Matrix-assisted Laser Desorption/ionization Time-of-flight Mass Research Articles

Proteomic features characterization of Hymenoptera venom allergy

BackgroundHymenoptera venom allergy is one of the most frequent causes of anaphylaxis. In its most severe form, the reaction to wasp and honey bee stings may be life-threatening. Therefore, immediate and proper diagnosis of venom allergy and implementation of suitable therapy are extremely important. Broadening the knowledge on the mechanism of the allergic reaction may contribute to the improvement of both diagnostic and treatment methods. Thus, this study aimed to discover changes in protein expression in serum of patients allergic to Hymenoptera (wasp and honeybee) venom and to point out proteins and peptides involved in the allergic inflammation.MethodsSerum proteomic patterns typical to allergic patients and healthy volunteers were obtained with MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) mass spectrometer. The spectra were processed, analyzed and compared using advanced bioinformatics tools. The discriminative peaks were subjected to identification with liquid chromatography coupled with tandem mass spectrometry.ResultsThis methodology allowed for the identification of four features differentiating between allergy and control groups. They were: fibrinogen alpha chain, coagulation factor XIII chain A, complement C4-A, and inter-alpha-trypsin inhibitor heavy chain H4. All of these proteins are involved in allergic inflammatory response.ConclusionsExtending the knowledge of the Hymenoptera venom sensitization will contribute to the development of novel, sensitive and specific methods for quick and unambiguous allergy diagnosis. Understanding the basis of the allergy at the proteomic level will support the improvement of preventive and therapeutic measures.

Characterization and immunostimulating effects on murine peritoneal macrophages of a novel protein isolated from Panax quinquefolius L

Ethnopharmacological relevancePanax quinquefolius L. has been used as a proverbial tonic in oriental countries for hundreds of years. It is used as a traditional medicinal herb to nourish vitality. Aim of the studyThe purpose of our study was to inquiry the activation effects on murine peritoneal macrophages of a novel protein separated from the roots of Panax quinquefolius L. Materials and methodsIn our work, a novel protein of the roots of American ginseng (AGNP) was separated and purified from the roots of Panax quinquefolius L. The characteristic was investigated with SDS-PAGE, high pressure gel filtration chromatography (HPGFC) and matrix-assisted laser desorption ionization/time-of-flight mass (MALDI-TOF-MS) spectrometry method. The method of neutral red was carried out to investigate the phagocytosis of peritoneal macrophages. And Griess method and colorimetry were executed to detect the level of nitric oxide and iNOS activity respectively. Tumor necrosis factor-α and interleukin-6 were analyzed by enzyme linked immunosorbent assay (ELISA). ResultsOur results demonstrated that the subunit molecular weight of AGNP determined by SDS-PAGE was 15kD and the content of proteins determined by Bradford assay was 2.31mg/mL. The molecular weight of the AGNP was15, 114Da both of electrophoresis and MS purity. And the result of HPGFC showed that the molecular weight of AGNP was 31,086Da, Immunological studied indicated that AGNP could conspicuously increase phagocytosis of macrophages, facilitate the nitric oxide production, Tumor necrosis factor-α and interleukin-6 production. What is more, AGNP dose-dependently stimulated NO formation through the up-regulation of iNOS activity. ConclusionsIn conclusion, AGNP had good immunoregulatory effects supporting the traditional claims and may provide a valuable therapeutic strategy to promoting immune function and metabolism.

Estudios de coste-efectividad con MALDI-TOF e impacto clínico

In general, new technologies usually increase laboratory costs due to the need for an initial investment. However, as occurred with MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) mass spectrometry, this increase is subsequently offset by the discontinued use of traditional technologies and by the benefits to patients of the new information generated. In the clinical microbiology laboratory, the identification time is reduced with the use of MALDI-TOF (by at least 24 hours) and turnaround is improved, allowing faster production of the microbiological report. This beneficial effect has mainly been studied with blood cultures in patients with bacteraemia. In these patients, the length of hospital stay has been reduced by 1.6-6.6 days, depending on the type of patient and the appropriateness of treatment. This leads to better antimicrobial use and a reduction in total hospital cost of up to 43% per patient. Another factor that has been analysed is the decrease in mortality due to better management of antimicrobial therapy. Future multicentre studies should include other factors such as hospital organisation changes and clinical activity arising in response to the efforts of the clinical microbiology laboratory to rapidly obtain information of clinical value.

Changes in the Matrix Markedly Enhance the Resolution and Accurate Identification of Human Pathogens by MALDI-TOF MS

The mass spectral profiles of three major/emerging Gram positive pathogens belonging to the genera Clostridium, Listeria and Propionibacterium were investigated using four MALDI-TOF (Matrix-Assisted Laser Desorption/ Ionization- Time of Flight) Mass Spectrometers from Waters, Shimadzu, Bruker and Ciphergen Biosystems. These instruments have been extensively used for developing a diagnostic platform for high throughput, low cost, near sample-free preparation for microbial identification. The results demonstrate the marked effect of spectral quality obtained by altering the matrix and the added value of simple preparative extraction procedures. While microbial spectral data are generally collected in the mass range 2 to 20 kDa for diagnostic signatures, most of the significant mass ions are <10 kDa. The change from the traditional use of CMBT (5-chloro-2-mercaptobenzothiazole) to DHB (2-(2, 5-DiHydroxyBenzoic acid) enabled successful identification of C. difficile whereas the substitution of CHCA (alpha-cyano-4-hydroxycinnamic acid) for DHB (2,5-dihydroxybenzoic acid) improved the identification of Listeria species. By contrast, the use of SELDI-TOF-MS (Surface Enhanced Laser Desorption/Ionization- Time of Flight- Mass Spectrometry), which employs affinity pre-capture of specific classes of molecules and analysed using sinapinic acid, showed a fivefold extension of the mass range of analytes, extending the potential for biomarker discovery and higher resolution strain typing of P. acnes.

Galactooligosaccharide Synthesis by Active β-Galactosidase Inclusion Bodies-Containing Escherichia coli Cells

In this study, galactooligosaccharide (GOS) was synthesized using active β-galactosidase (beta-gal) inclusion bodies (IBs)- containing Escherichia coli (E. coli) cells. Analysis by MALDI-TOF (matrix-assisted laser desorption/ionizationtime of flight) mass spectrometry revealed that a trisaccharide was the major constituent of the synthesized GOS mixture. Additionally, the optimal pH, lactose concentration, amounts of E. coli β-gal IBs, and temperature for GOS synthesis were 7.5, 500 g/l, 3.2 U/ml, and 37 °C, respectively. The total GOS yield from 500 g/l of lactose under these optimal conditions was about 32%, which corresponded to 160.4 g/l of GOS. Western blot analyses revealed that β-gal IBs were gradually destroyed during the reaction. In addition, when both the reaction mixture and E. coli β-gal hydrolysate were analyzed by high-performance thin-layer chromatography (HP-TLC), the trisaccharide was determined to be galactosyl lactose, indicating that a galactose moiety was most likely transferred to a lactose molecule during GOS synthesis. This GOS synthesis system might be useful for the synthesis of galactosylated drugs, which have recently received significant attention owing to the ability of the galactose molecules to improve the drugs solubility while decreasing their toxicity. β-Gal IB utilization is potentially a more convenient and economic approach to enzymatic GOS synthesis, since no enzyme purification steps after the transgalactosylation reaction would be required.

Comment on: Identification of antimicrobial activity among FDA-approved drugs for combating Mycobacterium abscessus and Mycobacterium chelonae

Sir, In the article titled ‘Identification of antimicrobial activity among FDA-approved drugs for combating Mycobacterium abscessus and Mycobacterium chelonae’ published in the July 2011 issue of JAC, it was reported that the FDA-approved drug metronidazole has potent activity (<0.015 mg/L; ∼87 nM) against the rapid-growing mycobacteria (RGM) Mycobacterium abscessus.1 This finding intrigued us and our clinical colleagues who treat M. abscessus-infected patients here at NIH. We therefore tried to repeat their experiment with M. abscessus ATCC 19977 and some recent patient isolates of M. abscessus to evaluate their susceptibility to metronidazole. We regret to report that we did not observe any MIC99, even at the highest concentration of the drug we tested (2 mM) after 48, 72 and 96 h. In accordance with the method published in the paper, we grew the M. abscessus ATCC 19977 (and four clinical isolates) from glycerol stock in 7H9 Middlebrook medium, passaged them once and let them grow to an OD650 of 0.2–0.4. The cell cultures were then diluted to an OD650 of 0.0002 in 7H9 medium and 50 μL of the cells was added to 50 μL of drug at various concentrations in a 96-well plate. They were incubated at 37°C for 48, 72 and 96 h and no killing of this RGM was observed (Figure 1a). Figure 1. (a) MICs of drugs for M. abscessus ATCC 19977 after 72 h. Each of the drugs was assayed in duplicate. MTZ, metronidazole. Each drug was serially diluted (50% each time) from well 1 to well 11, and well 12 does not have any drug. The concentration (in ... It is known that M. abscessus is susceptible to clarithromycin,2 and we therefore included this as a positive control in the experiment. PA-824, another nitroimidazole that is currently in Phase II clinical trials for the treatment of tuberculosis, has shown aerobic as well as anaerobic activity against the slow-growing mycobacteria Mycobacterium tuberculosis.3 It was also tested for activity against the RGM M. abscessus. The susceptibility of M. abscessus to clarithromycin clearly shows that there was no flaw in the experimental design, yet M. abscessus grew in the presence of 100 μM metronidazole and 50 μM PA-824 (Figure 1a). To investigate the susceptibility of M. abscessus ATCC 19977 and four clinical isolates to a higher concentration of metronidazole we determined the MIC99 starting with a 2 mM concentration of metronidazole, but we still did not observe any cidal effect of metronidazole even at such a high concentration (Figure 1b). In addition, no killing was observed with PA-824 at a concentration of 2 mM (data not shown). Strains tested have been identified as belonging to the M. abscessus group by partial sequencing of the secA gene4 and/or MALDI-TOF (matrix-assisted laser desorption ionization–time of flight) mass spectrometry.5 Metronidazole is known for its activity and has been used in the treatment of diseases caused by anaerobic microorganisms like Trichomonas vaginalis,6 Entamoeba histolytica7 and Clostridium difficile6 and microaerophilic bacteria like Helicobacter pylori.8 Though metronidazole is active against hypoxically adapted non-replicating M. tuberculosis, it is inactive against rapidly replicating M. tuberculosis under aerobic conditions.9 Metronidazole, which belongs to the class of 5-nitroimidazoles, has a single electron reduction potential of −0.4 to −0.5 V, which is out of the range normally accessible to the mammalian redox system. Under a limiting oxygen concentration in microbes the redox potential range is −0.42 V and lower and is suitable for the bioreductive activation of metronidazole. Upon single electron reduction of nitroimidazole, a nitro radical anion is produced,10 which could reduce oxygen in a microaerophilic organism to form superoxide.11 In the presence of transition elements like iron or copper, the hydrogen peroxide and superoxide react to form a hydroxyl radical by the Haber–Weiss reaction,10 causing inhibition of DNA synthesis and DNA strand breakage by this futile cycle. Metronidazole induces the expression of SOS response genes, which are responsible for DNA repair by the RecA pathway,10 as a result of which recA mutants in Escherichia coli and H. pylori are more susceptible to metronidazole treatment.12,13 Under normoxic conditions reduction of molecular oxygen by reduced metronidazole would be expected to render it harmless, as superoxide formed would be rapidly decomposed by the microbial superoxide dismutase.10 Our finding clearly demonstrates that metronidazole is inactive against M. abscessus under aerobic conditions, as has been reported. Metronidazole is potent against anaerobic organisms, but cannot be used in the treatment of infection with M. abscessus. Metronidazole does not kill RGM M. abscessus at a concentration of 2 mM (342.3 mg/L), which is more than 20 000-fold the concentration of what was reported by Chopra et al.1

Mass-spectrometry analysis of genetic markers of S. pneumoniae resistance to β-lactam antibiotics

Beta-lactam antibiotics remain the drugs of choice for treatment of S. pneumoniae infections in spite of growing level of resistance. The formation of S. pneumoniae resistance to these drugs is mediated by modifications of the penicillin-binding proteins (PBPs), the targets of the antibiotic action. A new approach to detection of mutations in PBP1A, 2B and 2X genes based on minisequencing reaction followed by MALDI-ToF (Matrix-Assisted Laser Desorption/Ionization Time of Flight) mass spectrometry was developed in this study. The evaluation of these mutations prevalence in clinical S. pneumoniae isolates (n = 194) with different susceptibility level to beta-lactam antibiotics was performed. Twenty-four different combinations of mutations in PBPs (genotypes) were detected. All isolates susceptible to penicillin (n = 49, MIC > or = 0.06 > or = gamma/ml) carried no mutations in all analyzed loci. For 145 S. pneumoniae isolates with reduced susceptibility to penicillin (MIC > 0.06 > or = gamma/ml) the mutations in PBPs were detected in 133 (91.7 %) cases that testify to high diagnostic sensitivity of such approach. The isolates with MIC > or = 4 > or = gamma/ml (n = 20) carried multiple mutations in all analyzed genes that confirms cumulative effects of penicillin resistance formation. However, it was not possible to associate observed mutations in PBPs genes with decrease of susceptibility to cefotaxime that allows suggesting the entire difference in molecular mechanisms of formation of resistance to penicillins and cephalosporins. The offered method of S. pneumoniae genotyping is suitable for susceptibility testing to penicillin of individual isolates and for molecular monitoring of the resistance determinants in population.

Synthesis and optical properties of tetrapyrazinoporphyrazine containing phenylene dendron unit as a peripheral substituent

A dendritic shell can create a distinct micro-environment within its core. It also has the advantage of possessing unique photochemical, photophysical, electrochemical, and catalytic properties. Polyphenylene dendrons, which are characterized by their shape-persistent structures and out-of-plane twisted phenyl components, have previously been successfully attached to various functional groups. We have recently developed a convenient method for synthesizing a new type of porphyrazine that contains both flexible (linear) and more rigid (dendritic) groups. The synthesis of this completely aromatic and dendronic structure is unique in that it is based on a [2+4] Diels-Alder cycloaddition of tetraphenylcyclopentadienone to an ethynyl compound, followed by the elimination of carbon monoxide. In this study, tetrapyrazinoporphyrazinato metal and metal-free complexes were prepared by mixing 2,3-dicyano-5-polyphenylpyrazines with magnesium in n-butanol. The synthesized tetrapyrazinoporphyrazines were characterized by UV-visible spectroscopy, MALDI-TOF-MS (matrix-assisted laser desorption/ionization time-of-flight mass) spectrometry, elemental analysis and 1 H NMR spectroscopy.

Synthesis of perialkynylated tetrapyrazinoporphyrazines and its Optical Properties

Some phthalocyanines soluble in organic solvents have been developed by peripheral introduction of substituent groups. We report a new method for preparation of the polyphenyl-substituted dicyanopyrazines based on the [2 + 4] Diels-Alder cycloaddition of the tetraphenylcyclopentadienone to an ethynyl compound. The synthesised tetrapyrazinoporphyrazinato metal complexes were characterised by UV-visible spectroscopy, MALDI-TOF-Ms (matrix-assisted laser desorption ionisation time-of-flight mass) spectroscopy, and 1H NMR spectroscopy.

Synthesis of polyphenylene dendrimeric tetrapyrazinoporphyrazines

In this study, new tetrapyrazinoporphyrazine derivatives containing both flexible (linear) and more rigid (dendritic) groups were synthesized. The absorption spectra of Mg-porphyrazines 5a and 5b showed that the splitted Q band at 653 and 736 nm caused the coupling of the transition moment between a pair (or more) of chromophores. The absorption spectra of metal-free porphyrazines 6 exhibited a well-resolved splitting of the Q x /Q y band in the 651–679 nm region and a broad B band in the 377 nm region. Synthesized tetrapyrazinoporphyrazine metal and metal-free complexes were characterized by UV–visible spectroscopy, MALDI-TOF-Ms (matrix-assisted laser desorption ionization time-of-flight mass) spectroscopy and 1H NMR spectroscopy.

Isolation, Phenotypic, Molecular and First-Time Proteomic Characterization of Human Mesenchymal Stem Cells (MSCs) Derived from Amniotic Fluid: Comparison to Bone Marrow MSCs.

Human mesenchymal stem cells (hMSCs) constitute a population of multipotent cells, easily expanded in culture and able to give rise to many lineages. These characteristics make MSCs a very attractive tool for developing new strategies for clinical applications based on cell therapy. So far, the most common source of MSCs has been the bone marrow (BM). However, identification and characterization of alternative sources of MSCs is of great importance. One such alternative source is the amniotic fluid (AF), which can be collected during scheduled amniocentesis without any ethical concerns.To this end, in the present study, we introduced an improved protocol for isolating and clonally expanding fetal MSCs from second trimester amniotic fluid (AF) and we further characterized these cells based on theirphenotype,pluripotency,differentiation potential andproteomic profile.The AF samples were obtained during routine amniocentesis and AF-MSCs were enriched by a modified culture protocol. The isolated MSCs expanded rapidly and exhibited differentiation potential into adipocytes and osteoblasts. More importantly, we showed that these cells can differentiate in vitro not only into cell types derived from mesoderm (adipocytes and osteoblasts) and ectoderm (neural cells) but also more interestingly into endoderm (hepatocytes) derived cells. Moreover, we documented that AF-MSCs express Oct-4 transcription factor, a marker of pluripotency, and we studied for the first time its expression over different passages by real time PCR and documented that it remained constant for at least 17 doublings.An extensive characterization of the phenotypic features of AF-MSCs by using a wide range of surface markers and flow cytometry, indicated that they are positive for all the mesenchymal stem cell markers such as CD90, CD105, CD73 and CD166 and generally exhibit a similar expression pattern to the BM-MSCs.To characterize these cells in more detail, we established the first proteomic database for human AF-MSCs. Using 2D-gel electrophoresis and matrix-assisted laser desorption ionisation-time of flight-mass (MALDI-TOF) spectrometry approach, we have generated for the first time the protein map of AF MSCs, by identifying 260 proteins and directly compared this protein profile with that of MSCs derived from BM. We further performed a similar analysis for BM-MSCs, identifying 170 different proteins and generating a reference map for these cells. The comparison of the proteomic pattern from both sources was similar. In general, 140 proteins were identified in AF-MSCs related to cell growth/maintenance, metabolism/energy pathways, protein metabolism, apoptosis, signal transduction and communication as well as transcription and transport, that are not present in BM-MSCs.The approach we initiated, is expected to facilitate systematic functional studies for these multipotent cells. One such approach could be the implementation of the proteomic analysis, during differentiation of AF-MSCs to cells derived from all three germ layers as shown in our study. Data derived from these approaches are expected to clarify the therapeutic potential of the MSCs.

Palmitoylation and Intracellular Domain Interactions Both Contribute to Raft Targeting of Linker for Activation of T Cells

Some transmembrane proteins must associate with lipid rafts to function. However, even if acylated, transmembrane proteins should not pack well with ordered raft lipids, and raft targeting is puzzling. Acylation is necessary for raft targeting of linker for activation of T cells (LAT). To determine whether an acylated transmembrane domain is sufficient, we examined raft association of palmitoylated and nonpalmitoylated LAT transmembrane peptides in lipid vesicles by a fluorescence quenching assay, by microscopic examination, and by association with detergent-resistant membranes (DRMs). All three assays detected very low raft association of the nonacylated LAT peptide. DRM association was the same as a control random transmembrane peptide. Acylation did not measurably enhance raft association by the first two assays but slightly enhanced DRM association. The palmitoylated LAT peptide and a FLAG-tagged LAT transmembrane domain construct expressed in cells showed similar DRM association when both were reconstituted into mixed vesicles (containing cell-derived proteins and lipids and excess artificial raft-forming lipids) before detergent extraction. We conclude that the acylated LAT transmembrane domain has low inherent raft affinity. Full-length LAT in mixed vesicles associated better with DRMs than the peptide. However, cells appeared to contain two pools of LAT, with very different raft affinities. Since some LAT (but not the transmembrane domain construct) was isolated in a protein complex, and the Myc- and FLAG-tagged forms of LAT could be mutually co-immunoprecipitated, oligomerization or interactions with other proteins may enhance raft affinity of one pool of LAT. We conclude that both acylation and other factors, possibly protein-protein interactions, target LAT to rafts.

Sample Preparation Optimization for the Analysis of Gliadins in Food by Matrixassisted Laser DesorptionIonization Timeofflight Mass Spectrometry

Analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS) of wheat gliadin extracts from distinct types of wheat varieties displays a similar characteristic gliadin mass pattern within the 30–40 kDa range. Direct observation of this pattern in food samples was used to identify gluten gliadins in food rapidly and easily. A modified sample preparation for gliadin analysis by MALDI/TOF-MS has improved sensitivity of gliadin detection by a factor of more than 50. This procedure allows the direct detection of complex mixtures of gluten gliadins in food sample extracts down to a concentration of 0.01 mg ml-1, i.e. down to levels which are toxic for coeliac patients. This highly sensitive improved method has been also exemplified by the facile detection of individual proteins at low concentrations (100–400 pmol ml-1). © 1997 by John Wiley & Sons, Ltd.

Highresolution Mass Spectrometry and Accurate Mass Measurements with Emphasis on the Characterization of Peptides and Proteins by Matrixassisted Laser DesorptionIonization Timeofflight Mass Spectrometry

Highresolution Mass Spectrometry and Accurate Mass Measurements with Emphasis on the Characterization of Peptides and Proteins by Matrixassisted Laser DesorptionIonization Timeofflight Mass Spectrometry

Chemical cross-linking of the human immunodeficiency virus type 1 Tat protein to synthetic models of the RNA recognition sequence TAR containing site-specific trisubstituted pyrophosphate analogues.

A chemical ligation procedure has been developed for the synthesis of oligoribonucleotides carrying a trisubstituted pyrophosphate (tsp) linkage in place of a single phosphodiester. Good yields of tsp were obtained when a single 2'-deoxynucleoside 5' to the tsp was used in the ligation reaction. A tsp linkage was found to be reasonably stable to hydrolysis but cleaved by treatment with ethylenediamine or lysine to give phosphoamidate adducts. A model human immunodeficiency virus type 1 (HIV-1) TAR RNA duplex containing an activated tsp was able to bind to HIV-1 Tat protein with only 3-fold reduced affinity and to a Tat peptide (residues 37-72) with identical affinity compared to that of an unmodified duplex. Tsps incorporated at sites previously identified as being in close proximity to Tat protein were able to cross-link to Tat peptide (37-72) to form a covalent phosphoamidate conjugate. Endopeptidase cleavage followed by MALDI-TOF (matrix-assisted laser desorption ionization time of flight) mass spectrometric analysis provided strong evidence that a TAR duplex containing a tsp replacing the phosphate at 38-39 had reacted specifically with Lys51 in the basic region of Tat peptide (37-72). The new chemical cross-linking method may be generally useful for identifying lysines in close proximity to phosphates in basic RNA-binding domains of proteins.