Illumina sequencing and assessment of new cost-efficient protocol for metagenomic-DNA extraction from environmental water samples.

In this study, the development and assessment of a modified, efficient, and cost-efficient protocol for mDNA (metagenomic DNA) extraction from contaminated water samples was attempted. The efficiency of the developed protocol was investigated in comparison to a well-established commercial kit (Epicentre, Metagenomic DNA Isolation Kit for Water). The comparison was in terms of degree of shearing, yield, purity, duration, suitability for polymerase chain reaction and next-generation sequencing in addition to the quality of next-generation sequencing data. The DNA yield obtained from the developed protocol was 2.6 folds higher than that of the commercial kit. No significant difference in the alpha (Observed species, Chao1, Simpson and PD whole tree) and beta diversity was found between the DNA samples extracted by the commercial kit and the developed protocol. The number of high-quality sequences of the samples extracted by the developed method was 20% higher than those obtained by the samples processed by the kit. The developed economic protocol successfully yielded high-quality pure mDNA compatible with complex molecular applications. Thus we propose the developed protocol as a gold standard for future metagenomic studies investigating a large number of samples.

Illumina sequencing and assessment of new cost-efficient protocol for metagenomic-DNA extraction from environmental water samples

Abstract In this study, the development and assessment of a modified, efficient, and cost-efficient protocol for mDNA (metagenomic DNA) extraction from contaminated water samples was attempted. The efficiency of the developed protocol was investigated in comparison to a well-established commercial kit (Epicentre, Metagenomic DNA Isolation Kit for Water). The comparison was in terms of degree of shearing, yield, purity, duration, suitability for polymerase chain reaction and next-generation sequencing in addition to the quality of next-generation sequencing data. The DNA yield obtained from the developed protocol was 2.6 folds higher than that of the commercial kit. No significant difference in the alpha (Observed species, Chao1, Simpson and PD whole tree) and beta diversity was found between the DNA samples extracted by the commercial kit and the developed protocol. The number of high-quality sequences of the samples extracted by the developed method was 20% higher than those obtained by the samples processed by the kit. The developed economic protocol successfully yielded high-quality pure mDNA compatible with complex molecular applications. Thus we propose the developed protocol as a gold standard for future metagenomic studies investigating a large number of samples.

Cost-effective and rapid lysis of Saccharomyces cerevisiae cells for quantitative western blot analysis of proteins, including phosphorylated eIF2α.

The common method for liberating proteins from Saccharomyces cerevisiae cells involves mechanical cell disruption using glass beads and buffer containing inhibitors (protease, phosphatase and/or kinase inhibitors), followed by centrifugation to remove cell debris. This procedure requires the use of costly inhibitors and is laborious, in particular when many samples need to be processed. Also, enzymatic reactions can still occur during harvesting and cell breakage. As a result low-abundance and labile proteins may be degraded, and enzymes such as kinases and phosphatases may still modify proteins during and after cell lysis. We believe that our rapid sample preparation method helps overcome the above issues and offers the following advantages: (a) it is cost-effective, as no inhibitors and breaking buffer are needed; (b) cell breakage is fast (about 15 min) since it only involves a few steps; (c) the use of formaldehyde inactivates endogenous proteases prior to cell lysis, dramatically reducing the risk of protein degradation; (d) centrifugation steps only occur prior to cell lysis, circumventing the problem of losing protein complexes, in particular if cells were treated with formaldehyde intended to stabilize and capture large protein complexes; and (e) since formaldehyde has the potential to instantly terminate protein activity, this method also allows the study of enzymes in live cells, i.e. in their true physiological environment, such as the short-term effect of a drug on enzyme activity. Taken together, the rapid sample preparation procedure provides a more accurate snapshot of the cell's protein content at the time of harvesting. Copyright © 2017 John Wiley & Sons, Ltd.

Different sample treatments for the determination of ICM-XR in fish samples followed by LC-HRMS.

Iodinated X-ray contrast media (ICM-XR) are a group of pharmaceuticals widely used in medicine. Due to their low biodegradation rate, which makes their removal at wastewater treatment plants difficult, and the high doses at which they are administered, they have been detected in aquatic environments. In the present paper, a method for the quantitative determination of a group of ICM-XR in different fish species was developed and validated for the first time. Two extraction techniques were compared: pressurised liquid extraction (PLE) and QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe), with PLE being selected, followed by liquid chromatography-high resolution mass spectrometry. In addition, several clean-up strategies were evaluated. The optimised method provided PLE recoveries ranging from 60% to 88% and limits of detection ranging from 5ng/g to 25ng/g (dry weight). The method was applied in order to evaluate the presence of the selected ICM-XR in different fish species.

Reducing the cost of semi-automated in-gel tryptic digestion and GeLC sample preparation for high-throughput proteomics.

Peptide generation by trypsin digestion is typically the first step in mass spectrometry-based proteomics experiments, including 'bottom-up' discovery and targeted proteomics using multiple reaction monitoring. Manual tryptic digest and the subsequent clean-up steps can add variability even before the sample reaches the analytical platform. While specialized filter plates and tips have been designed for automated sample processing, the specialty reagents required may not be accessible or feasible due to their high cost. Here, we report a lower-cost semi-automated protocol for in-gel digestion and GeLC using standard 96-well microplates. Further cost savings were realized by re-using reagent tips with optimized sample ordering. To evaluate the methodology, we compared a simple mixture of 7 proteins and a complex cell-lysate sample. The results across three replicates showed that our semi-automated protocol had performance equal to or better than a manual in-gel digestion with respect to replicate variability and level of contamination. In this paper, we also provide the Agilent Bravo method file, which can be adapted to other liquid handlers. The simplicity, reproducibility, and cost-effectiveness of our semi-automated protocol make it ideal for routine in-gel and GeLC sample preparations, as well as high throughput processing of large clinical sample cohorts.

Simultaneous extraction and cleanup of high-lipid organs from white sturgeon (Acipenser transmontanus) for multiple legacy and emerging organic contaminants using QuEChERS sample preparation.

The objective of this research was to utilize the QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method to extract a broad range of persistent organic pollutants from sturgeon organs (liver and gonad) as indicators of exposure. The analyte list was prioritized to include carcinogenic polyaromatic hydrocarbons (PAHs), the most commonly occurring polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs), persistent bioaccumulative and toxic chemicals (PBTs), and emergent contaminants of concern (ECCs) as indicators of human sewage exposure. White sturgeon (Acipenser transmontanus) were selected for this study to support a larger ecotoxicological study to monitor contaminants as an assessment of fish health. Organ tissues contained high lipid content with percentages of 15% and 34% for liver and gonad, respectively. Overall recoveries from fortified sturgeon tissues were high, 71-98% for PAHs, 60-107% for PBDEs and PCBs, 86-107% for PBT chemicals, and 88-107% for ECCs with the exception of octinoxate (28%) from liver tissues. Analyte recovery trends decreased as analyte lipophilicity and molecular weight increased. These recoveries demonstrate that extraction using QuEChERS can be used for screening of the most common bioaccumulating organic compounds in high lipid fish tissue using a single extraction and analysis.

Comparative assessment of genomic DNA extraction processes for Plasmodium: Identifying the appropriate method.

Plasmodium DNA, in addition to being used for molecular diagnosis of malaria, find utility in monitoring patient responses to antimalarial drugs, drug resistance studies, genotyping and sequencing purposes. Over the years, numerous protocols have been proposed for extracting Plasmodium DNA from a variety of sources. Given that DNA isolation is fundamental to successful molecular studies, here we review the most commonly used methods for Plasmodium genomic DNA isolation, emphasizing their pros and cons. A comparison of these existing methods has been made, to evaluate their appropriateness for use in different applications and identify the method suitable for a particular laboratory based study. Selection of a suitable and accessible DNA extraction method for Plasmodium requires consideration of many factors, the most important being sensitivity, cost-effectiveness and, purity and stability of isolated DNA. Need of the hour is to accentuate on the development of a method that upholds well on all these parameters.

Novel Set-Up for Low-Disturbance Sampling of Volatile and Non-volatile Compounds from Plant Roots.

Most studies on rhizosphere chemicals are carried out in substrate-free set-ups or in artificial substrates using sampling methods that require an air flow and may thus cause disturbance to the rhizosphere. Our study aimed to develop a simplified and inexpensive system that allows analysis of rhizosphere chemicals at experimentally less disturbed conditions. We designed a mesocosm in which volatile rhizosphere chemicals were sampled passively (by diffusion) without air- and water flow on polydimethylsiloxane-(PDMS) tubes. Dandelion (Taraxacum sect. ruderalia) was used as model plant; roots were left undamaged. Fifteen volatiles were retrieved from the sorptive material by thermal desorption for analysis by gas chromatography/mass spectrometry (GC/MS). Furthermore, three sugars were collected from the rhizosphere substrate by aqueous extraction and derivatized prior to GC/MS analysis. In order to study how the quantity of detected rhizosphere compounds depends on the type of soil or substrate, we determined the matrix-dependent recovery of synthetic rhizosphere chemicals. Furthermore, we compared sorption of volatiles on PDMS tubes with and without direct contact to the substrate. The results show that the newly designed mesocosm is suitable for low-invasive extraction of volatile and non-volatile compounds from rhizospheres. We further highlight how strongly the type of substrate and contact of PDMS tubes to the substrate affect the detectability of compounds from rhizospheres.

Electromechanical cell lysis using a portable audio device: enabling challenging sample preparation at the point-of-care.

Audio sources are ubiquitously available on portable electronic devices, including cell phones. Here we demonstrate lysis of Mycobacterium marinum and Staphylococcus epidermidis bacteria utilizing a portable audio device coupled with a simple and inexpensive electromagnetic coil. The resulting alternating magnetic field rotates a magnet in a tube with the sample and glass beads, lysing the cells and enabling sample preparation for these bacteria anywhere there is a cell phone, mp3 player, laptop, or other device with a headphone jack.

A rapid LC-MS/MS quantification of peramivir using a simple and inexpensive sample precipitation: application to PK.

AIM: Peramivir is a newly approved selective neuraminidase inhibitor designed to inhibit influenza virus infection. METHODOLOGY/RESULTS: We report a robust and sensitive method utilizing simple precipitation extraction with LC-MS/MS for the high-throughput quantification. Addition of 0.06 M of ammonium formate and 0.1% formic acid in mobile phase could help reduce the matrix effect. This method uses 100 µl of plasma and covers a linear concentration range from 5 to 10,000 ng/ml. Other validation parameters are also evaluated and meet regulatory expectations by US FDA guidelines. CONCLUSION: The developed HPLC-MS/MS method has been successfully applied to support a clinical pharmacokinetic study. The strategy presented here can be applied elsewhere and may be useful for other amphiphilic drugs.

Simplified sample preparation in the simultaneous measurement of whole blood antimony, bismuth, manganese, and zinc by inductively coupled plasma mass spectrometry.

OBJECTIVES: We developed and validated a simplified sample preparation for the analysis of antimony (Sb), bismuth (Bi), manganese (Mn), and zinc (Zn) in whole blood. This simplification included a reduction in sample volume, removal of a lengthy acidic digestion, and optimization of the internal standard. DESIGN AND METHODS: Measurement of Sb, Bi, Mn and Zn in whole blood was conducted using inductively coupled-plasma mass spectrometry. Method performance characteristics, including intra- and inter-assay imprecision, accuracy, linearity, AMR, sensitivity, carryover, sample stability and assay stability were determined in accordance with clinical laboratory standards. In addition, analytical and clinical recoveries were assessed to investigate comparability between goat blood matrix and pooled patient blood. RESULTS: Established assay performance characteristics included inter- and intra-assay imprecision <4.5% and carryover of <0.04% for all four elements, analytical measurement range of 1 to 25 µg/L (Sb and Bi), 1 to 80 µg/L (Mn), and 50 to 1500 µg/dL (Zn), limit of quantification of 1 µg/L (Sb, Bi, Mn) and 50 µg/dL (Zn) (coefficient of variation <14%), proportional bias of 0.96 and constant bias of -0.28 (Sb), 0.94 and -0.45 (Bi), 1.07 and -0.37 (Mn) and 0.96 and +18.05 (Zn) based upon repeat patient samples, proficiency testing samples, and comparison to an outside reference laboratory. CONCLUSION: This method overcomes the laborious acidic heat digestion previously used and replaces it with a simplified sample preparation involving an alkaline dilution. The method requires minimal sample preparation with the dilution of alkaline diluent and is validated to quantify Sb and Bi from 1 to 25 µg/L, Mn from 1 to 80 µg/L, and Zn from 50 to 1500 µg/dL in whole blood.

Nonreductive chemical release of intact N-glycans for subsequent labeling and analysis by mass spectrometry.

A novel strategy is proposed, using cost-saving chemical reactions to generate intact free reducing N-glycans and their fluorescent derivatives from glycoproteins for subsequent analysis. N-Glycans without core α-1,3-linked fucose are released in reducing form by selective hydrolysis of the N-type carbohydrate-peptide bond of glycoproteins under a set of optimized mild alkaline conditions and are comparable to those released by commonly used peptide-N-glycosidase (PNGase) F in terms of yield without any detectable side reaction (peeling or deacetylation). The obtained reducing glycans can be routinely derivatized with 2-aminobenzoic acid (2-AA), 1-phenyl-3-methyl-5-pyrazolone (PMP), and potentially some other fluorescent reagents for comprehensive analysis. Alternatively, the core α-1,3-fucosylated N-glycans are released in mild alkaline medium and derivatized with PMP in situ, and their yields are comparable to those obtained using commonly used PNGase A without conspicuous peeling reaction or any detectable deacetylation. Using this new technique, the N-glycans of a series of purified glycoproteins and complex biological samples were successfully released and analyzed by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS), demonstrating its general applicability to glycomic studies.

A quick, easy, cheap, effective, rugged, and safe sample pretreatment and liquid chromatography with tandem mass spectrometry method for the simultaneous quantification of 33 mycotoxins in Lentinula edodes.

Lentinula edodes, one of the most cultivated edible fungi in the world, are usually neglected for mycotoxins contamination due to the initial thinking of its resistance to mycotoxingenic molds. In the present study, a sensitive and reliable liquid chromatography with tandem mass spectrometry method was developed for the simultaneous quantification of 33 mycotoxins in L. edodes. Targeted mycotoxins were extracted using a quick, easy, cheap, effective, rugged, and safe procedure without any further clean-up step, and analyzed by liquid chromatography with tandem mass spectrometry on an Agilent Poroshell 120 EC-C18 column (100 × 3 mm, 2.7 µm) with a linear gradient elution program using water containing 5 mM ammonium acetate and methanol as the mobile phase. After validation by determining linearity (R(2) > 0.99), sensitivity (LOQ ≤ 20 ng/kg), recovery (73.6-117.9%), and precision (0.8-19.5%), the established method has been successfully applied to reveal the contamination states of various mycotoxins in L. edodes. Among the 30 tested samples, 22 were contaminated by various mycotoxins with the concentration levels ranging from 3.3-28,850.7 µg/kg, predicting that the edible fungus could be infected by the mycotoxins-producing fungi. To the best of our knowledge, this is the first report about real mycotoxins contamination in L. edodes.

A Combined Fabrication and Instrumentation Platform for Sample Preparation.

While potentially powerful, access to molecular diagnostics is substantially limited in the developing world. Here we present an approach to reduced cost molecular diagnostic instrumentation that has the potential to empower developing world communities by reducing costs through streamlining the sample preparation process. In addition, this instrument is capable of producing its own consumable devices on demand, reducing reliance on assay suppliers. Furthermore, this instrument is designed with an "open" architecture, allowing users to visually observe the assay process and make modifications as necessary (as opposed to traditional "black box" systems). This open environment enables integration of microfluidic fabrication and viral RNA purification onto an easy-to-use modular system via the use of interchangeable trays. Here we employ this system to develop a protocol to fabricate microfluidic devices and then use these devices to isolate viral RNA from serum for the measurement of human immunodeficiency virus (HIV) viral load. Results obtained from this method show significantly reduced error compared with similar nonautomated sample preparation processes.

Preconcentration of total mercury from river water by anion exchange mechanism.

A simple and cheap analytical technique was developed for the measurement of total mercury in river water samples using inductively coupled plasma-mass spectrometry (ICP-MS). It is based on the direct complexation of mercury ions using iodide and a cationic surfactant in water for its subsequent solid-phase extraction. Mercury ions are retained on the silica phase as ion pairs in the presence of iodide ions and dodecyltrimethylammonium bromide. Parameters having influential influence on the retention of Hg(II) were investigated: sample flowrate, eluent type, sample volume, iodide and surfactant concentrations. The retained mercury ions are stripped off from silica phase using 10 mL of 8 mol L(-1) HNO3 and quantified by ICP-MS. An enrichment factor of 50 was achieved with a maximum adsorption capacity of 718 µg Hg(II) g(-1). The limit of detection of Hg(II) was 8 pg mL(-1). The developed method was applied for the determination of total mercury in river and tap-water samples.

Endo-ß-N-acetylglucosaminidase H de-N-glycosylation in a domestic microwave oven: application to biomarker discovery.

Sample preparation is the rate-limiting step in glycan analysis workflows. Among all of the steps, enzymatic digestions, which are usually performed overnight, are the most time-consuming. In the current study, we report an economical and fast preparation of N-glycans from serum, including microwave-assisted enzymatic digestion in the absence of denaturing chemicals and solvents during the release. To this end, we used a household microwave oven to accelerate both pronase and endo-ß-N-acetylglucosaminidase H (Endo H) digestions. Purification was then performed using self-made SP20SS and carbon tips. We were able to prepare samples in 55 min instead of 21 h. Finally, the method was applied in the context of oncological biomarker discovery exemplarily to ovarian and colon cancer. We observed a significant downregulation of sialylated hybrid structures in ovarian cancer samples using capillary electrophoresis-laser-induced fluorescence (CE-LIF). Furthermore, sepsis, a systemic inflammatory response syndrome, was also included in the study to understand whether the changes observed in ovarian cancer patients were due to the cancer itself or to the inflammation that usually accompanies its development. Because sialylated hybrid structures were upregulated in sepsis samples, the downregulation of these structures in ovarian cancer is specific to the cancer itself and, therefore, could be used as a biomarker.

A modified QuEChERS method as sample treatment before the determination of isoflavones in foods by ultra-performance liquid chromatography-triple quadrupole mass spectrometry.

This paper reports the development of an analytical method for the determination of isoflavones in legumes using LC-MS/MS. A modified approach of the QuEChERS methodology was used to extract the analytes from the food samples. The proposed method includes a two-step extraction process and allows the determination of isoflavones in pulses without the need of a clean-up step. Use of this methodology for the extraction of natural occurring substances provides advantages such as simplicity and ease of use, especially taking into account the complexity of food matrices. The method was applied successfully for the determination of eight isoflavones, including aglycones and glucosides, in legumes of Spanish origin (chickpeas, lentils and beans from the region of Castilla y León). The target compounds were the glucosides daidzin, glycitin and genistin, and the aglycones daidzein, glycitein, genistein, formononetin, and biochanin A. The detection limits were in the 0.7 µg L(-1) to 1.5 µg L(-1) range for formononetin and glycitin respectively. Recoveries ranged from 72% to 119%, and standard deviations lower than 25% were obtained for the inter-day precision. The method described is precise, selective and not time-consuming.

Selective extraction and determination of vitamin B12 in urine by ionic liquid-based aqueous two-phase system prior to high-performance liquid chromatography.

A rapid and simple extraction technique based on aqueous two-phase system (ATPS) was developed for separation and enrichment of vitamin B(12) in urine samples. The proposed ATPS-based method involves the application of the hydrophilic ionic liquid (IL) 1-hexyl-3-methylimidazolium chloride and K(2)HPO(4). After the extraction procedure, the vitamin B(12)-enriched IL upper phase was directly injected into the high performance liquid chromatography (HPLC) system for analysis. All variables influencing the IL-based ATPS approach (e.g., the composition of ATPS, pH and temperature values) were evaluated. The average extraction efficiency was 97% under optimum conditions. Only 5.0 mL of sample and a single hydrolysis/deproteinization/extraction step were required, followed by direct injection of the IL-rich upper phase into HPLC system for vitamin B(12) determination. A detection limit of 0.09 µg mL(-1), a relative standard deviation (RSD) of 4.50% (n=10) and a linear range of 0.40-8.00 µg mL(-1) were obtained. The proposed green analytical procedure was satisfactorily applied to the analysis of samples with highly complex matrices, such as urine. Finally, the IL-ATPS technique could be considered as an efficient tool for the water-soluble vitamin B(12) extraction.

Automated tagging of pharmaceutically active thiols under flow conditions using monobromobimane.

The thiol-specific derivatization reagent monobromobimane (MBB) is applied--for the first time--under flow conditions. Sequential injection analysis allows the handling of precise volumes of the reagent in the micro-liter range. The effect of the main chemical and instrumental variables was investigated using captopril (CAP), N-acetylcysteine (NAC) and penicillamine (PEN) as representative pharmaceutically active thiols. Previously reported hydrolysis of MBB due to interaction with nucleophilic components of the buffers was avoided kinetically under flow conditions. The proposed analytical scheme is suitable for the fluorimetric determination of thiols at a sampling rate of 36 h(-1).

Portable surface-enhanced Raman scattering sensor for rapid detection of aniline and phenol derivatives by on-site electrostatic preconcentration.

A portable surface-enhanced Raman scattering (SERS) sensor is developed and applied to simultaneous detection of aniline and phenol derivatives in a label-free way with an electrostatic preconcentration technique to amplify the signals. A SERS-active substrate, silver-electrodeposited screen-printed electrodes (Ag-SPEs), is used for qualification and quantification of polar organic pollutants. Observation of SERS spectra at different potentials indicates that polar pollutants are selectively adsorbed on the Ag-SPEs at a given potential, suggesting that Ag-SPEs could selectively attract polar pollutants to an oppositely charged electrode at different potentials. Optimum SERS-active substrate was obtained when a potential of -0.15 V vs Ag/AgCl was applied on the SPEs in 0.1 M AgNO(3) solution for 10 min. Moreover, the effects of experimental variables such as the electrodeposition time and potential of Ag and preconcentration time of polar molecules on the SERS signals are presented. Under optimum conditions and with a 785 nm laser, the method is effective over a wide range of concentration (1 nM to 1 µM) for aniline and phenol derivatives. The novel method described herein presents a new detection regime for environmental pollutant analysis and also demonstrates simultaneous multiplexed detection of polar organic pollutants using convenient Ag-SPEs.