Cavity enhanced absorption spectroscopy of multiple trace gas species using a supercontinuum radiation source.

Supercontinuum radiation sources are attractive for spectroscopic applications owing to their broad wavelength coverage, which enables spectral signatures of multiple species to be detected simultaneously. Here we report the first use of a supercontinuum radiation source for broadband trace gas detection using a cavity enhanced absorption technique. Spectra were recorded at bandwidths of up to 100 nm, encompassing multiple absorption bands of H(2)O, O(2) and O(2)-O(2). The same instrument was also used to make quantitative measurements of NO(2) and NO(3). For NO(3) a detection limit of 3 parts-per-trillion in 2 s was achieved, which corresponds to an effective 3sigma sensitivity of 2.4 x 10(-9) cm(-1)Hz(-1/2). Our results demonstrate that a conceptually simple and robust instrument is capable of highly sensitive broadband absorption measurements.

Aroma release and delivery following the consumption of beverages.

Processes controlling aroma release and delivery during and after the consumption of a beverage were studied using real-time physiological and aroma release measurements. The key processes were as follows. During swallowing, a portion of the buccal gas phase was transferred first to the throat and then to the nasal passages via the tidal breath flow. This mechanism accounted for the sharp pulse of aroma seen at the beginning of the swallow breath and on subsequent swallows. The persistence effect was due to liquid-air partition from beverage coated on the throat and was dependent on the concentration of volatile compounds in the beverage. Lipid in the beverage caused a decrease in the intensity of volatile compounds on the breath, but the presence of a thickening agent had no effect on persistence.

A novel precursor ion discovery method on a hybrid quadrupole orthogonal acceleration time-of-flight (Q-TOF) mass spectrometer for studying protein phosphorylation.

A tandem quadrupole time-of-flight (Q-TOF) mass spectrometer has been programmed such that phosphorylated peptides can automatically be discovered and identified in a way similar to that of the use of precursor ion or neutral loss scanning, but without the need to scan the quadrupole mass filter. Instead, the method capitalizes on the innate capability of the Q-TOF to record mass spectra and product ion spectra quickly, with good sensitivity and with good mass accuracy. Alternate mass spectra, with and without fragmentation, are recorded at high and low collision energy with the quadrupole operating in wideband mode. The method of analysis is both compatible with and dependant on liquid chromatography for separation of complex mixtures. The method has been demonstrated by searching for the neutral loss of 98 Da (H3PO4) from phosphoserine and phosphothreonine residues, or for the phosphorylated immonium ion at m/z 216 from phosphotyrosine. The method also incorporates acquisition of the product ion spectrum from any candidate precursor ions, thereby allowing confirmation of the neutral loss or product ion and providing additional sequence information to assist identification of the protein and assign the site of phosphorylation.

Should we give detailed advice and information booklets to patients with back pain? A randomized controlled factorial trial of a self-management booklet and doctor advice to take exercise for back pain.

STUDY DESIGN: Randomized controlled factorial trial. OBJECTIVE: To assess the effectiveness of a booklet and of physician advice to take regular exercise. SUMMARY OF BACKGROUND DATA: Educational booklets are one of the simplest interventions for back pain but have not been shown to alter pain and function. Although there is evidence that advice to mobilize is effective, doctors have also been advised to encourage regular exercise-but there is no evidence that such advice alone improves outcomes. METHOD: Eight doctors from six practices randomized 311 patients with a new episode of back pain using sealed numbered opaque envelopes to receive a detailed self-management booklet, advice to take regular exercise, both, or neither. All groups were advised to mobilize and to use simple analgesia. Patients were telephoned during the first week after entry into the study, and after 3 weeks to assess a validated numerical pain/function score (0 = no pain normal activities to 100 = extreme pain no normal activities). Patients also returned a postal questionnaire in the first week with the Aberdeen pain and function scale, a knowledge score, and a reliable satisfaction scale (mean score of 4 items: 0 = not satisfied to 100 = extremely satisfied). RESULTS: Pain/function scores were obtained in 239 (77%) patients. There were interactions between exercise and booklet groups for both pain/function scores and the Aberdeen scale, which are unlikely to have been chance findings (P = 0.009 and P = 0.012, respectively). In comparison with the control group, there were reductions in the pain/function score in the first week with a booklet (-8.7, 95% CI -17.4 to -0.03) or advice to exercise (-7.9; -16.7 to 0.8) but much less effect with both together (-0.08, -9.0 to 8.9). Similarly, the Aberdeen scale was lower in the booklet group (-3.8, -7.7 to 0.07) and in the exercise advice group (-5.3; -9.3 to -1.38) but much less with both combined (-1.9, -5.8 to 2.1). There was no significant difference between groups in pain/function scores by week 3, when 58% reported being back to normal. Satisfaction was increased in booklet (7.9, 1.3 to 14.4) and exercise groups (7.4, 0.8 to 13.9)), and a booklet also increased knowledge (Kruskal-Wallis chi2 27.2, P = 0.001). CONCLUSION: Doctors can increase satisfaction and moderately improve functional outcomes in the period immediately after the consultation when back pain is worst, by using very simple interventions: either by endorsing a self-management booklet or by giving advice to take exercise. Previous studies suggest that simple advice and the same written information provide reinforcement. This study supports evidence that it may not be helpful to provide a detailed information booklet and advice together, where the amounts or formats of information differ.

A new approach for dynamics of enzyme-catalyzed glutathione conjugation by electrospray quadrupole/time-of-flight mass spectrometry.

The dynamics of enzyme-catalyzed glutathione conjugation was studied by electrospray quadrupole/time-of-flight (Q-TOF) mass spectrometry with a nanospray interface. After incubation of human glutathione S-transferase A1-1 (GT) with glutathione (GSH) and an electrophilic substrate, electrospray indicated the presence of enzyme/product adducts such as [2GT + product], [2GT + GSH' + product], and [2GT + 2 products] as well as [2GT] and [2GT + GSH']. The relative abundance of GT/product adduct ions increased with incubation time. The wide m/z range of detection (m/z 300-5000) allowed the observation of product, suggested to be released from enzyme/product adducts, in the same mass spectrum. The noncovalent complexes of GT/product were completely replaced by GT/inhibitor complexes following the addition of GT inhibitor to the incubation mixture. Furthermore, a collision-activated decomposition analysis of these ion species provided us with useful information to interpret or identify ion species. The results suggest that electrospray Q-TOF mass spectrometry is a powerful approach for studying the dynamics of the enzyme reaction as well as the structure of enzyme complexes at high sensitivity.

Analysis of urinary nucleosides. II. Comparison of mass spectrometric methods for the analysis of urinary nucleosides.

Qualitative and quantitative analyses of urinary nucleosides have diagnostic potential as tumour markers. We have developed separation techniques linked to mass spectrometric detection in order to overcome the problems associated with past identification and quantitation methods. The three methods of analysis utilised were: gas chromatography/mass spectrometry (GC/MS), high-performance liquid chromatography/ion-trap mass spectrometry (HPLC/ITMS) and capillary liquid chromatography/triple quadrupole mass spectrometry (CapLC/TQMS). Here we compare the relative effectiveness of each of the techniques for subsequent application in the systematic study of urinary nucleoside profiles in cancer patients. All three methods proved to be valuable techniques for such urinary nucleoside analyses, and a combination rather than one single choice is concluded as the ideal.

Proteome study of colorectal carcinogenesis.

Development of cancer is a complex process involving multiple changes in gene expression. To unravel these alterations, a proteome approach aimed at the identification of qualitative and quantitative changes in protein composition, including their post-translational modifications, attracts great attention. Our study was focused on the identification of proteins whose amount is altered in the course of malignant transformation of colon mucosa. Proteins extracted from tissue specimens or cell lysates were separated by two-dimensional gel electrophoresis (2-DE). Comparative analyses of 2-DE protein patterns were done using computerized image analysis. Selected proteins exhibiting statistically significant abundance alterations comparing healthy and diseased tissues were identified by mass spectrometry. Globally, we have found 57 proteins that exhibited either a significant decrease or increase in amount in pathological tissues, and 18 of these were annotated by mass spectrometry. The alterations in the expression of nine proteins were common for both precancerous and neoplastic tissues suggesting their role in colon tumorigenesis. The epithelial origin of all identified spots was checked in two cell lines Caco-2 and DLD-1 originating from well-differentiated and poorly differentiated colon carcinoma, respectively.

Facile sequencing of oligosaccharides by matrix-assisted laser desorption/ionisation on a hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer.

N-Linked oligosaccharide mixtures released from a number of standard glycoproteins were derivatised with 3-acetylamino-6-acetylaminoacridine (AA-Ac) using reductive amination. Analysis of these mixtures using an experimental matrix-assisted laser desorption/ionisation (MALDI) hybrid quadrupole orthogonal acceleration time-of-flight (Q-TOF) mass spectrometer provided detailed information about the mass distribution of the glycan derivatives. Collision-induced dissociation of the singly protonated [M + H](+) ions also gave rise to a number of product ions produced by the sequential cleavage of the glycosidic linkages. As fragmentation of the positively charged species occurred predominantly in one direction, i.e., from the non-reducing end of the glycan to the AA-Ac moiety, a considerable amount of information could be obtained with ease about the sequence in which the sugar residues were attached to one another. This derivatisation procedure and mass spectrometric methodology were applied successfully to neutral and acidic glycans released from proteins separated by gel electrophoresis.

Identification of proteins in human prostate tumor material by two-dimensional gel electrophoresis and mass spectrometry.

Protein patterns in cells collected from benign prostatic tissues and prostate carcinomas were analyzed using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. Polypeptide expression was evaluated by computer-assisted image analysis (PDQUEST). Proteins expressed by prostate tumors were identified via in-gel digestion and subsequent matrix-assisted laser desorption/ionization mass spectrometry. In addition to cytoskeletal and mitochondrial proteins, a 40-kDa protein was identified as prostatic acid phosphatase (PAP). PAP expression decreased approximately twofold between benign and malignant tissue. Increased expression of heat shock protein 70 and decreased expression of tropomyosin 1 were also observed in the malignant tissue. The analysis of prostate material by two-dimensional gel electrophoresis and mass spectrometry shows that particular proteins are of interest as markers of disease.

O-glycan analysis of natural human neutrophil gelatinase B using a combination of normal phase-HPLC and online tandem mass spectrometry: implications for the domain organization of the enzyme.

Gelatinase B is a matrix metalloproteinase (MMP-9) expressed under strict control by many cell types including neutrophils, monocytes, macrophages, and tumor cells. MMP-9 is a key mediator in the physiological maintenance of the extracellular matrix both in tissue remodeling and development, while uncontrolled enzyme activity contributes to pathologies such as cancer and inflammation. Neutrophils release MMP-9 from granules in response to IL-8 stimulation. Human MMP-9 has three potential N-linked glycosylation sites and contains a Ser/Pro/Thr rich domain, known as the type V collagen-like domain, which is expected to be heavily O-glycosylated. Indeed, approximately 85% of the total sugars on human neutrophil MMP-9 are O-linked. This paper presents the detailed analysis of picomole amounts of these O-glycans using a novel HPLC-based strategy for O-glycan analysis that provides linkage and arm specific information in addition to monosaccharide sequence. The initial structural assignments were confirmed using HPLC with online MS/MS fragmentation analysis. Twelve sugars were identified that contained from two to nine monosaccharide residues. Most of these contained type 2 core structures with Galbeta1-4GlcNAc (N-acetyl lactosamine) extensions, with or without sialic acid or fucose. The O-glycans were modeled using the oligosaccharide structural database. On the basis of the structure of gelatinase A (MMP-2), a model of MMP-9 suggests that the type V collagen-like domain in gelatinase B is located on a loop remote from the active site. Fourteen potential O-glycosylation sites are multiply presented on this loop of 52 amino acids. Many of the O-glycans identified contain terminal galactose residues that may provide recognition epitopes. Importantly, heavy glycosylation of this loop region, absent in gelatinase A, has considerable implications for the domain organization of MMP-9.

Noncovalent associations of glutathione S-transferase and ligands: a study using electrospray quadrupole/time-of-flight mass spectrometry.

Human glutathione S-transferase A1-1 was observed predominantly as dimeric ions (51 kDa) during electrospray mass spectrometric analysis from aqueous solution at pH 7.4, in keeping with the known dimeric structure in solution. When analyses were performed on solutions of the enzyme containing glutathione (GSH), noncovalent adducts of protein dimer and one or two ligand molecules were observed; each mass increment, which exceeded the mass of GSH alone, was provisionally interpreted to indicate concomitant association of two water molecules per bound GSH. Noncovalent adducts of ligand and protein dimer were similarly observed for oxidized glutathione and for two glutathione inhibitors, both incorporating substituted thiol structures. In these instances, the mass increments exactly matched the ligand masses, suggesting that the apparent concomitant binding of water was associated with the presence in the ligand of a free thiol group. Collisionally activated decomposition during tandem mass spectrometry analyses of noncovalent adducts incorporating protein dimer and ligands yielded initially the denuded dimer; at higher collision energies the monomer and a protein fragment were formed.

Analysis of oligosaccharides by microbore high-performance liquid chromatography.

A 1-mm microbore hydrophilic interaction column has been used for the separation of 2-aminoacridone (2-AMAC)-derivatized glycan mixtures, released from naturally occurring and recombinant proteins. Primary structure identification of the 2-AMAC glycan derivatives was carried out by HPLC using fluorescence and mass spectrometric detection. In some cases, enzymatic digestion of the 2-AMAC glycans was applied to confirm glycan structure. This strategy is considerably more rapid than methods normally used in glycan analysis, which involves manual collection of separated oligosaccharide derivatives and analysis of individual fractions by mass spectrometry.

Structural characterization of chemically derivatized oligosaccharides by nanoflow electrospray ionization mass spectrometry.

Oligosaccharides released from several glycoproteins were derivatized with either 4-aminobenzoic acid 2-(diethylamino)ethyl ester (ABDEAE) (Yoshino, K.; et al. Anal. Chem. 1995, 67, 4028-4031) or 2-aminopyridine. The resulting derivatives were analyzed on a nanoflow electrospray ionization (ESI) quadrupole-inlet time-of-flight mass spectrometer using the low-energy collision-induced dissociation technique. In the MS/MS spectra, the oxonium (b or internal series) and y series ions, which are derived from the multiply charged precursor ions, were predominant and were used for the structural readout. Some oxonium ions that were observed in the low-mass region, but that were not found in the PSD analyses (Mo, W.; et al. Anal. Chem. 1998, 70, 4520-4526), rendered a more detailed structural insight. The oxonium ions at m/z 512.2, which are derived from the fucosylated oligosaccharides of immunoglobulin Y and thyroglobulin, were observed, suggesting that fucosylation had occurred proximal to the outer nonreducing terminus. In addition, the data herein show that structural elucidation can be routinely achieved at a low sample concentration. For the case of ABDEAE derivatives, this can be achieved at the 50 fmol/microL level and with the actual sample consumption at the attomole level using nanoflow ESI MS/MS.

Structural characterisation of N-linked glycan mixtures by precursor ion scanning and tandem mass spectrometric analysis

2-Aminoacridone (2-AMAC) labelled N-linked glycan pools were analysed directly by a hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer (Q-Tof) in the precursor ion scanning and tandem mass spectrometry (MS/MS) modes. The use of a precursor ion scanning strategy on this instrument provides a rapid and sensitive method of screening glycan mixtures, without prior separation by chromatographic methods. It allows facile and preliminary characterisation of glycans into different classes, for example, high-mannose or complex glycans. Preliminary sequencing information for each glycan is obtained in the initial precursor ion scanning mode, but further sequencing information of selected glycans can be obtained using the MS/MS mode. Copyright 1999 John Wiley & Sons, Ltd.

Randomized controlled study of ultrasound therapy in the management of acute lateral ligament sprains of the ankle joint.

OBJECTIVE: To determine the efficacy of a fixed dose of ultrasound energy to treat acute lateral ligament sprains of the ankle joint. STUDY DESIGN: Double-blind randomised controlled trial. SETTING: Accident and Emergency department of University Teaching Hospital. SUBJECTS: Patients presenting at Accident and Emergency with ankle injuries. INTERVENTION: Ultrasound or placebo, and Tubigrip. OUTCOME MEASURES: Pain measured with visual analogue scales, swelling using a tape measure, range of movement using a fluid-filled goniometer, and weight bearing using two scales simultaneously. RESULTS: Patients in both groups improved symptomatically. There were no statistically significant differences between groups in any outcome measure. Within groups, statistically significant differences were detected in pain perceived, and range of movement (dorsiflexion). CONCLUSION: At the dose and duration used, ultrasound therapy is no better than placebo in the management of lateral ligament injuries.

Changes in surfactant-associated protein mRNA profile in growth-restricted fetal sheep.

To test the hypothesis that chronic placental insufficiency resulting in fetal growth restriction causes an increase in fetal lung surfactant-associated protein (SP) gene expression, we embolized chronically catheterized fetal sheep (n = 6) daily using nonradioactive microspheres in the abdominal aorta for 21 days (between 0.74 and 0.88 of gestation) until fetal arterial oxygen content was reduced by approximately 40-50%. Control animals (n = 7) received saline only. Basal fetal plasma cortisol concentration was monitored. At the end of the experiment, fetal lung tissues were collected, and ratios of tissue levels of SP-A, SP-B, and SP-C mRNA to 18S rRNA were determined by standard Northern blot analysis. Total DNA content of fetal lungs was reduced by 30% in the embolized group compared with control group (P = 0.01). There was a 2.7-fold increase in fetal lung SP-A mRNA (P < 0.05) and a 3.2-fold increase in SP-B mRNA (P < 0.01) in the chronically embolized group compared with those in the control group. SP-A and SP-B mRNA tissue levels were highly correlated with the mean fetal plasma cortisol levels on days 20-21 (r = 0.90, P < 0.01 for SP-A mRNA and r = 0.94, P < 0.01 for SP-B mRNA). SP-C mRNA tissue levels were not significantly affected by placental insufficiency. We conclude that fetal growth restriction due to placental insufficiency is associated with alterations in fetal lung SP, suggesting enhanced lung maturation that was highly dependent on the degree of increase in fetal plasma cortisol levels.

Structural characterization by tandem mass spectrometry of the posttranslational polyglycylation of tubulin.

Polyglycylation is a posttranslational modification specific to tubulin. This modification was originally identified in highly stable microtubules from Paramecium cilia. As many as 34 posttranslationally added glycine residues have been located in the C-terminal domains of Paramecium alpha- and beta-tubulin. In this study, post source decay matrix-assisted laser desorption/ionization mass spectrometry (PSD MALDI MS) and electrospray ionization on a hybrid quadrupole orthogonal time-of-flight tandem mass spectrometer (ESI Q-TOF MS/MS) were both used to demonstrate that a single molecule of beta-tubulin, from either dynamic cytoplasmic microtubules or stable axonemal microtubules, can be glycylated on each of the last four C-terminal glutamate residues Glu437, Glu438, Glu439, and Glu441 in the sequence 427DATAEEEGEFEEEGEQ442. In both dynamic and stable microtubules the most abundant beta-tubulin isoform contains six posttranslationally added glycine residues: two glycine residues on both Glu437 and Glu438 and one glycine residue on both Glu439 and Glu441. The number and relative abundance of glycylated isoforms of beta-tubulin in both cytoplasmic and axonemal microtubules were compared by MALDI MS.1 The abundance of the major glycylated isoforms in axonemal tubulin decreases regularly with glycylation levels from 6 to 19 whereas it drops abruptly in cytoplasmic tubulin with glycylation levels from 6 to 9. However, the polyglycine chains are similarly distributed on the four C-terminal glutamate residues of cytoplasmic and axonemal tubulin. The polyglycylation results in bulky C-terminal domains with negatively charged surfaces, all surrounding the microtubular structure.

Profiling of 2-aminoacridone derivatised glycans by electrospray ionization mass spectrometry.

Three different types of N-glycans, high-mannose, complex and hybrid, have been derivatised with 2-aminoacridone and analysed by electrospray mass spectrometry and tandem mass spectrometry (MS/MS) using a hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer, fitted with a nanoflow electrospray ion source. Relatively simple MS/MS fragmentation patterns have been observed for each type of glycan, allowing rapid elucidation of the order in which the monosaccharide residues making up these glycans are linked to one another.

Mass spectrometric analysis of 2-deoxyribonucleoside and 2'-deoxyribonucleotide adducts with aldehydes derived from lipid peroxidation.

An important emerging issue in chemical carcinogenesis is the role that products of endogenous metabolism play in formation of covalently modified DNA. One example is the formation of alpha, beta-unsaturated aldehydes as a result of endogenous and drug-stimulated lipid peroxidation. Malondialdehyde (MDA), crotonaldehyde (CR), 2-hexenal (HX), and 4-hydroxy-2-nonenal (HNE) react covalently with 2'-deoxyguanosine (dG) and 2'-deoxyadenosine (dA) residues on DNA to form promutagenic cyclic adducts that may be important in the etiology of cancer in humans and animals. The accurate quantification of such adducts provides a powerful tool in molecular epidemiology for assessing carcinogenic risks from various lifestyle choices (e.g. diet, drug use) in humans. 32P-Postlabeling is recognized as one of the most sensitive methods available for detection of DNA adducts in human tissues, but without adequate validation such methodology can yield inaccurate quantitative measurements. We have used LC separations in conjunction with electrospray ionization MS and tandem MS (triple quadrupole and hybrid quadrupole-orthogonal acceleration time of flight analyzers) to characterize MDA-, CR-, HX- and HNE-modified dG and nucleotide (3'- and 5'-monophosphate; 3',5'-bisphosphate) adducts. These data have been used to validate 32P-postlabeling methods for quantification of low level MDA-dG adducts formed in DNA of human and animal tissues. Availability of reliable methods for quantification of endogenous DNA damage in humans and animals is essential for determining unknown etiologies of cancer and for the assessment of cancer risks in humans.

Why do GPs perform investigations?: The medical and social agendas in arranging back X-rays.

OBJECTIVE: We aimed to determine the most important medical and psychosocial reasons GPs report for requesting back X-rays. METHODS: All GPs in a single health district were mailed a questionnaire and asked to document their reasons for requesting back X-rays. RESULTS: A total of 166/236 (70%) of GPs responded. There were 445 comments (mean 2.7 per doctor): 319 (72%) were medical indications (mean 1.9 per doctor) and 126 (28%) psychosocial reasons (mean 0.8 per doctor). GPs' medical criteria for requesting back X-rays were mainly in line with current guidelines. The most common psychosocial reasons were patient satisfaction (17%), work related (14%) and reassurance (8%). CONCLUSION: GPs' reported medical criteria for arranging back X-rays are mainly 'appropriate', but psychosocial reasons-especially patient satisfaction and reassurance-are also likely to be important factors. If psycho-social agendas are important in ordering investigations, then clinical guidelines which discuss only medical criteria may not be effective in reducing 'inappropriate' investigations.