Mass Spectrometry Spectroscopy Research Articles

Clinical and Epidemiological Metabonomics

Metabonomics is an omics approach to identify and monitor metabolic characteristics, changes, and phenotypes with respect to various synergetic factors such as environment, life style, diet, and potential pathophysiological processes. Recently, metabonomics has also opened up a possibility for functional genomics in large genome-wide studies. Mass spectrometry (MS) and proton nuclear magnetic resonance (NMR) spectroscopy are the two key experimental technologies in the field. Recent advancements are numerous, and methodologies currently exist that allow for automated high-throughput experimentation in a very cost-effective manner. Technical developments have evoked an increasing number of metabonomics applications in clinical and epidemiological disciplines to understand the complex molecular foundations of various diseases. Techniques with the aim of assessing large numbers of metabolites that are substrates, intermediates, or products in various metabolic pathways are particularly relevant in the risk assessment of metabolic conditions like diabetes and vascular diseases. It has also been envisioned that metabonomics approaches may overtake standard analytical measurements of individual metabolites and eventually lead to holistic multimetabolic risk phenotyping in the early detection of high-risk individuals for various metabolic diseases. Thus, metabonomics offers potential means to move away from single biomarkers and thresholds in clinical medicine. And, furthermore, even though the current medical biomarkers and thresholds are necessary benchmarks for clinical practice, it will be crucial to let the future diagnostics to follow from new science. Good predictions are necessary for effective prevention; an important step towards this is expected to happen when complex traits will not anymore be limited by clinical diagnoses and definitions but will be handled as multivariate continuous dimensions. Consequently, clinical and epidemiological metabonomics is likely to be one of the key new omics areas to assist translational research in the near future. Here, the Journal of Biomedicine and Biotechnology presents this special issue, 2011. Although only representing a small fraction of the contribution of metabonomics to various biomedical disciplines, the papers presented here show the increasing potential of metabonomics approaches to complement clinical and epidemiological research. To start the special issue, we have five contributions that review several aspects of metabonomics research. M. G. Barderas et al. discuss the concepts of metabolic profiling, fingerprinting, and footprinting focusing on the area of cardiovascular diseases. E. Silvestri and coworkers discuss the timely and relevant issue of integrative analyses of related omics data sets in order to furnish new insights not accessible through one-dimensional data. Further, I. Kouskoumvekaki and G. Panagiotou give an extensive up-to-date overview on the database resources on metabolic pathways and metabolomes and also summarize the statistical tools often used for the analysis of metabonomics data. Y. Kim and S. Milner deal with the challenges related to bioactive food components in cancer research and prevention. Moreover, M. Sofia and coauthors review the potential use of exhaled breath condensate as a suitable matrix for NMR-based metabonomics of airway diseases. This special issue contains two methodology reports. G. Kastenmuller and coworkers have developed a freely accessible methodology to facilitate automated and standardized analysis for quantitative metabolic data, covering the steps from data acquisition to biological interpretation. M. C. Martinez-Bisbal and colleagues, using magnetic resonance microscopy, present a critical and valuable interpretation on the high-resolution magic angle spinning data of human tumor tissues. The six original research papers in this special issue start with the paper by J. Willmann et al. who demonstrate the advantage of combining high-pressure liquid chromatography, MS, and NMR spectroscopy in analyzing three different types of extracts of a common membrane component phosphatidylcholine. Then, D. F. Brougham et al. focus on the use of artificial neural networks in classifying proton NMR spectroscopic data recorded on whole-cell culture samples of four different lung carcinoma cell lines, displaying different drug-resistance patterns. In a mouse study, D. Otter et al. apply MS-based nontargeted urinary metabolite profiling to identify biomarkers of colon inflammation. In another mouse study, J. Zhou et al. provide evidence that NMR metabonomics may be sensitive enough to detect small differences in the composition of maternal plasma and thus be helpful for identifying biomarkers of birth defects. Additionally, in low-birth weight and high-birth weight piglets P. M. Nissen and coworkers combine NMR and MS to reveal a relationship between birth weight and plasma inositols, suggesting their potential role in fetal programming of type 2 diabetes. L. Guo et al. end this special issue in providing the latest epidemiologic information on the etiology distribution of neurodevelopmental disabilities in Chinese, and by demonstrating, on the basis of 8-year clinical experience, that MS-based pediatric metabonomics is clinically helpful. Overall, we hope that this special issue hints at the rising potential of metabonomics approaches in clinical and epidemiological research and translational medicine. Mika Ala-Korpela Veikko Salomaa Olav M. Kvalheim

Systems level studies of mammalian metabolomes: the roles of mass spectrometry and nuclear magnetic resonance spectroscopy

The study of biological systems in a holistic manner (systems biology) is increasingly being viewed as a necessity to provide qualitative and quantitative descriptions of the emergent properties of the complete system. Systems biology performs studies focussed on the complex interactions of system components; emphasising the whole system rather than the individual parts. Many perturbations to mammalian systems (diet, disease, drugs) are multi-factorial and the study of small parts of the system is insufficient to understand the complete phenotypic changes induced. Metabolomics is one functional level tool being employed to investigate the complex interactions of metabolites with other metabolites (metabolism) but also the regulatory role metabolites provide through interaction with genes, transcripts and proteins (e.g. allosteric regulation). Technological developments are the driving force behind advances in scientific knowledge. Recent advances in the two analytical platforms of mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy have driven forward the discipline of metabolomics. In this critical review, an introduction to metabolites, metabolomes, metabolomics and the role of MS and NMR spectroscopy will be provided. The applications of metabolomics in mammalian systems biology for the study of the health-disease continuum, drug efficacy and toxicity and dietary effects on mammalian health will be reviewed. The current limitations and future goals of metabolomics in systems biology will also be discussed (374 references).

Expression and identification of a small recombinant beefy meaty peptide secreted by the methylotrophic yeast Pichia pastoris

A tasty peptide, beefy meaty peptide (BMP), which was initially isolated from beef digested by papain, has potential as a new flavor enhancer. BMP is a small peptide consisting of eight amino acids. In this study, DNA encoding BMP was synthesized and cloned into the expression vector pPICZαA to obtain the recombinant expression vector pPICZαA-BMP. The recombinant vector was linearized and then integrated into the genome of Pichia pastoris strain X-33 by electroporation. The recombinant strain P.p2 was then incubated in BMGY medium and then induced in BSM medium for expression of BMP. The fermentation broth was centrifuged and the supernatant was purified by gel-filtration chromatography. Purified recombinant BMP was then tested by high performance liquid chromatography-mass spectrometry (HPLC-MS). After optimization of the culturing process, the yield of BMP reached 10 mg/L in the clarified broth. The results of mass spectrometry/mass spectrometry (MS/MS) showed that recombinant BMP secreted from P. pastoris had a molecular weight of 873.7 Da rather than the expected 847 Da. We speculate that the last amino acid, Ala, of the BMP sequence was replaced with Pro in the BMP fragment. Key words: beefy meaty peptide, Pichia pastoris, peptide expression, HPLC-MS.

Identification of liver proteins and their roles associated with carbon tetrachloride-induced hepatotoxicity

Carbon tetrachloride (CCl(4)) is a common hepatotoxin used in experimental models to elicit liver injury. To identify the proteins involved in CCl(4)-induced hepatotoxicity, two-dimensional gel electrophoresis was employed followed by mass spectrometry - mass spectrometry (MS/MS) to study the differentially expressed proteins during CCl(4) exposure in the Fischer 344 rat liver proteome for 5 weeks. Ten spots with notable changes between the Control and CCl(4) groups were successfully identified. Among them, four proteins with significant up-regulation, namely calcium-binding protein 1, protein disulfide isomerase, mitochondrial aldehyde dehydrogenase precursor, and, glutathione-S-transferase mu1 and six proteins with significant down-regulation, namely catechol-O-methyltransferase, hemoglobin-alpha-2-chain, hemopexin precursor, methionine sulfoxide reductase A, catalase and carbonic anhydrase 3, were identified. The data indicates that CCl(4) causes hepatotoxicity by depleting oxygen radical scavengers in the hepatocytes. In this rat model, we profiled hepatic proteome alterations in response to CCl(4) intoxication. The findings should facilitate understanding of the mechanism of CCl(4)-induced liver injury.

Marked Variability of Monacolin Levels in Commercial Red Yeast Rice Products

Red yeast rice (RYR) is a widely available dietary supplement used by millions of patients as an alternative therapy for hyperlipidemia. It contains 14 active compounds called monacolins that inhibit hepatic cholesterol synthesis. Although studies have suggested that some formulations of RYR may be effective and safe for lipid lowering, monacolin levels are not standardized among marketed products and are generally not published on labels. We evaluated monacolin levels in 12 commercial RYR formulations and tested for citrinin, a mycotoxin that is nephrotoxic in animals. Each formulation of RYR was labeled "600 mg/capsule" of active product. Analyses for monacolins and citrinin were performed between August 2006 and June 2008 using high-performance liquid chromatography with mass spectroscopy-mass spectroscopy detection. Laboratory analyses of RYR products were conducted by ConsumerLab.com, White Plains, New York. There was marked variability in the 12 RYR products in total monacolins (0.31-11.15 mg/capsule), monacolin K (lovastatin) (0.10-10.09 mg/capsule), and monacolin KA (0.00-2.30 mg/capsule). Four products had elevated levels of citrinin. We found striking variability in monacolin content in 12 proprietary RYR products and the presence of citrinin in one-third of the formulations tested. Although RYR may have potential as an alternative lipid-lowering agent, our findings suggest the need for improved standardization of RYR products and product labeling. Until this occurs, physicians should be cautious in recommending RYR to their patients for the treatment of hyperlipidemia and primary and secondary prevention of cardiovascular disease.

Integrated Phosphoproteomics Analysis of a Signaling Network Governing Nutrient Response and Peroxisome Induction

Phosphorylation of proteins is a key posttranslational modification in cellular signaling, regulating many aspects of cellular responses. We used a quantitative, integrated, phosphoproteomics approach to characterize the cellular responses of the yeast Saccharomyces cerevisiae to the fatty acid oleic acid, a molecule with broad human health implications and a potent inducer of peroxisomes. A combination of cryolysis and urea solubilization was used to minimize the opportunity for reorientation of the phosphoproteome, and hydrophilic interaction liquid chromatography and IMAC chemistries were used to fractionate and enrich for phosphopeptides. Using these approaches, numerous phosphorylated peptides specific to oleate-induced and glucose-repressed conditions were identified and mapped to known signaling pathways. These include several transcription factors, two of which, Pip2p and Cst6p, must be phosphorylated for the normal transcriptional response of fatty acid-responsive loci encoding peroxisomal proteins. The phosphoproteome data were integrated with results from genome-wide assays studying the effects of signaling molecule deletions and known protein-protein interactions to generate a putative fatty acid-responsive signaling network. In this network, the most highly connected nodes are those with the largest effects on cellular responses to oleic acid. These properties are consistent with a scale-free topology, demonstrating that scale-free properties are conserved in condition-specific networks.

Alternate Dissociation Pathways Identified in Charge-Reduced Protein Complex Ions

Tandem mass spectrometry (MS) of large protein complexes has proven to be capable of assessing the stoichiometry, connectivity, and structural details of multiprotein assemblies. While the utility of tandem MS is without question, a deeper understanding of the mechanism of protein complex dissociation will undoubtedly drive the technology into new areas of enhanced utility and information content. We present here the systematic analysis of the charge state dependent decay of the noncovalently associated complex of human transthyretin, generated by collision-induced dissociation (CID). A crown ether based charge reduction approach was applied to generate intact transthyretin tetramers with charge states ranging from 15+ to 7+. These nine charge states were subsequently analyzed by means of tandem MS and ion mobility spectrometry. Three different charge-dependent mechanistic regimes were identified: (1) common asymmetric dissociation involving ejection of unfolded monomers, (2) expulsion of folded monomers from the intact tetramer, and (3) release of C-terminal peptide fragments from the intact complex. Taken together, the results presented highlight the potential of charge state modulation as a method for directing the course of gas-phase dissociation and unfolding of protein complexes.

Characterisation of commercially available linear alkylbenzenesulfonates by LC-SPE-NMR/MS (liquid chromatography-solid phase extraction-nuclear magnetic resonance spectroscopy-mass spectroscopy)

Commercially available linear alkylbenzenesulfonates (LASs) are a mixture of various homologues and isomers, leading to 20 major species. In this work we investigated the commercial product by liquid chromatography-solid phase extraction-nuclear magnetic resonance spectroscopy-mass spectrometry (LC-SPE-NMR/MS). The commercial product was separated into 17 fractions by liquid chromatography (LC). After chromatographic separation, 5% of the flow was split to a mass spectrometer (MS) while 95% was send to post-column solid phase extraction cartridges for enrichment of the analytes (LC-SPE). After elution from the SPE-cartridges a NMR-spectrometer equipped with a cryo-probe was used for the characterisation of the different LASs species. For the first time 1H-1D and H-H-COSY spectra for 14 LASs species out of 20 major isomers are presented, whereas the 6 remaining species are detected as mixtures in 3 1H-1D and H-H-COSY spectra. These data were used to correlate the chromatographic retention of the LASs isomers to the substitution pattern of the alkyl chain.

Up-regulation of hnRNP A1, Ezrin, tubulin β-2C and Annexin A1 in sentinel lymph nodes of colorectal cancer

To investigate the early metastasis-associated proteins in sentinel lymph node micrometastasis (SLNMM) of colorectal cancer (CRC) through comparative proteome. Hydrophobic protein samples were extracted from individual-matched normal lymph nodes (NLN) and SLNMM of CRC. Differentially expressed protein spots were detected by two-dimensional electrophoresis and image analysis, and subsequently identified by matrix assisted laser desorption/ionization-time of flight mass spectrometry-mass spectrometry and Western blotting, respectively. Forty proteins were differentially expressed in NLN and SLNMM, and 4 metastasis-concerned proteins highly expressed in SLNMM were identified to be hnRNP A1, Ezrin, tubulin β-2C and Annexin A1. Further immunohistochemistry staining of these four proteins showed their clinicopathological characteristics in lymph node metastasis of CRC. Variations of hydrophobic protein expression in NLN and SLNMM of CRC and increased expression of hnRNP A1, Ezrin, tubulin β-2C and Annexin A1 in SLNMM suggest a significantly elevated early CRC metastasis.

Reactivity of platinum-based antitumor drugs towards a Met- and His-rich 20mer peptide corresponding to the N-terminal domain of human copper transporter 1

Cellular uptake of platinum-based antitumor drugs is a critical step in the mechanism of the drug action and associated resistance, and deeper understanding of this step may inspire development of novel methods for new drugs with reduced resistance. Human copper transporter 1 (hCtr1), a copper influx protein, was recently found to facilitate the cellular entry of several platinum drugs. In the work reported here, we constructed a Met- and His-rich 20mer peptide (hCtr1-N20) corresponding to the N-terminal domain of hCtr1, which is the essential domain of hCtr1 for transporting platinum drugs. The interactions of the peptide with cisplatin and its analogues, including transplatin, carboplatin, oxaliplatin, and [Pt(L: -Met)Cl(2)], were explored at the molecular level. Electrospray ionization (ESI) mass spectrometry (MS) data revealed that all of the platinum(II) complexes used in present study can bind to hCtr1-N20 in 1:1 and 2:1 stoichiometry. Four Met residues should be involved in binding to cis-platinum complexes on the basis of the tandem MS spectrometry and previously reported data. Time-dependent 2D [(1)H,(15)N] heteronuclear single quantum coherence NMR spectra indicate the reaction of cisplatin with hCtr1-N20 is a stepwise process. The intermediate, however, is transient, which is consistent with the ESI-MS results. Time-dependent ESI-MS data revealed that the geometry and the properties of both the leaving and the nonleaving groups of platinum(II) complexes play essential roles in controlling the reactivity and formation of the final products with hCtr1-N20.

Defining the Structural Basis of Human Plasminogen Binding by Streptococcal Surface Enolase

The flesh-eating bacterium group A Streptococcus (GAS) binds and activates human plasminogen, promoting invasive disease. Streptococcal surface enolase (SEN), a glycolytic pathway enzyme, is an identified plasminogen receptor of GAS. Here we used mass spectrometry (MS) to confirm that GAS SEN is octameric, thereby validating in silico modeling based on the crystal structure of Streptococcus pneumoniae alpha-enolase. Site-directed mutagenesis of surface-located lysine residues (SEN(K252 + 255A), SEN(K304A), SEN(K334A), SEN(K344E), SEN(K435L), and SEN(Delta434-435)) was used to examine their roles in maintaining structural integrity, enzymatic function, and plasminogen binding. Structural integrity of the GAS SEN octamer was retained for all mutants except SEN(K344E), as determined by circular dichroism spectroscopy and MS. However, ion mobility MS revealed distinct differences in the stability of several mutant octamers in comparison with wild type. Enzymatic analysis indicated that SEN(K344E) had lost alpha-enolase activity, which was also reduced in SEN(K334A) and SEN(Delta434-435). Surface plasmon resonance demonstrated that the capacity to bind human plasminogen was abolished in SEN(K252 + 255A), SEN(K435L), and SEN(Delta434-435). The lysine residues at positions 252, 255, 434, and 435 therefore play a concerted role in plasminogen acquisition. This study demonstrates the ability of combining in silico structural modeling with ion mobility-MS validation for undertaking functional studies on complex protein structures.

Identification of two pistachio allergens, Pis v 1 and Pis v 2, belonging to the 2S albumin and 11S globulin family

IgE-mediated allergic reactions to pistachio appear to be occurring more frequently; however, little is known about its allergenic proteins. We attempted to identify pistachio allergens and to clone the encoding genes. Pistachio proteins were extracted and separated by SDS-PAGE. Immunolabelling was performed with sera from 28 pistachio-allergic individuals. Proteins of interest were further analysed by Edman sequencing and mass spectrometry/mass spectrometry (MS/MS). In parallel, a cDNA library was generated from immature pistachios and screened with primers designed on the basis of internal sequences and peptide spectra. Full-length cDNA clones were isolated from the library and sequenced. Recombinant proteins were expressed and tested with sera from pistachio-allergic patients. Nineteen out of 28 patients (68%) showed IgE binding to a 7 kDa protein fraction, while 14 (50%) showed specific IgE to a 32 kDa protein fraction. Analysis by Edman sequencing and MS/MS revealed that these proteins were homologue to the cashew nut allergens Ana o 3 and Ana o 2, respectively. Screening of the pistachio cDNA library resulted in isolation of novel protein cDNAs. Open-reading frame translation provided the complete amino acid sequences of two new allergenic pistachio proteins. Recombinant proteins were recognized by six out of six selected patients. Therefore, these new allergens were named Pis v 1 and Pis v 2 by the Allergen Nomenclature Subcommittee. Novel allergens in pistachio, Pis v 1 and Pis v 2, which belong to 2S albumin and 11S globulin family, respectively, were isolated and the genes encoding these allergens were identified.

Antifungal Azole Metabolites: Significance in Pharmaceutical and Biomedical Analysis

Antifungal Azole Metabolites: Significance in Pharmaceutical and Biomedical Analysis Individualised therapy and factors determining such variability among patients are confusing to both physicians and their patients because of the observed therapeutic, metabolic and toxic response. The same is true about antifungal azoles. They are under the influence and become targets of metabolic drug-drug interactions where more than one active form of the drug may be involved. The clinical relevance of these interactions may vary upon the azole involved and upon the intention of drug administration. The pharmacodynamics and pharmacokinetics of azole drugs as indicated by the reviewed data make the need for characterization of all their metabolites even more evident. The health care systems also emphasize the identification and quantitation of the metabolites for a comprehensive understanding of the biological safety of individual metabolites, thus, revealing the need and scope of bioanalytical research in metabolite and toxicity profiling of drugs. Availability of protocols for qualitative and quantitative characterization of all metabolites will have many applications for therapeutic drug monitoring, bioequivalence, toxicological and all related studies. Identification of metabolites may be done by a variety of chromatographic and spectroscopic techniques, either alone or in combination with other techniques. Conventional liquid chromatography has been exploited widely in the field of metabolite profiling. The arrival of hyphenated techniques has revolutionized metabolite profiling, by not only separating but also generating data for the structural identification of metabolites as well. Among all techniques, the most exploited are Liquid Chromatography-Mass Spectroscopy, Nuclear Magnetic Resonance spectroscopy, Liquid Chromatography-Nuclear Magnetic Resonance spectroscopy, Liquid Chromatography-Nuclear Magnetic Resonance spectroscopy-Mass Spectroscopy and Extraction-Nuclear Magnetic Resonance spectroscopy. This compilation provides a tool for the metabolic, bioanalytical and biomedical understanding of antifungal azole metabolites.

Desorption Electrospray Ionization Mass Spectrometry for Trace Analysis of Agrochemicals in Food

Desorption electrospray ionization (DESI) is applied to the rapid, in situ, direct qualitative and quantitative (ultra)trace analysis of agrochemicals in foodstuffs. To evaluate the potential of DESI mass spectrometry (MS) in toxic residue testing in food, 16 representative multiclass agricultural chemicals (pesticides, insecticides, herbicides, and fungicides) were selected (namely, ametryn, amitraz, azoxystrobin, bitertanol, buprofezin, imazalil, imazalil metabolite, isofenphos-methyl, malathion, nitenpyram, prochloraz, spinosad, terbuthylazine, thiabendazole, and thiacloprid). The DESI-MS experiments were performed using 3 microL of solution spotted onto conventional smooth poly(tetrafluoroethylene) (PTFE) surfaces, with examination by MS and tandem mass spectrometry (MS/MS) using an ion trap mass spectrometer. Optimization of the spray solvent led to the use of acetonitrile/water (80:20) (v/v), with 1% formic acid. Most of the compounds tested showed remarkable sensitivity in the positive ion mode, approaching that attainable with conventional direct infusion electrospray mass spectrometry. To evaluate the potential of the proposed approach in real samples, different experiments were performed including the direct DESI-MS/MS analysis of fruit peels and also of fruit/vegetable extracts. The results proved that DESI allows the detection and confirmation of traces of agrochemicals in actual market-purchased samples. In addition, MS/MS confirmation of selected pesticides in spiked vegetable extracts was obtained at absolute levels as low as 1 pg for ametryn. Quantitation of imazalil residues was also undertaken using an isotopically labeled standard. The data obtained were in agreement with those from the liquid chromatography mass spectrometry (LC-MS) reference method, with relative standard deviation (RSD) values consistently below 15%. The results obtained demonstrate the sensitivity of DESI as they meet the stringent European Union pesticide regulation requirements (maximum residue levels) for a large percentage of the studied compounds.

Monitoring the Tanford transition in β‐lactoglobulin by 8‐anilino‐1‐naphthalene sulfonate and mass spectrometry

The fluorescent dye 8-anilino-1-naphthalene sulfonate (ANS) is known to interact with proteins by conformation-specific hydrophobic interactions and rather nonspecific electrostatic interactions. To which category the complexes detectable by mass spectrometry (MS) belong is still the subject of debate. Here, the Tanford transition in beta-lactoglobulin (BLG) is exploited as an experimental device to expose hydrophobic binding sites by an increase in pH, rather than, as usually done, by lowering the pH. Complex formation is monitored by electrospray ionization (ESI)-MS and fluorescence spectroscopy. Both techniques reveal stronger ANS binding to BLG at pH 7.9 than at pH 5.9, suggesting that dye binding inside the calyx, which is known to be hydrophobically driven in solution, can contribute to the complexes detected by ESI-MS. Electrostatic interactions between the protein and the ANS sulfonate group can only be weaker at pH 7.9 than at pH 5.9, supporting the interpretation of the results by the protein conformational change. Lysozyme is used as a negative control, which shows no variation in the interaction with ANS in the same range of pH, in the absence of conformational changes. However, comparison of MS and fluorescence data at variable pH for BLG and myoglobin (Mb) suggests that conformation-specific ANS binding to proteins is detectable by ESI-MS only inside well-structured cavities of folded structures, like the BLG calyx and apoMb heme pocket. Indeed, ANS interactions with highly dynamic structures or molten globules, although detectable in solution, are easily lost in the gas phase.

A Comparison of IEF and MSMS for Clinical Hemoglobinopathy Screening in 40,000 Newborns

Currently, newborn haemoglobinopathy screening is carried out using HPLC or isoelectric focusing (IEF). We have previously described a rapid and specific electrospray mass spectrometry–mass spectrometry (MSMS) technique, using multiple reaction monitoring (MRM) based peptide analysis, for simultaneous detection of the clinically significant haemoglobinpathies; haemoglobin (Hb)S, HbC, HbE, HbDPunjab and HbOArab. Here we report the results of a comparison of 40,000 newborn blood spots screened by both IEF and MSMS.For both IEF and MSMS analysis, blood spots (3.2mm) were punched into separate 96 well plates. IEF was performed using the Resolve haemoglobin test kit (PerkinElmer Life Sciences, Waltham, USA) and Isoscan imaging system. For MSMS analysis, the blood spots were digested for 30min at 37°C with a trypsin reagent, and diluted in mobile phase (acetonitrile: water, 50:50, with 0.025% formic acid. Sample, 2μl, was injected directly into the mobile phase (flow rate 80μl/min) and analysed, in positive ion mode, using a Sciex API4000 (Applied Biosystems, Warrington, UK). Specific MRM transitions for HbS, HbC, HbE, HbDPunjab, HbOArab, normal beta, alpha, gamma and delta chains were acquired; total acquisition time per sample was 60 sec. This enabled identification of sickling disorders and thalassaemia major, as well as assessment of transfusion state and potential identification of HbLepore and HbBarts.40,000 blood spot samples for routine newborn haemoglobinopathy screening were analysed in parallel. HbS was detected in 199 samples; 8 were HbS/HbF only and 3 HbSC. HbC was detected in 39 samples, HbDPunjab in 52, HbE in 48. No HbOArab or HbLepore mutations were detected by either method. There have been no discrepancies between the analytical techniques. Using MSMS, mutation positive samples can be re-run in product ion scan mode to provide peptide sequence and hence unequivocal confirmation of the haemoglobin variant. In addition, 5,000 samples were analysed on a Sciex API4000 Q trap; using the information dependent acquisition facility provided “real time” peptide sequencing thus removing the requirement for re-injection.Sample preparation is very quick and simple for both methods, but the consumable costs associated with the MSMS technique are <10% of those for IEF. The capital cost of MSMS can be offset by high throughput and/or integration with current inherited metabolic disease screening by MSMS. The specificity of the MSMS analysis implies that haemoglobinopathy detection can be limited to specified conditions, based on agreed screening policy, and can eliminate the need for costly and time consuming second line testing.This study demonstrates that newborn haemoglobinopathy screening can be carried out rapidly, easily, and cost effectively using MSMS technology. It enables rationalisation of technology platforms in newborn screening by consolidating screening for haemoglobinopathies and inherited metabolic diseases onto MSMS.

Study on Different Proteomics of Denuded tongue coating

Objective To investigate the microbiostatic mechanism of denuded tongue coating, Proteomic methods were used to find differential expression proteins in denuded tongue. Methods The total protein of normal or denuded tongue coating was separated by means of immobilized pH gradient-based two-dimensional gel electrophoresis (2-DE). The differential expression protein was analyzed by using ImageMaster 2D analysis software. Image analysis software was used for comparative analysis to find differential expression proteins; Mass spectrometry (mass spectrometry) was applied to get the corresponding peptide fingerprint (peptide mass finger print_ PMF). Search the database to identify differences in protein.Results Establish two-dimensional gel electrophoresis for normal and denuded tongue; Segregation analysis on four groups of proteome of tongue coating showed that gel could be detected in the protein spots at 1082=t=105 and 1143±140 respectively; Compared with normal group, there are eight points of protein expression being regulated upward and seven points down.Altogether 9 differences in protein expression were identified. Conclusion The sound resolution and repeatability Two-dimensional electrophoresis silver staining pattern were established; Some differences in protein expression related to denuded tongue were identified. Key words: Tongue coating: Proteomics: Two-dimensional gel electrophoresis: Differences in protein expression profile: Methodology

Determination of paralytic shellfish poisoning toxins in Norwegian shellfish by liquid chromatography with fluorescence and tandem mass spectrometry detection

A novel extraction and clean-up method has been developed for the determination of paralytic shellfish poisoning (PSP) toxins in shellfish samples. Raw shellfish material was extracted with an acidic acetonitrile/water (80:20, v/v) solution, whilst being homogenised. During the homogenisation the sample extraction solution was cooled with ice water. Subsequently, the extract was frozen at −20 °C for at least 4 h. During freezing, two layers were formed, only the lower predominantly aqueous layer was used for the determination. The final extract solution was cleaned-up using a combination of Oasis HLB and Carbograph activated carbon SPE columns. The developed extraction and clean-up methods combined with gradient elution liquid chromatography (LC)-mass spectrometry/mass spectrometry (MS/MS) has resulted in a method which can determine the analogs GTX 1-5, C1-2, DcGTX 2-3, DcSTX, Neo, STX in a single analysis with an overall detection limit of 313 μg STXdiHCL-eq./kg shellfish meat. The use of the developed extraction method with post-column high performance liquid chromatography (HPLC) with fluorescence detection (FLD) method provided an overall limit of detection of 89 μg STXdiHCL-eq./kg shellfish meat for the same toxins. Both post-column HPLC-FLD and LC–MS/MS was used to investigate the Norwegian PSP toxin profile. It was found that the PSP toxins could be detected in shellfish samples from the Norwegian coastline for 10 months of the year, from March till December. The toxin profile consisted mainly of the carbamate toxins, GTX 1-4, Neo and STX, in terms of both concentrations and contribution to the overall toxicity. In addition, several of the n-sulfo-carbamoyl toxins were either detected in the samples at relatively low concentrations or their presence in the samples were indicated but could not be confirmed by the post-column HPLC-FLD and LC–MS/MS analyses.

Determination of Fatty Acid Ethyl Esters (FAEE) and Ethyl Glucuronide (EtG) in Hair: A Promising Way for Retrospective Detection of Alcohol Abuse During Pregnancy?

The retrospective detection of alcohol consumption during pregnancy is an important part of the diagnosis of the fetal alcohol syndrome. A promising way to solve this problem can be the determination of fatty acid ethyl esters (FAEE) or/and ethyl glucuronide (EtG) in hair of the mothers. In this article, the present state in analytical determination and interpretation of FAEE and EtG concentrations in hair are reviewed. Both FAEE and EtG are minor metabolites of ethanol and as direct alcohol markers very specific for alcohol. They are durably deposited in hair, which enables taking advantage of the long diagnostic time window of this sample material. In the last years, specific and sensitive methods for determination of both alcohol markers in hair were developed. Headspace solid phase microextraction in combination with gas chromatography-mass spectroscopy after hair extraction with an n-heptane/dimethylsulfoxide mixture proved to be a favorable technique for determination of four characteristic FAEE (ethyl myristate, ethyl palmitate, ethyl oleate, and ethyl stearate). EtG is extracted from hair by water and analyzed either by gas chromatography-mass spectroscopy with negative chemical ionization after cleanup with solid phase extraction and derivatization with pentafluoropropionic anhydride or by liquid chromatography-mass spectroscopy-mass spectroscopy. The detection limits of the single FAEE as well as of EtG are in the range of 1 to 10 pg/mg. FAEE as well as EtG were determined in a larger number of hair samples of teetotalers, social drinkers, patients in alcohol withdrawal treatment, and death cases with previous known heavy drinking. From the results, the following criteria were derived: strict abstinence is excluded or improbable at C FAEE >0.2 ng/mg or C EtG >7 pg/mg. Moderate social drinkers should have C FAEE <0.5 ng/mg and C EtG <25 pg/mg; above these values, alcohol abuse is probable. Until now, there has been no evaluation in context of FAS diagnosis; however, a successful application for this purpose can be expected from the good experience in driving ability examination.

なぜリフレクトロンモードでＭＳ／ＭＳ様の測定が可能なのか

By measurement of post-source decay (PSD) product ion mass spectrum, matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOFMS) enable mass spectrometry/mass spectrometry (MS/MS)-like analysis to obtain structural information. We will explain the basics of MS/MS-like analysis in reflectron mode of MALDI TOFMS by comparing post-source decay product ion mass spectrum acquired by the standard reflectron and the curved field reflectron.