Comparison and optimization of strategies for a more profound profiling of the sialylated N-glycoproteomics in human plasma using metal oxide enrichment

Glycosylation is an important posttranslational modification of proteins and plays a crucial role in both cellular functions and secretory pathways. Sialic acids (SAs), a family of nine-carbon-containing acidic monosaccharides, often terminate the glycan structures of cell surface molecules and secreted glycoproteins and perform an important role in many biological processes. Hence, a more profound profiling of the sialylated glycoproteomics may improve our knowledge of this modification and its effects on protein functions. Here, we systematically investigated different strategies to enrich the SA proteins in human plasma using a newly developed technology that utilizes titanium dioxide for sialylated N-glycoproteomics profiling by mass spectrometry. Our results showed that using a combination of a filter-aided sample preparation method, TiO₂ chromatography, multiple enzyme digestion, and two-dimensional reversed-phase peptide fractionation led to a more profound profiling of the SA proteome. In total, 982 glycosylation sites in 413 proteins were identified, among which 37.8 % were newly identified, to establish the largest database of sialic acid containing proteins from human plasma.

Evaluation of the toxicity of ZnO nanoparticles to Chlorella vulgaris by use of the chiral perturbation approach

The toxicity of ZnO nanoparticles (NPs) has been widely investigated because of their extensive use in consumer products. The mechanism of the toxicity of ZnO NPs to algae is unclear, however, and it is difficult to differentiate between particle-induced toxicity and the effect of dissolved Zn²⁺. In the work discussed in this paper we investigated particle-induced toxicity and the effects of dissolved Zn²⁺ by using the chiral perturbation approach with dichlorprop (DCPP) as chiral perturbation factor. The results indicated that intracellular zinc is important in the toxicity of ZnO NPs, and that ZnO NPs cause oxidative damage. According to dose–response curves for DCPP and the combination of ZnO NPs with (R)-DCPP or (S)-DCPP, the toxicity of DCPP was too low to perturb the toxicity of ZnO NPs, so DCPP was suitable for use as chiral perturbation factor. The different glutathione (GSH) content of algal cells exposed to (R)-DCPP or (S)-DCPP correlated well with different production of reactive oxygen species (ROS) after exposure to the two enantiomers. Treatment of algae with ZnO NPs and (R)-DCPP resulted in reduced levels of GSH and the glutathione/oxidized glutathione (GSH/GSSG) ratio in the cells compared with the control. Treatment of algae with ZnO NPs and (S)-DCPP, however, resulted in no significant changes in GSH and GSH/GSSG. Moreover, trends of variation of GSH and GSH/GSSG were different when algae were treated with ZnSO₄·7H₂O and the two enantiomers. Overall, the chiral perturbation approach revealed that NPs aggravated generation of ROS and that released Zn²⁺ and NPs both contribute to the toxicity of ZnO NPs.

Microfluidic robotic device coupled with electrochemical sensor field for handling of paramagnetic micro-particles as a tool for determination of plant mRNA

The expression of genes responsible for the biosynthesis of stress proteins corresponds to the exposition of an organism to abiotic and/or biotic stress. We utilize two types of paramagnetic particles for isolation of total mRNA from early somatic embryos of Norway Spruce (Picea abies /L./ Karst.) and maize plants (Zea mays L.) treated with cadmium(II) ions. The paramagnetic particles were evaluated for analysis of real samples, and poly-adenine was used as a model mRNA. Various approaches (from non-automatic to fully automatic) were tested in terms of handling the particles.

Profiling and semiquantitative analysis of the cell surface proteome in human mesenchymal stem cells

Mulitpotent mesenchymal stem cells (MSCs) derived from human bone marrow are promising candidates for the development of cell therapeutic strategies. MSC surface protein profiles provide novel biological knowledge concerning the proliferation and differentiation of these cells, including the potential for identifying therapeutic targets. Basic fibroblast growth factor (bFGF) affects cell surface proteins, which are associated with increased growth rate, differentiation potential, as well as morphological changes of MSCs in vitro. Cell surface proteins were isolated using a biotinylation-mediated method and identified using a combination of one-dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis and mass spectrometry. The resulting gel lines were cut into 20 bands and digested with trypsin. Each tryptic fragment was analyzed by liquid chromatography–electrospray ionization tandem mass spectrometry. Proteins were identified using the Mascot search program and the International Protein Index human database. Noble MSC surface proteins (n?=?1,001) were identified from cells cultured either with (n?=?857) or without (n?=?667) bFGF-containing medium in three independent experiments. The proteins were classified using FatiGO to elucidate their function. We also confirmed the proteomics results using Western blotting and immunofluorescence microscopic analysis. The nature of the proteins identified makes it clear that MSCs express a wide variety of signaling molecules, including those related to cell differentiation. Among the latter proteins, four Ras-related Rab proteins, laminin-R, and three 14-3-3 proteins that were fractionated from MSCs cultured on bFGF-containing medium are implicated in bFGF-induced signal transduction of MSCs. Consequently, these finding provide insight into the understanding of the surface proteome of human MSCs.

Occurrence of venlafaxine residues and its metabolites in marine mussels at trace levels: development of analytical method and a monitoring program

Coastal areas are subject to growing pressures and impacts because of the increase in human activities. Lipophilic organic contaminants, such as polycyclic aromatic hydrocarbons (PAHs) or polychlorinated biphenyls (PCBs), have been monitored for decades within monitoring programs. However, until now, little information on the detection of so-called “emerging contaminants” such as hydrophilic organic compounds in the marine environment and no data on its metabolites or transformation products in marine organisms is available. In this report, a sensitive analytical methodology for identification and confirmation of venlafaxine (VEN) residues and five of its main metabolites in the marine mussels Mytilus galloprovincialis was validated. The sample preparation procedure was based on the Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) approach. An analytical method was developed to quantify these compounds at trace levels by liquid chromatography coupled to high-resolution mass spectrometry. The method was then applied to marine mussels collected from the Mediterranean Sea in southeastern France. Residues of the antidepressant VEN were occasionally detected at ng/g dw level. In addition, the approach allowed us to identify several transformation products in the analyzed samples. N-desmethylvenlafaxine (NDV) was the most frequently detected metabolite followed by N,O-di-desmethylvenlafaxine (NODDV).

Sorption of platinum on immobilized microorganisms for its on-line preconcentration and chemiluminescent determination in water samples

Fungi of the type Aspergillus sp. were immobilized on a cellulosic resin and used as a biosorbent for the on-line preconcentration and separation of Pt(IV) ions prior to their chemiluminescent determination via flow injection analysis. Biosorption and elution conditions were optimized, and the results compared to biosorbents based on the use of Chlorella vulgaris algae and Saccharomyces cerevisiae yeast in terms of preconcentration and selective retention of Pt(IV). The immobilized fungi presented here have a high potential for use in platinum biosorption. The procedure exhibits the currently lowest limit of detection (0.02 ng mL^?1 of Pt) and very high selectivity. The procedure was applied to the determination of Pt(IV) in river water, road run-off, and wastewater samples.

Mass Discrimination in High-Mass MALDI-MS

In high-mass matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), the accessible m/z range is limited by the detector used. Therefore, special high-mass detectors based on ion conversion dynodes (ICDs) have been developed. Recently, we have found that mass bias may exist when such ICD detectors are used [Weidmann et al., Anal. Chem. 85(6), 3425–3432 (2013)]. In this contribution, the mass-dependent response of an ICD detector was systematically studied, the response factors for proteins with molecular weights from 35.9 to 129.9 kDa were determined, and the reasons for mass bias were identified. Compared with commonly employed microchannel plate detectors, we found that the mass discrimination is less pronounced, although ions with higher masses are weakly favored when using an ICD detector. The relative response was found to depend on the laser power used for MALDI; low-mass ions are discriminated against with higher laser power. The effect of mutual ion suppression in dependence of the proteins used and their molar ratio is shown. Mixtures consisting of protein oligomers that only differ in mass show less mass discrimination than mixtures consisting of different proteins with similar masses. Furthermore, mass discrimination increases for molar ratios far from 1. Finally, we present clear guidelines that help to choose the experimental parameters such that the response measured matches the actual molar fraction as closely as possible.

Toxicological study of pesticides in air and precipitations of Paris by means of a bioluminescence method

A detailed toxicological study on several pesticides, including chlorothalonil, cyprodynil, dichlobénil, pendimethaline, trifluraline, and α-endosulfan, present at trace levels in air and total atmospheric precipitations of Paris is presented. The pesticides contained in the atmospheric samples, collected during sampling campaigns in February–March 2007, are identified and quantified by a high-performance liquid chromatographic (HPLC)-UV detection method. The toxicity measurements are performed by means of the Microtox® bioluminescence method, based on the evaluation of the bioluminescence inhibition of the Vibrio fischeri marine bacteria at two exposure times to the pesticide solutions. The specific toxicity, corresponding to the particular toxicity of the compound under study and represented by the EC₅₀ parameter, is determined for these pesticides. Also, the global toxicity, which is the toxicity of all micro-pollutants present in the sample under study, is estimated for the extracts of air and atmospheric precipitation (rainwater) samples. The specific toxicities strongly vary with the nature of the pesticide, the EC₅₀ parameter values being comprised between 0.17 and 0.83 mg/mL and 0.15 and 0.66 mg/mL, respectively, for exposure times of 5 and 15 min. The importance of the atmospheric samples’ global toxicity and the respective contribution of the toxic potency of the various pesticides contained in these samples are discussed.

Using ICP-qMS to trace the uptake of nanoscale titanium dioxide by microalgae–potential disadvantages of vegetable reference material

As nanoscale materials have gained in economic importance over recent years, concerns about accumulation in the environment and, consequently, analysis of nanoparticles in biological material have increasingly become the focus of scientific research. A nanomaterial used in a wide range of food, consumer and household products is titanium dioxide (nTiO₂). Monitoring of nTiO₂ via determination of elemental titanium (Ti) can be very challenging because of a variety of possible interferences. This work describes problems during the development of a quantification method for titanium dioxide (TiO₂) using inductively coupled plasma-quadrupole mass spectrometry (ICP-qMS). To evaluate the analytical method, certified vegetable reference material NCS DC 73349 was used. Interestingly, measurements of NCS DC 73349 seemed to result in acceptable recovery values—however, this was without considering interferences or conceivable differences in the natural isotopic abundance of the certified titanium calibration solution and NCS DC 73349. Actually, recoveries were lower than initially assumed. The potential interferences causing augmented recovery could be attributed to the presence of the elements sulfur (S) and phosphorus (P), which were able to form oxide ions and nitrogen-interfering species. The effect of such interfering cluster ions could be prevented by dry ashing as a sample preparation step, to evaporate S and P, before digestion with aqua regia in a high-pressure asher (HPA). Final practicability of the analysis method was proved by monitoring the uptake of nTiO₂ by the microalgae Scenedesmus acutus in an environmental exposure study.

The coupling of rapidly synergistic cloud point extraction with thermospray flame furnace atomic absorption spectrometry

Rapidly synergistic cloud point extraction (RS-CPE) was coupled with thermospray flame furnace atomic absorption spectrometry (TS-FF-AAS) to result in new CPE patterns and accelerated (1?min) protocols. It is demonstrated, for the case of copper (II) ion, that TS-FF-AAS improves the sampling efficiency and the sensitivity of FAAS determinations. Problems of nebulization associated with previous methods based on the coupling of FAAS and RS-CPE are overcome. TS-FF-AAS also improves sensitivity and gives a limit of detection for copper of 0.20?μg?L^-1, which is better by a factor of 32. Compared to direct FAAS, the factor is 114.

Efficient sample proteolysis based on a microchip containing a glass fiber core with immobilized trypsin

Trypsin was immobilized on cellulose-coated glass fibers via a condensation reaction between the aldehyde groups of the oxidized cellulose and the primary amino groups of trypsin. A piece of the modified fiber was inserted into the main channel of a poly(methyl methacrylate) microchip to form a microfluidic proteolytic bioreactor. Scanning electron microscopy of the cross section of the fiber revealed a rough film on the surface of the fiber glass. The performance of the bioreactor was demonstrated by the tryptic digestion of hemoglobin and cytochrome c, where the time for digestion was reduced to <10?s. The digests were identified by MALDI-TOF-MS to obtain peptide mass fingerprint spectra. The results indicated that the digestion in the microfluidic bioreactor is comparable to that of a 12-h solution tryptic digest and thus provides a promising platform for the high throughput identification of proteins.

Determination of some trace elements by flame atomic absorption spectrometry after preconcentration and separation by Escherichia coli immobilized on multiwalled carbon nanotubes

We have immobilized living and non-living Escherichia coli (E. coli) bacteria on multiwalled carbon nanotubes (MWCNT) and used such materials as a biosorbent for the separation and preconcentration of copper, cobalt, cadmium and nickel prior to their determination by flame atomic absorption spectrometry (FAAS). E. coli bacteria cells were mixed with MWCNTs in a 1:1 ratio, dried and placed at the tip of a 50-mL syringe. The ions were retained on the sorbent and then eluted by drawing and ejecting back the sample (or standard solution) and an eluent, respectively. The effects of various experimental parameters on the sorption and elution were investigated. The analytes were quantitatively retained (at pH values of 7) and eluted (with 0.5 M nitric acid) with high precision, the RSD being <5%. The performances of the new sorbents were compared using certified reference materials. The sorbent modified with living E. coli has a higher adsorption capacity and displays somewhat better recoveries compared to sorbent based on non-living E. coli. Both sorbents were successfully used for the separation and preconcentration of copper, cobalt, cadmium and nickel prior to their determination by flame atomic absorption spectrometry.

Part I: characterization of the extracellular proteome of the extreme thermophile Caldicellulosiruptor saccharolyticus by GeLC-MS2

The proteome of extremely thermophilic microorganisms affords a glimpse into the dynamics of microbial ecology of high temperature environments. The secretome, or extracellular proteome of these microorganisms, no doubt harbors technologically important enzymes and other thermostable biomolecules that, to date, have been characterized only to a limited extent. In the first of a two-part study on selected thermophiles, defining the secretome requires a sample preparation method that has no negative impact on all downstream experiments. Following efficient secretome purification, GeLC-MS² analysis and prediction servers suggested probable protein secretion to complement experimental data. In an effort to define the extracellular proteome of the extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus, several techniques were considered regarding sample processing to achieve the most in-depth analysis of secreted proteins. Order of operation experiments, all including the C₁₈ bead technique, demonstrated that two levels of sample purification were necessary to effectively desalt the sample and provide sufficient protein identifications. Five sample preparation combinations yielded 71 proteins and the majority described, as enzymatic and putative uncharacterized proteins, anticipate consolidated bioprocessing applications. Nineteen proteins were predicted by Phobius, SignalP, SecretomeP, or TatP for extracellular secretion, and 11 contained transmembrane domain stretches suggested by Phobius and transmembrane hidden Markov model. The sample preparation technique demonstrating the most effective outcome for C. saccharolyticus secreted proteins in this study, involved acetone precipitation followed by the C₁₈ bead method in which 2.4% (63 proteins) of the predicted proteome was identified, including proteins suggested to have secretion and transmembrane moieties.

Analytical Properties of Solid-substrate Electrospray Ionization Mass Spectrometry

Conventional electrospray ionization mass spectrometry (ESI-MS) uses a capillary for sample loading and ionization. Along with the development of ambient ionization techniques, ESI-MS using noncapillary emitters has attracted more interest in recent years. Following our recent report on ESI-MS using wooden tips (Anal. Chem. 83, 8201–8207 (2011)), the technique was further investigated and extended in this study. Our results revealed that the wooden tips could serve as a chromatographic column for separation of sample components. Sequential and exhaustive ionization was observed for proteins and salts on wooden tips with salts ionized sooner and proteins later. Nonconductive materials that contain microchannels/pores could be used as tips for ESI-MS analysis with sample solutions loaded to the sharp-ends only, since rapid diffusion of sample solutions by capillary action would enable the tips to become conductive. Tips of inert materials such as bamboo, fabrics, and sponge could be used for sample loading and ionization, while samples such as tissue, mushroom, and bone could form tips to induce ionization for direct analysis with application of a high voltage.

Analysis of a Soluble (UreD:UreF:UreG)2 Accessory Protein Complex and Its Interactions with Klebsiella aerogenes Urease by Mass Spectrometry

Maturation of the nickel-containing urease of Klebsiella aerogenes is facilitated by the UreD, UreF, and UreG accessory proteins along with the UreE metallo-chaperone. A fusion of the maltose binding protein and UreD (MBP-UreD) was co-isolated with UreF and UreG in a soluble complex possessing a (MBP-UreD:UreF:UreG)₂ quaternary structure. Within this complex a UreF:UreF interaction was identified by chemical cross-linking of the amino termini of its two UreF protomers, as shown by mass spectrometry of tryptic peptides. A pre-activation complex was formed by the interaction of (MBP-UreD:UreF:UreG)₂ and urease. Mass spectrometry of intact protein species revealed a pathway for synthesis of the urease pre-activation complex in which individual hetero-trimer units of the (MBP-UreD:UreF:UreG)₂ complex bind to urease. Together, these data provide important new insights into the structures of protein complexes associated with urease activation.

FAAS slurry analysis of lead and copper ions preconcentrated on titanium dioxide nanoparticles coated with a silver shell and modified with cysteamine

We report on the separation and preconcentration of lead(II) and copper(II) ions using silver-coated titanium dioxide nanoparticles modified with cysteamine, and their determination by slurry analysis via flame atomic absorption spectrometry. The ions were adsorbed via a conventional batch technique, and the ion-loaded slurry was separated and directly introduced into the spectrometer, thereby eliminating a number of drawbacks. The effects of pH, amount of sorbent, slurry volume, sample volume and other ions on the recovery were investigated. Under optimized experimental conditions, copper and lead can be recovered within the 95% confidence level in certificated waste water, but also in spiked sea water samples. The technique is fast, simple, and leads to complete elution. The limit of detection (3δ, at n?=?10) was 0.37 μg L^?1 for Cu(II), and 0.38 μg L^?1 for Pb(II).

The determination of pharmaceutical residues in cooked and uncooked marine bivalves using pressurised liquid extraction, solid-phase extraction and liquid chromatography–tandem mass spectrometry

An optimised and validated method for the determination of pharmaceutical residues in blue mussels (Mytilus spp.) is presented herein, as well as an investigation of the effect of cooking (by steaming) on any potential difference in human exposure risk. Selected pharmaceuticals included two non-steroidal anti-inflammatory drugs (diclofenac and mefenamic acid), an antibiotic (trimethoprim), an anti-epileptic (carbamazepine) and a lipid regulator (gemfibrozil). An in vivo exposure experiment was set up in the laboratory in which mussels were exposed either directly by injection (10 ng) or daily through spiked artificial seawater (ASW) over 96 h. In liquid matrices, pharmaceutical residues were either determined using liquid chromatography–tandem mass spectrometry (LC-MS/MS) directly, or in combination with solid-phase extraction (SPE) for analyte concentration purposes. The extraction of pharmaceuticals from mussel tissues used an additional pressurised liquid extraction step prior to SPE and LC-MS/MS. Limits of quantification of between 2 and 46 ng L^?1 were achieved for extracted cooking water and ASW, between 2 and 64 μg L^?1 for ASW in exposure tanks, and between 4 and 29 ng g^?1 for mussel tissue. Method linearities were achieved for pharmaceuticals in each matrix with correlation coefficients of R ²?>?0.975. A selection of exposed mussels was also cooked (via steaming) and analysed using the optimised method to observe any effect on detectable concentrations of parent pharmaceuticals present. An overall increase in pharmaceutical residues in the contaminated mussel tissue and cooking water was observed after cooking.

Identification of selenium in the leaf protein of sunflowers by a combination of 2D-PAGE and laser ablation ICP-MS

We report on a method for the identification of selenium-containing proteins in an extract of sunflower leafs. It is based on the separation of the proteins by 2-dimensional gel electrophoresis, followed by detection of selenium via laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The laser system was operated in a raster mode at 100?μm?s^-1 and proved to be an efficient alternative in the search for selenoproteins in the spots of the gels. The instrumental parameters were optimized in terms of plasma energy and application of optimal reaction cell conditions, and the detection of the mass ⁸⁰Se¹⁶O⁺ which enabled the elimination of interfering species. Selenium was identified in 9.6% of the analyzed spots, indicating its random incorporation into the primary structure of the proteins.

Rapid and sensitive detection of bacteria via platinum-labeled antibodies and on-particle ionization and enrichment prior to MALDI-TOF mass spectrometry

We introduce a rapid and sensitive approach to study the interactions of an affinity probe with the bacterial wall. Immunoglobulin was immobilized on platinum nanoparticles, and the resulting probe nanoparticles bind to bacterial walls as confirmed by transmission electron microscopy. A MALDI-MS assay was developed that can detect ~10⁵ cfu mL^?1 of S. marcescens and E. coli. This approach enables simple, rapid and straightforward detection of bacterial proteins, with high resolution and sensitivity, and without the requirement for tedious washing/separation steps.

Solid-phase extraction method for the isolation of plant thionins from European mistletoe, wheat and barley using zirconium silicate embedded in poly(styrene-co-divinylbenzene) hollow-monoliths

Thionins are cysteine-rich, biologically active small (～5 kDa) and basic proteins occurring ubiquitously in the plant kingdom. This study describes an efficient solid-phase extraction (SPE) method for the selective isolation of these pharmacologically active proteins. Hollow-monolithic extraction tips based on poly(styrene-co-divinylbenzene) with embedded zirconium silicate nano-powder were designed, which showed an excellent selectivity for sulphur-rich proteins owing to strong co-ordination between zirconium and the sulphur atoms from the thiol-group of cysteine. The sorbent provides a combination of strong hydrophobic and electrostatic interactions which may help in targeted separation of certain classes of proteins in a complex mixture based upon the binding strength of different proteins. European mistletoe, wheat and barley samples were used for selective isolation of viscotoxins, purothionins and hordothionins, respectively. The enriched fractions were subjected to analysis by matrix-assisted laser desorption/ionisation–time-of-flight mass spectrometer to prove the selectivity of the SPE method towards thionins. For peptide mass-fingerprint analysis, tryptic digests of SPE eluates were examined. Reversed-phase high-performance liquid chromatography hyphenated to diode-array detection was employed for the purification of individual isoforms. The developed method was found to be highly specific for the isolation and purification of thionins.