Analytica Chimica Acta (v.956, #C)

Elucidating rhizosphere processes by mass spectrometry – A review by Ariana Rugova; Markus Puschenreiter; Gunda Koellensperger; Stephan Hann (1-13).
The presented review discusses state-of-the-art mass spectrometric methods, which have been developed and applied for investigation of chemical processes in the soil-root interface, the so-called rhizosphere. Rhizosphere soil's physical and chemical characteristics are to a great extent influenced by a complex mixture of compounds released from plant roots, i.e. root exudates, which have a high impact on nutrient and trace element dynamics in the soil-root interface as well as on microbial activities or soil physico-chemical characteristics. Chemical characterization as well as accurate quantification of the compounds present in the rhizosphere is a major prerequisite for a better understanding of rhizosphere processes and requires the development and application of advanced sampling procedures in combination with highly selective and sensitive analytical techniques. During the last years, targeted and non-targeted mass spectrometry-based methods have emerged and their combination with specific separation methods for various elements and compounds of a wide polarity range have been successfully applied in several studies. With this review we critically discuss the work that has been conducted within the last decade in the context of rhizosphere research and elemental or molecular mass spectrometry emphasizing different separation techniques as GC, LC and CE. Moreover, selected applications such as metal detoxification or nutrient acquisition will be discussed regarding the mass spectrometric techniques applied in studies of root exudates in plant-bacteria interactions. Additionally, a more recent isotope probing technique as novel mass spectrometry based application is highlighted.Display Omitted
Keywords: Rhizosphere; Root exudates; Mass spectrometry; Separation techniques;

Magnesium oxide microspheres functionalized with phenyltrichlorosilane (PTS-MgO) were synthesized by surface modification through silanization reaction, which was confirmed by Fourier transform infrared (FT-IR), scanning electron microscopy (SEM), X-ray diffraction (XRD), thermogravimetry analysis (TGA) and N2 adsorption-desorption. The result indicated that PTS-MgO not only possessed the ability of enhancing the retention with PAHs, but also weakening the interference from chlorinated compounds. As a sorbent for the matrix solid-phase dispersion (MSPD) extraction, PTS-MgO was used to selectively extract seven dioxin-like polycyclic aromatic hydrocarbons (DL-PAHs) from soil samples. Various parameters affecting the recoveries of seven DL-PAHs were investigated and optimized, such as sorbent/sample mass ratio, grinding time, rinsing and eluting conditions. Under the optimized conditions, the developed method combining MSPD with HPLC-FLD exhibited good sensitivity (0.02–0.12 ng g−1 detection of limits) and linearity (linear correlation coefficient greater than 0.9997). Satisfactory recoveries with DL-PAHs spiked at two levels (10 and 80 ng g−1) were obtained in the range of 72.2–113.1% with RSD < 9.6%, indicating that PTS-MgO had a potential in MSPD extraction of DL-PAHs in soils. Additionally, the proposed MSPD-HPLC-FLD method was also verified by detecting seven DL-PAHs in the standard reference soil. Based on the developed method, DL-PAHs in soil samples were detected with the concentration ranging from 70.08 to 555.05 ng g−1 dry weight (dw). The total toxic equivalency quotients (TEQ) of seven DL-PAHs varied from 9.93 to 143.94 ng TEQ/g dw.Display Omitted
Keywords: Matrix solid-phase dispersion; Phenyltrichlorosilane; Magnesium oxide; Dioxin-like polycyclic aromatic hydrocarbons;

Probing tumor extracellular metabolites is a vitally important issue in current cancer biology. In this study an analytical system was constructed for the in vivo monitoring of mouse tumor extracellular hydrogen peroxide (H2O2), lactate, and glucose by means of microdialysis (MD) sampling and fluorescence determination in conjunction with a smart sequential enzymatic derivatization scheme—involving a loading sequence of fluorogenic reagent/horseradish peroxidase, microdialysate, lactate oxidase, pyruvate, and glucose oxidase—for step-by-step determination of sampled H2O2, lactate, and glucose in mouse tumor microdialysate. After optimization of the overall experimental parameters, the system's detection limit reached as low as 0.002 mM for H2O2, 0.058 mM for lactate, and 0.055 mM for glucose, based on 3 μL of microdialysate, suggesting great potential for determining tumor extracellular concentrations of lactate and glucose. Spike analyses of offline-collected mouse tumor microdialysate and monitoring of the basal concentrations of mouse tumor extracellular H2O2, lactate, and glucose, as well as those after imparting metabolic disturbance through intra-tumor administration of a glucose solution through a prior-implanted cannula, were conducted to demonstrate the system's applicability. Our results evidently indicate that hyphenation of an MD sampling device with an optimized sequential enzymatic derivatization scheme and a fluorescence spectrometer can be used successfully for multi-analyte monitoring of tumor extracellular metabolites in living animals.Display Omitted
Keywords: Enzymatic derivatization; Glucose; Hydrogen peroxide; Lactate; Tumor extracellular fluid;

A facile method for the preparation of stable isotopically labeled peptides was developed by means of filter-assisted tryptic 16O/18O water labeling, which could be directly applied to the determination of hepatitis B virus infection from human serum with tandem mass spectrometry. Tryptic peptides of hepatitis B surface antigen or hepatitis B e antigen from different subtypes of hepatitis B virus were synthesized with traditional solid-phase peptide synthesis as potential biomarkers. Trypsin catalyzed oxygen-18 exchange at their amidated c-terminus of arginine or lysine residue. The protease catalyzed oxygen-18 to oxygen-16 back exchange reaction was eliminated due to the complete removal of trypsin by the centrifugal filter containing a thin membrane associated with molecular weight cut-off of 10 KDa. The synthetic isotopic peptides were spiked into trichloroacetic acid/acetone precipitated human serum as internal standards and were selectively detected with multiplexed parallel reaction monitoring on a hybrid quadrupole-orbitrap mass spectrometer. The limit of detection for all synthetic peptides were in the range of 0.09 fmol–1.13 fmol. The results indicated that the peptide YLWEWASVR derived from hepatitis B surface antigen was quantified approximately 200 fmol per μl serum and may serve as a diagnostic biomarker for the detection of hepatitis B virus infected disease.Display Omitted
Keywords: Filter-assisted enzymatic labeling; Hepatitis B virus; Mass spectrometry; Parallel reaction monitoring; Stable isotopically labeled peptides;

The co-feature ratio, a novel method for the measurement of chromatographic and signal selectivity in LC-MS-based metabolomics by Albert Elmsjö; Jakob Haglöf; Mikael K.R. Engskog; Marika Nestor; Torbjörn Arvidsson; Curt Pettersson (40-47).
Evaluation of analytical procedures, especially in regards to measuring chromatographic and signal selectivity, is highly challenging in untargeted metabolomics. The aim of this study was to suggest a new straightforward approach for a systematic examination of chromatographic and signal selectivity in LC-MS-based metabolomics.By calculating the ratio between each feature and its co-eluting features (the co-features), a measurement of the chromatographic selectivity (i.e. extent of co-elution) as well as the signal selectivity (e.g. amount of adduct formation) of each feature could be acquired, the co-feature ratio. This approach was used to examine possible differences in chromatographic and signal selectivity present in samples exposed to three different sample preparation procedures. The capability of the co-feature ratio was evaluated both in a classical targeted setting using isotope labelled standards as well as without standards in an untargeted setting.For the targeted analysis, several metabolites showed a skewed quantitative signal due to poor chromatographic selectivity and/or poor signal selectivity. Moreover, evaluation of the untargeted approach through multivariate analysis of the co-feature ratios demonstrated the possibility to screen for metabolites displaying poor chromatographic and/or signal selectivity characteristics. We conclude that the co-feature ratio can be a useful tool in the development and evaluation of analytical procedures in LC-MS-based metabolomics investigations. Increased selectivity through proper choice of analytical procedures may decrease the false positive and false negative discovery rate and thereby increase the validity of any metabolomic investigation.Display Omitted
Keywords: Metabolomics; Co-feature ratio (CFR); Data evaluation; Method evaluation; Selectivity; LC-MS;

A set of 90 mer long ssDNA candidates, with different degrees of cytosine (C-levels) (% and clusters) was analyzed for their function as suitable Ag-nanocluster (AgNC) nucleation scaffolds. The sequence (P4) with highest C-level (42.2%) emerged as the only candidate supporting the nucleation process as evident from its intense fluorescence peak at λ660 nm. Shorter DNA subsets derived from P4 with only stable hairpin structures could support the AgNC formation. The secondary hairpin structures were confirmed by PAGE, and CD studies. The number of base pairs in the stem region also contributes to the stability of the hairpins. A shorter 29 mer sequence (Sub 3) (ΔG = −1.3 kcal/mol) with 3-bp in the stem of a 7-mer loop conferred highly stable AgNC. NAD+ strongly quenched the fluorescence of Sub 3-AgNC in a concentration dependent manner. Time resolved photoluminescence studies revealed the quenching involves a combined static and dynamic interaction where the binding constant and number of binding sites for NAD+ were 0.201 L mol−1 and 3.6, respectively. A dynamic NAD+ detection range of 50–500 μM with a limit of detection of 22.3 μM was discerned. The NAD+ mediated quenching of AgNC was not interfered by NADH, NADP+, monovalent and divalent ions, or serum samples. The method was also used to follow alcohol dehydrogenase and lactate dehydrogenase catalyzed physiological reactions in a turn-on and turn-off assay, respectively. The proposed method with ssDNA-AgNC could therefore be extended to monitor other NAD+/NADH based enzyme catalyzed reactions in a turn-on/turn-off approach.Display Omitted
Keywords: Silver nanocluster; Nicotinamide adenine dinucleotide; Fluorescence assay; Detection;

The analysis of DNA methylation and MTase activities is very important in the early clinical diagnosis of cancer, on purposes of providing insights into the mechanism of gene repression and developing novel drugs of treating methylation-related diseases. Combining the dendritic rolling circle amplification and Mg2+-dependent DNAzyme with a function of catalyzing the generation of a fluorophore-labeled nucleic acid acting as readout signal for the analyses, a new fluorescent method for DNA methyltransferase detection was reported. In the presence of DNA methyltransferases (MTase), the methylation-responsive sequence of double-stranded DNA probe was methylated and then cleaved by the methylation-sensitive restriction endonuclease DpnI. The cleaved hybrid DNA probe then functioned as a signal primer to initiate the dendritic rolling circle amplification reaction, containing a circular DNA and a structurally tailored hairpin structure. Subsequently, the circular nucleic acid template produced a complementary sequence to the Mg2+-dependent DNAzyme and a sequence identical to the loop region of the co-added hairpin structure. At last, a fluorescence readout signal was afforded by the DNAzyme-catalyzed cleavage of a fluorophore/quencher-modified substrate. This method enabled the analysis of the target MTase with a detection limit up to 0.36 U mL−1, and a dynamic range was obtained from 1.0 to 10 U mL−1. Moreover, the proposed strategy was successfully applied in real sample assay. With this assay, the inhibitors of MTase were evaluated and screened which might be helpful for the discovery of anticancer drugs.Display Omitted
Keywords: DNA methyltransferase; Dendritic rolling circle amplification; Fluorescence; DNA; DNAzyme;

A novel colorimetric biosensor, which employs DNAzyme-functionalized magnetic beads (MBs) as recognition probe, enzyme-assisted catalytic oxidation of TMB (3,3′,5,5’-tetramethylbenzidine sulfate) as signal and DNA hybridization chain reaction as amplification strategy, has been developed for detecting trace uranyl ion (UO2 2+) in seafood and aqueous environment with high sensitivity and specificity. We demonstrated that UO2 2+ can specifically cleave DNAzyme immobilized on MBs surface to release a short single-strand DNA (primer), and the released primer trigger DNA hybridization chain reaction to form a long one dimensional DNA concatamer on the MBs surface. The resulting long DNA concatamer could capture a large amount of HRP to generate the one UO2 2+-to-multiple HRP amplification effect. Upon the addition of TMB-H2O2 solution, the HRP-tagged DNA concatamer-MBs conjugates could catalyze the H2O2-mediated oxidation of TMB, and thus results in a color change from colorless to blue in solution. This provided a sensitive and selective sensing platform for the visual or colorimetric detection of UO2 2+. The proposed biosensor has high sensitivity and strong anti-interference capability, it can be used to detect as low as 2.5 ppb (9.25 nM) of UO2 2+ by naked-eye observation and 0.09 ppb (0.33 nM) of UO2 2+ by UV-visible spectrometry with no interference of other ions and a RSD ≤ 6% (n = 5). With the help of this method, we have successfully determined trace UO2 2+ in fish muscle and river water with a recovery of 93–106%. High sensitivity and specificity, as well operation convenience, low cost and strong resistibility to the matrix, which makes our method a potential approach for the on-site detection of UO2 2+ in seafood and aqueous environment.Display Omitted
Keywords: Uranyl ion; DNAzyme; Colorimetric biosensor; Magnetic beads; DNA hybridization chain reaction;