RESUMEN
Counterfeit watches are products of illicit activity and contain traces of their production and distribution. Traces provide pertinent information through one of their fundamental characteristics: the ability to reveal links between specimens or cases. The aim of this study was to develop an analytical strategy to obtain the elemental composition of watchcases, by analysing a selection of 35 counterfeit watches. We propose a methodology based on multivariate statistical analysis of chemical results that discriminates between watches from common and different origins, and, ultimately, classifies them into chemical groups. All watchcases were analysed using inductively coupled plasma mass spectrometry (ICP-MS), providing representative descriptive data on the composition of watchcases. Several multivariate approaches were assessed, considering different scenarios, each using a different set of variables. It appeared that the model that performed best in terms of classification criteria could be misleading, especially in an exploratory context that focuses on the production of intelligence. At the end of the day, hierarchical cluster analysis (HCA) allowed us to classify the specimens into 14 chemical classes. Information gained through chemical analysis revealed several links between the specimens. This initial study was performed on a very limited number of watches. Although still in the developmental stage, our approach exhibits promising capabilities and encourages chemical profiling of counterfeit watches on larger scale.
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To date, the development of bioreactors for the study of red blood cells (RBCs, daily transfused in the case of disease or hemorrhage) has focused on hematopoietic stem cells. Despite the fact that mature RBCs are enucleated and do not expand, they possess complex cellular and metabolic pathways, as well as post-translation modification signaling and gas-exchange regulation. In order to dynamically study the behavior of RBCs and their signaling pathways under various conditions, a small-scale perfusion bioreactor has been developed. The most advanced design developed here consists of a fluidized bed of 7.6 mL containing 3·10(9) cells and perfused at 8.5 µL/min. Mimicking RBC storage conditions in transfusion medicine, as a proof-of-concept, we investigated the ex vivo aging of RBCs under both aerobic and anaerobic conditions. Hence, RBCs stored in saline-adenine-glucose-mannitol (SAGM) were injected in parallel into two bioreactors and perfused with a modified SAGM solution over 14 days at room temperature under air or argon. The formation of a fluidized bed enabled easy sampling of the extracellular medium over the storage period used for the quantitation of glucose consumption and lactate production. Hemolysis and microvesiculation increased during aging and were reduced under anaerobic (argon) conditions, which is consistent with previously reported findings. Glucose and lactate levels showed expected trends, i.e., decreased and increased during the 2-week period, respectively; whereas extracellular glucose consumption was higher under aerobic conditions. Metabolomics showed depletion of glycolsis and pentose phosphate pathway metabolites, and an accumulation of purine metabolite end-products. This novel approach, which takes advantage of a fluidized bed of cells in comparison to traditional closed bags or tubes, does not require agitation and limit shear stress, and constantly segragates extracellular medium from RBCs. It thus gives access to several difficult-to-obtain on- and off-line parameters in the extracellular medium. This dynamic bioreactor system does not only allow us to probe the behavior of RBCs under different storage conditions, but it also could be a powerful tool to study physiological or pathological RBCs exposed to various conditions and stimuli.
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High-resolution mass spectrometry (HRMS) has been associated with qualitative and research analysis and QQQ-MS with quantitative and routine analysis. This view is now challenged and for this reason, we have evaluated the quantitative LC-MS performance of a new high-resolution mass spectrometer (HRMS), a Q-orbitrap-MS, and compared the results obtained with a recent triple-quadrupole MS (QQQ-MS). High-resolution full-scan (HR-FS) and MS/MS acquisitions have been tested with real plasma extracts or pure standards. Limits of detection, dynamic range, mass accuracy and false positive or false negative detections have been determined or investigated with protease inhibitors, tyrosine kinase inhibitors, steroids and metanephrines. Our quantitative results show that today's available HRMS are reliable and sensitive quantitative instruments and comparable to QQQ-MS quantitative performance. Taking into account their versatility, user-friendliness and robustness, we believe that HRMS should be seen more and more as key instruments in quantitative LC-MS analyses. In this scenario, most targeted LC-HRMS analyses should be performed by HR-FS recording virtually "all" ions. In addition to absolute quantifications, HR-FS will allow the relative quantifications of hundreds of metabolites in plasma revealing individual's metabolome and exposome. This phenotyping of known metabolites should promote HRMS in clinical environment. A few other LC-HRMS analyses should be performed in single-ion-monitoring or MS/MS mode when increased sensitivity and/or detection selectivity will be necessary.
Asunto(s)
Cromatografía Liquida/métodos , Espectrometría de Masas en Tándem/métodos , Límite de DetecciónRESUMEN
The aim of this work was to identify Lactobacillus johnsonii NCC533 (La1) surface molecules mediating attachment to intestinal epithelial cells and mucins. Incubation of Caco-2 intestinal epithelial cells with an L. johnsonii La1 cell wall extract led to the recognition of elongation factor Tu (EF-Tu) as a novel La1 adhesin-like factor. The presence of EF-Tu at the surface of La1 was confirmed by analysis of purified outer surface protein extract by immunoblotting experiments, by electron microscopy, and by enzyme-linked immunosorbent assays of live bacteria. Furthermore, tandem mass spectrometry analysis proved that EF-TU was expressed at the La1 surface as an intact molecule. Using recombinant La1 EF-Tu protein, we were able to determine that its binding to intestinal cells and to mucins is pH dependent. Competition experiments suggested that EF-Tu has an important role in La1 mucin binding capacity. In addition, immunomodulation studies performed on HT29 cells showed that EF-Tu recombinant protein can induce a proinflammatory response in the presence of soluble CD14. Our in vitro results indicate that EF-Tu, through its binding to the intestinal mucosa, might participate in gut homeostasis.
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Adhesión Bacteriana , Pared Celular/metabolismo , Intestinos/microbiología , Lactobacillus/patogenicidad , Mucinas/metabolismo , Factor Tu de Elongación Peptídica/metabolismo , Adhesinas Bacterianas/química , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/inmunología , Adhesinas Bacterianas/metabolismo , Secuencia de Aminoácidos , Animales , Células CACO-2 , Línea Celular , Humanos , Inflamación , Interleucina-8/metabolismo , Intestinos/química , Intestinos/citología , Lactobacillus/metabolismo , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Factor Tu de Elongación Peptídica/química , Factor Tu de Elongación Peptídica/genética , Factor Tu de Elongación Peptídica/inmunología , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismoRESUMEN
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-Tof-MS) has recently become a popular and versatile method to analyze macromolecules from biological origin. In this paper, we will review the application of MALDI-Tof-MS in clinical chemistry and biology. MALDI-Tof-MS is used in clinical chemistry, e.g. disease markers can be identified with MALDI-MS analysis in combination with 1-D and 2-D gel electrophoresis separations thanks to either peptide mass fingerprinting (PMF) or peptide sequence tag (PST) followed by data base searching. In microbiology, MALDI-Tof-MS is employed to analyze specific peptides or proteins directly desorbed from intact viruses, bacteria and spores. The capability to register biomarker ions in a broad m/z range, which are unique and representative for individual microorganisms, forms the basis of taxonomic identification of bacteria by MALDI-Tof-MS. Moreover, this technique can be applied to study either the resistance of bacteria to antibiotics or the antimicrobial compounds secreted by other bacterial species. More recently, the method was also successfully applied to DNA sequencing (genotyping) as well as screening for mutations. High-throughput genotyping of single-nucleotide polymorphisms has the potential to become a routine method for both laboratory and clinical applications. Moreover, posttranscriptional modifications of RNA can be analyzed by MALDI using nucleotide-specific RNAses combined with further fragmentation by post source decay (PSD).
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Química Clínica/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Alérgenos/análisis , Enfermedad de Alzheimer/líquido cefalorraquídeo , Enfermedad de Alzheimer/diagnóstico , Artritis Reumatoide/diagnóstico , Artritis Reumatoide/inmunología , Bacterias/química , Biomarcadores/análisis , Análisis Mutacional de ADN/métodos , Humanos , Estructura Molecular , Neoplasias/química , Neoplasias/diagnóstico , Proteínas/análisis , Análisis de Secuencia de ADN/métodos , Esporas/química , Virus/químicaRESUMEN
Quantification of o-tyrosine, o-nitrotyrosine, and o,o'dityrosine from cat urine samples was achieved by LC/ electrospray ionization-MS/MS (LC/ESI-MS/MS) using an isotope dilution technique in multiple reaction monitoring mode before butylation of o,o'-dityrosine and after butylation of o-tyrosine and o-nitrotyrosine. This novel approach of amino acids butylation enhanced the MS response by a factor of 7-fold for o-tyrosine and 6-fold for o-nitrotyrosine and decreased the overall chemical background noise. Butylation of o,o'-dityrosine resulted in a lower MS response as a result of the formation of both mono- and doubly butylated species. The mean recovery for the oxidized amino acids was estimated at 73 +/- 2%. The limits of quantitation of NO2-Tyr butyl ester, o-Tyr butyl ester, and di-Tyr in cat urine samples were calculated at 14.5, 28.2 and 140.9 nM, respectively. The oxidized amino acids levels in cat urine extracts ranged from 157 to 250 ng/day for o-Tyr and from 3,289 to 11,803 ng/day for di-Tyr. NO2-Tyr was found in only two urine extracts at levels below 58 ng/day. A certain trend of correlation was observed between o,o'-dityrosine and o-tyrosine when comparing these values against their respective creatinine amounts. A comparison of the data gathered from the ThermoFinnigan TSQ 7000 and Micromass Q-TOF instruments revealed several advantages of using the Q-TOF regarding the exact mass measurement, a lower ion suppression effect and the possibility to perform analyses in full scan product ion mode. These results demonstrate that a Q-TOF instrument can be a good alternative to classical triple quadrupole for quantitative purposes on a relatively small linear dynamic range (4 orders of magnitude for the Q-TOF, as compared to 6 for the triple quadrupole).
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Espectrometría de Masa por Ionización de Electrospray/instrumentación , Tirosina/análogos & derivados , Tirosina/orina , Animales , Gatos , Cromatografía Líquida de Alta Presión , Isótopos , Espectrometría de Masa por Ionización de Electrospray/normasRESUMEN
Infant formula powders were analyzed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) to assess the whey proteins quality, which may be altered by the heat treatment used during the processing conditions. Lactosylation was found to be the major chemical modification occurring in whey proteins. In parallel, a two-dimensional (2-D) gel electrophoresis was performed on the milk sample and the entire protein patterns were analyzed by nano-ESI-MS after cutting the different gel spots and in-gel trypsin digestion. A highly selective and specific tandem MS technique has been developed to characterize and localize up to ten lactosylation sites in beta-lactoglobulin (beta-Lg) and alpha(S2)-casein. alpha-Lactalbumin (alpha-La), with five lactosylated peptides, was found to be an interesting protein marker in the milk powder sample to detect chemical modification induced by the processing/storage conditions.
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Electroforesis en Gel Bidimensional/métodos , Alimentos Infantiles/análisis , Lactosa/química , Proteínas de la Leche/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Animales , Caseínas/química , Humanos , Lactante , Lactalbúmina/química , Lactoglobulinas/química , Nanotecnología , Polvos , Proteína de Suero de LecheRESUMEN
Egg mass extract was used to characterize regulatory peptides, involved in the successive steps of egg-laying of the cuttlefish Sepia officinalis. Among these peptides, a C-terminally amidated hexapeptide revealed a sperm-attracting activity. MALDI-TOF MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry) and Edman degradation led to a peptide of m/z 596.6 and the following primary sequence: Pro-Ile-Asp-Pro-Gly-Val-CO(NH2). From concentrations as low as 10(-17)M, the PIDPGVamide was able to attract freshly dissected spermatozoa. Nano-ESI-Q-TOF MS (nano-electrospray ionization-quadrupole-time-of-flight mass spectrometry) analysis established the quantitative occurrence of this peptide in different egg structures. The PIDPGVamide appears to be synthesized in oocytes during vitellogenesis and released by the embedded oocytes in the external media during egg-laying to facilitate fertilization by increasing chances of gamete collision. This novel peptide called SepSAP for Sepia sperm attracting peptide is the first sperm-attracting peptide, identified in mollusks or even in protostomians.
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Moluscos/fisiología , Oligopéptidos/farmacología , Transporte Espermático/efectos de los fármacos , Espermatozoides/fisiología , Animales , Bioensayo , Quimiotaxis , Relación Dosis-Respuesta a Droga , Femenino , Cinética , Masculino , Modelos Biológicos , Oligopéptidos/análisis , Oligopéptidos/química , Oocitos/química , Oviductos/efectos de los fármacos , Oviductos/fisiología , Espectrometría de Masa por Ionización de Electrospray , Espermatozoides/citologíaRESUMEN
OBJECTIVE: To identify a new autoantigen/autoantibody population in rheumatoid arthritis (RA) sera. METHODS: Following a population-based recruitment effort, 255 patients with very early arthritis (median disease duration 4 months) were studied using different clinical, biologic, and radiologic assessments. After a followup period of 1 year, patients were classified as having RA (n = 145), non-RA rheumatic diseases (n = 70), and undifferentiated arthritis (n = 40). Patients' sera were analyzed by one-dimensional (1D) and 2D Western blotting. The recognized 50-kd protein was analyzed by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS). RA serum reactivities were evaluated against the recombinant protein synthesized by an in vitro coupled transcription-translation system. RESULTS: On 1D Western blots, 36 of the 145 RA sera bound to a 50-kd polypeptide. On 2D Western blots, anti-50-kd+ RA sera recognized a triplet of isoelectric point 6.5-7.0 and a molecular mass of 50 kd. The 3 spots of the triplet were analyzed by MALDI-TOF MS and were shown to correspond to human alpha-enolase. A goat anti-enolase antiserum, which recognized a band comigrating with the 50-kd antigen on 1D Western blots, gave a labeling pattern on 2D Western blots similar to that observed with anti-50-kd+ RA sera. Among the 36 RA sera that identified alpha-enolase in protein maps, only 8 recognized the recombinant (unmodified) alpha-enolase. The specificity of anti-alpha -enolase antibodies for RA was 97.1%. Half of the anti-alpha -enolase-positive RA patients were negative for both rheumatoid factor and antifilaggrin antibodies. The presence of anti-alpha-enolase antibodies was the greatest predictive factor of radiologic progression in the first 66 RA patients included. CONCLUSION: Autoantibodies to alpha-enolase, an enzyme of the glycolytic pathway, are present in the sera of patients with very early RA and have potential diagnostic and prognostic value for RA.