the range tested (0-1.5 muM, 0-15 pmol injected). For quantification of d0 and d6 in blood components following a standard solvent extraction, a stable-isotope-labelled internal standard (d3-alpha-tocopherol) was employed. To counter matrix ion suppression effects, standard response curves were generated following identical solvent extraction procedures to those of the samples. Within-day and between-day precision were determined for quantification of d0- and d6-labelled alpha-tocopherol in each blood component and both averaged 3-10%. Accuracy was assessed by comparison with a standard high-performance liquid chromatography (HPLC) method, achieving good correlation (r(2) = 0.94), and by spiking with known concentrations of alpha-tocopherol (98% accuracy). Limits of detection and quantification were determined to be 5 and 50 fmol injected, respectively. The assay was used to measure the appearance and disappearance of deuterium-labelled alpha-tocopherol in human blood components following deuterium-labelled (d6) RRR-alpha-tocopheryl acetate ingestion. The new LC/TOFMS method was found to be sensitive, required small sample volumes, was reproducible and robust, and was capable of high throughput when large numbers of samples were generated. Copyright (C) 2003 John Wiley Sons, Ltd.

Vacuum-induced bubble formation in liquid-tempered chocolate

Bubble inclusion is one of the fastest growing operations practiced in the food industry. A variety of aerated foods is currently available in supermarkets, and newer products are emerging all the time. This paper aims to combine knowledge on chocolate aeration with studies performed on bubble formation and dispersion characteristics. More specifically, we have investigated bubble formation induced by applying vacuum. Experimental methods to determine gas hold-up (volume fraction of air), bubble section distributions along specific planes, and chocolate rheological properties are presented. This study concludes that decreasing pressures elevate gas hold-up values due to an increase in the number of bubble nuclei being formed and release of a greater volume of dissolved gases. Furthermore, bubbles are observed to be larger at lower pressures for a set amount of gas because the internal pressure needs to be in equilibrium with the surrounding pressures. Temperature-induced changes to the properties of the chocolate have less of an effect on bubble formation. On the other hand, when different fats and emulsifiers are added to a standard chocolate recipe, milk fat was found to increase, significantly, the gas hold-up values and the mean bubble-section diameters. It is hypothesized that this behavior is related to the way milk fats, which contain different fatty acids to cocoa butter, crystallize and influence the setting properties of the final product. It is highlighted that apparent viscosity values at low shear rate, as well as setting behavior, play an important role in terms of bubble formation and entrainment.

An optimized reverse-phase high performance liquid chromatographic method for evaluating percutaneous absorption of glucosamine hydrochloride

A relatively simple, selective, precise and accurate high performance liquid chromatography (HPLC) method based on a reaction of phenylisothiocyanate (PITC) with glucosamine (GL) in alkaline media was developed and validated to determine glucosamine hydrochloride permeating through human skin in vitro. It is usually problematic to develop an accurate assay for chemicals traversing skin because the excellent barrier properties of the tissue ensure that only low amounts of the material pass through the membrane and skin components may leach out of the tissue to interfere with the analysis. In addition, in the case of glucosamine hydrochloride, chemical instability adds further complexity to assay development. The assay, utilising the PITC-GL reaction was refined by optimizing the reaction temperature, reaction time and PITC concentration. The reaction produces a phenylthiocarbarnyl-glucosamine (PTC-GL) adduct which was separated on a reverse-phase (RP) column packed with 5 mu m ODS (C-18) Hypersil particles using a diode array detector (DAD) at 245 nm. The mobile phase was methanol-water-glacial acetic acid (10:89.96:0.04 v/v/v, pH 3.5) delivered to the column at 1 ml min(-1) and the column temperature was maintained at 30 degrees C Using a saturated aqueous solution of glucosamine hydrochloride, in vitro permeation studies were performed at 32 +/- 1 degrees C over 48 h using human epidermal membranes prepared by a heat separation method and mounted in Franz-type diffusion cells with a diffusional area 2.15 +/- 0.1 cm(2). The optimum derivatisation reaction conditions for reaction temperature, reaction time and PITC concentration were found to be 80 degrees C, 30 min and 1 % v/v, respectively. PTC-Gal and GL adducts eluted at 8.9 and 9.7 min, respectively. The detector response was found to be linear in the concentration range 0-1000 mu g ml(-1). The assay was robust with intra- and inter-day precisions (described as a percentage of relative standard deviation, %R.S.D.) < 12. Intra- and inter-day accuracy (as a percentage of the relative error, %RE) was <=-5.60 and <=-8.00, respectively. Using this assay, it was found that GL-HCI permeates through human skin with a flux 1.497 +/- 0.42 mu g cm(-2) h(-1), a permeability coefficient of 5.66 +/- 1.6 x 10(-6) cm h(-1) and with a lag time of 10.9 +/- 4.6 h. (c) 2005 Elsevier B.V. All rights reserved.

A pilot randomised, open, uncontrolled, clinical study of two dosages of St John's wort (Hypericum perforatum) herb extract (LI-160) as an aid to motivational/behavioural support in smoking cessation

There is an association between smoking and depression, yet the herbal antidepressant St John's wort (Hypericum perforatum L.: SJW) herb extract has not previously been investigated as an aid in smoking cessation. In this open, uncontrolled, pilot study, 28 smokers of 10 or more cigarettes per day for at least one year were randomised to receive SJW herb extract (LI-160) 300mg once or twice daily taken for one week before and continued for 3 months after a target quit date. In addition, all participants received motivational/behavioural support from a trained pharmacist. At 3 months, the point prevalence and continuous abstinence rates were both 18%, and at 12 months were 0%. Fifteen participants (54%) reported 23 adverse events up to the end of the 3-month follow-up period. There was no statistically significant difference in the frequency of adverse events for participants taking SJW once or twice daily (p > 0.05). Most adverse events were mild, transient and non-serious. This preliminary study has not provided convincing evidence that a SJW herb extract plus individual motivational/behavioural support is likely to be effective as an aid in smoking cessation. However, it may be premature to rule out a possible effect on the basis of a single, uncontrolled pilot study, and other approaches involving SJW extract may warrant investigation.

Individual variation in psychological and psychomotor symptoms following smoking cessation: the implications for treatment

Some commonly experienced signs and symptoms occur during abstinence from tobacco, but specific signs and symptoms and their intensity vary greatly from individual to individual. The aim of this study was to re-examine psychological and psychomotor symptoms in smokers in the general population, and to explore the individual variation in these. Quitting smokers (n = 123) reported their experiences pre- and post-cessation, on a questionnaire developed for the study. Analysis of variance and frequency analysis showed significant decreases between pre- and post-cessation on positive experiences (F = 9.81, p < 0.0001) but no significant change on negative experiences, suggesting a loss of pleasure rather than increased negative affect upon quitting. The variance of the pre- to post-cessation difference score suggested wide variation in the reporting of withdrawal symptoms. These results lead us to consider the implications for treatment, using cognitive therapies and moderating the significant emphasis that is at present put on withdrawal.

Attribution and the effects of expectancy: how beliefs can influence the experiences of smoking cessation

Research on smoking cessation has found consistencies and similarities during abstinence, but also that the specific signs and symptoms and their intensity vary greatly from individual to individual. One possible source of this variation is the cognitions associated with quitting. We investigated the experiences and associated cognitions in normal cessation by asking quitting smokers to rate their experiences on a questionnaire and to indicate the most likely reason for each experience. Statistical analyses confirmed that attributions to abstinence were significantly higher for increased negative experiences, and there were significantly more reattributions than would be found by chance for items associated with smoking abstinence. Significantly more attributions to abstinence were made by clinic attendees and significantly more attributions of negative experiences to abstinence were made by unaided quitters using self-help materials. These results can be interpreted in the context of attribution theory; quitters may use the cognitions available to them to attribute their negative experiences to quitting. Consequently, counsellors could use cognitive therapy to alter their clients' expectations and explanations of their experiences, and emphasise the positive outcomes of cessation. (C) 2002 Elsevier Science Ltd. All rights reserved.

Investigation of liquid MALDI and optimization for instrument tuning and quantitative measurements

The success of Matrix-assisted laser desorption / ionisation (MALDI) in fields such as proteomics has partially but not exclusively been due to the development of improved data acquisition and sample preparation techniques. This has been required to overcome some of the short comings of the commonly used solid-state MALDI matrices such as - cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB). Solid state matrices form crystalline samples with highly inhomogeneous topography and morphology which results in large fluctuations in analyte signal intensity from spot to spot and positions within the spot. This means that efficient tuning of the mass spectrometer can be impeded and the use of MALDI MS for quantitative measurements is severely impeded. Recently new MALDI liquid matrices have been introduced which promise to be an effective alternative to crystalline matrices. Generally the liquid matrices comprise either ionic liquid matrices (ILMs) or a usually viscous liquid matrix which is doped with a UV lightabsorbing chromophore [1-3]. The advantages are that the droplet surface is smooth and relatively uniform with the analyte homogeneously distributed within. They have the ability to replenish a sampling position between shots negating the need to search for sample hot-spots. Also the liquid nature of the matrix allows for the use of additional additives to change the environment to which the analyte is added.

Liquid crystal phase formation by biopolymers

This review discusses liquid crystal phase formation by biopolymers in solution. Lyotropic mesophases have been observed for several classes of biopolymer including DNA, peptides, polymer/peptide conjugates, glycopolymers and proteoglycans. Nematic or chiral nematic (cholesteric) phases are the most commonly observed mesophases, in which the rod-like fibrils have only orientational order. Hexagonal columnar phases are observed for several systems (DNA, PBLG, polymer/peptide hybrids) at higher concentration. Lamellar (smectic) phases are reported less often, although there are examples such as the layer arrangement of amylopectin side chains in starch. Possible explanations for the observed structures are discussed. The biological role of liquid crystal phases for several of these systems is outlined. Commonly, they may serve as a template to align fibrils for defined structural roles when the biopolymer is extruded and dried, for instance in the production of silk by spiders or silkworms, or of chitin in arthropod shells. In other cases, liquid crystal phase formation may occur in vivo simply as a consequence of high concentration, for instance the high packing density of DNA within cell nuclei.

Small-angle x-ray scattering study of flow alignment of a thermotropic liquid crystal in the nematic and smectic phases

WThe capillary flow alignment of the thermotropic liquid crystal 4-n-octyl-4′-cyanobiphenyl in the nematic and smectic phases is investigated using time-resolved synchrotron small-angle x-ray scattering. Samples were cooled from the isotropic phase to erase prior orientation. Upon cooling through the nematic phase under Poiseuille flow in a circular capillary, a transition from the alignment of mesogens along the flow direction to the alignment of layers along the flow direction (mesogens perpendicular to flow) appears to occur continuously at the cooling rate applied. The transition is centered on a temperature at which the Leslie viscosity coefficient α3 changes sign. The configuration with layers aligned along the flow direction is also observed in the smectic phase. The transition in the nematic phase on cooling has previously been ascribed to an aligning-nonaligning or tumbling transition. At high flow rates there is evidence for tumbling around an average alignment of layers along the flow direction. At lower flow rates this orientation is more clearly defined. The layer alignment is ascribed to surface-induced ordering propagating into the bulk of the capillary, an observation supported by the parallel alignment of layers observed for a static sample at low temperatures in the nematic phase.