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June 1, 2005
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June 1, 2005
Abstract
For some years now, scientists have been spending a lot of effort in developing methods to analyse and compare complex protein samples. One of the goals of such global analyses of what is known as proteomes is to discover specific protein markers–or fingerprints of protein markers−from various types of affected biological samples. Considering the battery of technologies currently available, mass spectrometry (MS) constitutes an essential tool in proteomics. We describe here the type of MS instrumentation that is currently dedicated to proteomics research. We also describe the major experimental workflows that are typically used in proteomics today, with a focus on those incorporating MS as a major analysis tool.
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June 1, 2005
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Cardiovascular disease (CVD) is the major cause of death in the Western hemisphere. Oxidative stress is involved in the pathophysiology of cancer, neurodegenerative conditions and CVD. Lipid peroxidation is one of the oxidative modifications possible in biological systems. The isoprostanes are derivatives of one specific lipid, i.e. , arachidonic acid, after lipid peroxidation. Several isoprostanes have been identified in biological tissues and fluids, among them 8-iso prostaglandin F 2α (8-iso-PGF 2α , 8-epi-PGF 2α , iPF 2α -III, 15-F 2t -IsoP) and its metabolite, 2,3-dinor-4,5-dihydro-8-iso-PGF 2α . The isoprostanes are reliable in vivo markers of lipid peroxidation in humans: they are endogenously formed, characteristic in structure, ubiquitous in nature, stable in- and ex vivo and reliably quantitatable. In this Review, different analytical approaches will be discussed including immunologic, chromatographic and spectrometric techniques with the main emphasis on mass spectrometry. Analysis of isoprostanes applying radio immunoassay (RIA), enzyme immunoassay (ElA), high performance-liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-tandem MS), gas chromatography-mass spectrometry (GC-MS) and GC-tandem MS will be exemplified in the field of cardiovascular research. Results from several clinical studies are included indicating the validity of isoprostanes as surrogate parameters of oxidative stress in cardiovascular disease.
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June 1, 2005
Abstract
The term “clinical proteomics” refers to the application of available proteomics technologies to current areas of clinical investigation. The ability to simultaneously and comprehensively examine changes in large numbers of proteins in the context of disease or other changes in physiological conditions holds great promise as a tool to unlock the solutions to difficult clinical research questions. Proteomics is a rapidly growing field that combines high throughput analytical methodologies such as two-dimensional gel electrophoresis and SELDI mass spectrometry methods with complex bioinformatics to study systems biology–the system of interest is defined by the investigator. Even with all its potential, however, studies must be carefully designed in order to differentiate true clinical differences in protein expression from differences originating from variation in sample collection, variation in experimental condition, and normal biological variability. Proteomic analyses are already widely in use for clinical studies ranging from cancer to other diseases such as cardiovascular disease, organ transplant, and pharmacodynamic studies.
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June 1, 2005
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Therapeutic drug monitoring of antipsychotic drugs has become more and more important in recent years, and a well-organized therapeutic drug monitoring service with fast turn-around times is very important. Therefore, an analytical method coupling high-performance liquid chromatography to mass spectrometry with electrospray ionization in the positive mode was established in our laboratory. Amitriptyline, citalopram, clomipramine (including norclomipramine), desipramine, dibenzepin, doxepin (including nordoxepin), escitalopram, flupentixol, fluphenazine, fluvoxamine, imipramine, nortriptyline, opipramol, pipamperone, reboxetine, thioridazine, trimipramine and zuclopenthixol were separated on a reversed-phase C18 column. The mobile phase consisted of acetonitrile and ammonium acetate buffer pH 4. Because of different organic solvents used for the liquid-liquid extraction and the different volumes of mobile phases in which the residues were dissolved, four analytical systems had to be applied. Within the run-time of maximal 10 minutes the 13 antidepressant and five neuroleptic drugs were baseline separated on the corresponding mass-to-charge track. The calibration range of each drug was linear, all between-day coefficients of variation were below 7% and the recovery rates were between 60 and 103%. Using 1 ml of serum, the lower limits of quantification were between 1.2 and 54 nmol/l for the different drugs and below the therapeutic range for each of the different drugs.
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June 1, 2005
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In this study, we present a versatile new procedure for the analysis of transferrin and its isoforms isolated from human body fluids such as serum, plasma, and cerebrospinal fluid. This method is based on a three-step procedure: (i) isolation of transferrins using anion-exchange chromatography with UV detection; (ii) concentration of the transferrin fraction; (iii) detection of the transferrins with liquid chromatography-electrospray mass spectrometry. Pre-analytical sample procedures can be omitted and no immunoaffinity columns or transferrin-specific immunoassays were used. Anticoagulants such as heparin, EDTA, citrate, and oxalate do not interfere with our analysis. According to their respective molecular masses, up to ten different isoforms of transferrin could be identified in a serum sample from a patient with a congenital disorder of glycosylation type Ia (CDG-Ia). The method was successfully applied to different pathological samples from patients with CDG-Ia, CDG-Ib, CDG-Ic, CDG-Ie, CDG-If, and CDG-IIa. Additionally, samples from alcohol consumers that were found with turbidimetric immunoassay to contain increased levels of carbohydrate-deficient transferrin were analyzed.
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June 1, 2005
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Proteins are essential biomolecules which are frequently involved in major pathological syndromes and are widely used as diagnostic markers or therapeutic agents. The emergence of proteomics will doubtless further increase the significance of proteins both in the clinic and in the life sciences in general. Our main objective is to offer innovative solutions to what we like to call the “post-proteomics era”. To achieve our goal, we intend to develop novel approaches and technologies for in vivo metabolic studies of proteins using mass spectrometry (MS), focusing on pharmacokinetics and pharmacodynamics. Using goserelin as a model, we have successfully developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the detection and quantification of an intact analogue of luteinizing hormone-releasing hormone (LHRH) in small volumes of rat plasma samples at concentrations ranging from 0.3 to 405.0 μg/l. To this end, a microbore reversed-phase-HPLC system was coupled on-line to a tandem high resolution quadrupole time-of-flight (Q-TOF) instrument fitted with an electrospray ion source and operated in LC-MS/MS mode. External calibration was used and the high resolution was crucial to discard contaminating signals, which would not have been possible with the more conventional triple quadrupole mass spectrometers operated in a static mode. For low sample amounts, calibration curves were constructed corresponding to rat plasma levels of 0.3 to 16.4 μg/l and found to be of third order with a coefficient of determination greater than 0.999. The relative standard deviation was found to be lower than 15%. A lower limit of detection (LLOD) of 0.17 μg/l and a lower limit of quantification (LLOQ) of 0.3 μg/l were determined.
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June 1, 2005
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A specific method has been developed for the quantitative determination of nicotine and its major metabolite cotinine in plasma or serum of active and passive smokers. Deuterium-labelled nicotine and cotinine were used as internal standards. The amounts of nicotine and cotinine present in a sample of plasma or serum were extracted with a simple extraction procedure (liquid-liquid or solid-phase extraction). The extracts were analysed by gas chromatography coupled with mass spectrometry using ion-trap detection. The analysis was done in positive chemical ionisation with methanol as the liquid reagent. The method has been demonstrated to be linear up to 1000 μg/l. Limits of quantification for nicotine and cotinine are 10 and 5 μg/l, respectively with liquid-liquid extraction, and 1 μg/l for each of the compounds with solid-phase extraction. The present method has been applied to several real cases.
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June 1, 2005
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A reliable and sensitive procedure is presented for the analysis of nitrofuran metabolite residues in foods of animal origin, like poultry and shrimp. The method is based on a one-step extraction/derivatization, followed by solid-phase extraction clean-up with polymeric phase cartridges. The separations were carried out by liquid chromatography coupled with tandem mass spectroscopy detection. Results from the full validation of the procedure and from a survey of food available from the Swiss market, with the analysis of 236 samples of shrimp, poultry, fish and rabbit, are presented and demonstrate that this method is satisfactory in order to control nitrofuran residues in foods of animal origin.
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June 1, 2005
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Methadone (MTD) is a chiral drug widely used for the treatment of opioid dependence for which a rapid analytical method for the determination of each enantiomer would be advantageous. In order to improve method sensitivity and to automate the entire analytical process, a column-switching configuration has been developed. An online extraction system coupled to a cellulose-based chiral stationary phase (CSP), namely Chiralcel OJ-R, was used and detection was performed by mass spectrometry. Fifty μl of plasma were injected into the liquid chromatography-mass spectrometry (LC-MS) system after addition of acetonitrile (ACN) containing methadone deuterated D9 (MTD-D 9 ) (internal standard) and centrifugation. For the rapid extraction step, a large particle size support was selected. A baseline separation of MTD enantiomers was obtained in less than 12 min. Trueness and precision were evaluated with control samples at 500 ng/ml of (R,S)-methadone. Trueness values were 106.6% and 103.0% for (R)-MTD and (S)-MTD, respectively, with a coefficient of variation inferior to 2.5% for both compounds. Finally, a good concordance was found using this method for analysis of plasma samples from patients in maintenance treatment as compared to a previously described HPLC-UV method after liquid-liquid extraction.
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June 1, 2005
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Strains of mice that are susceptible to autoimmunity have provided experimental models to analyze the molecular basis for the complex multifactorial inheritance of human autoimmune disease. In this study proteins associated with collagen-induced arthritis (CIA) in mice were experimentally identified using a global proteomics approach. Two-dimensional gels of proteins from inflamed and non-inflamed joints showed a distinguished protein profile visualizing about 530 Coomassie-stained protein spots in the pH 4–7 range. A total of 76 spots were identified by peptide mass fingerprinting with good confidence. They included proteins of cytoskeletal origin, chaperones, enzymes and also some signal transduction molecules. Comparison to gels from non-inflamed paws pointed to proteins that were differentially expressed between the control and diseased state. Ferritin light chain and antioxidant protein 2 were slightly more abundant, lymphoid enhancer binding factor 1 slightly, but significantly, less abundant in inflamed paws. Fourteen of the identified proteins were the products of genes that had increased transcript levels in the diseased state. However, on the protein level no significant differences were found in comparison to the controls. This study provides us with the framework for more detailed approaches to understanding the complex disease arthritis that go beyond global proteomics.
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June 1, 2005
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Bile acids play a pivotal role in the metabolism of cholesterol and lipids. Their blood concentrations are important prognostic and diagnostic indicators of hepatobiliary and intestinal dysfunction. This class of molecules comprises a heterogeneous group of compounds with a common cholesterol scaffold. Recently, the introduction of liquid chromatography coupled to tandem mass spectrometry methods has revealed an innovative path in the quantisation of specific bile acids in biological specimens. A robust and sensitive method has been developed based on high performance liquid chromatography separation coupled to an electrospray triple-quadrupole mass spectrometer. Human plasma samples were analysed on a C18 reverse-phase column. The elution profiles were monitored in multiple reaction-monitoring mode, quantifying and identifying each analyte by its own unique precursor to product patterns. A linear correlation over a broad range of bile acid concentrations (0.1–100 μM) was observed. The average recovery period for all of the analysed bile acids was 98±3%. Intra-day and inter-day precision averages were 2% and 5.4%, respectively. The determination was achieved within a single chromatographic run for all unconjugated, glycine-and taurine-conjugated isomeric forms of bile acids. As a proof of principle this method has been validated on a small subset of cholestatic patients (n = 7) and compared to appropriate clinical controls (n = 10). Based upon our encouraging experimental results, the described HPLC separation coupled to tandem mass spectrometry method for the analysis of bile acids in biological samples is deemed a robust and accurate procedure. Consequently, we propose this technique as a suitable candidate method for the identification and quantitation of bile acids in routine analysis.
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June 1, 2005
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Organic acid analysis is used for the early detection/exclusion and for the follow-up of inherited disorders of amino acid and organic acid metabolism. Urinary organic acid concentrations in 417 healthy Caucasian children (1 day to 17 years of age) were determined after liquid solid extraction, as their trimethylsilyl derivatives, by gas chromatography and mass spectrometry. Concentrations of most of the organic acids adjusted for creatinine tend to decrease with age. No differences were found between gender except for the Krebs cycle intermediates in the older age groups. In neonates, the immaturity of the neonatal kidney led to a much larger variation of organic acid levels when related to creatinine. The low number of subjects (n = 36−52) per age class resulted in large 95% confidence intervals of the percentiles used for decision. This must be taken into account when using the data for exclusion or diagnosis of disorders.
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July 27, 2005
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