Preliminary studies on selenium-containing proteins in Brassica juncea by size exclusion chromatography and fast protein liquid chromatography coupled to ICP-MS

An approach for screening and resolving selenium-containing plant proteins was developed based on the combination of sample preparation and multi-dimensional liquid chromatography coupled to ICP-MS. Different protein extraction protocols were investigated. A 24 h dodecylsulfate-mediated protein extraction in a sonication bath followed by acetone precipitation was found to be optimal. The use of different protein precipitate solubilizing agents (sodium dodecyl sulfate media and Tris-HCl buffer) demonstrates possible fractionation of the selenium-containing proteins. Selenium-containing protein screening and fractionation were carried out by means of SEC-ICP-MS. High molecular weight selenium containing proteins were solubilized with a sodium dodecyl sulfate-containing buffer, whereas the low molecular weight compounds were released into a Tris-HCl buffer. Size exclusion chromatography-fast protein liquid chromatography coupled to ICP-MS allowed separation and detection of several selenium-containing proteins in Se-supplemented wild type Brassica juncea plant, a fast growing selenium accumulator.     

Confirmation and determination of carboxylic acids in root exudates using LC-ESI-MS

Reversed-phase liquid chromatography with UV detection is of limited applicability in the separation and identification of carboxylic acids because of the column's poor separation efficiency and the non-selective nature of the UV detector. To address this issue, RP-LC with electrospray ionization mass spectrometry has been explored for the confirmation and determination of carboxylic acids in plant root exudates, with ESI-MS providing structural information, high selectivity, and high sensitivity. The separation of 10 carboxylic acids (pyruvic, lactic, malonic, maleic, fumaric, succinic, malic, tartaric, trans-aconitic, and citric acid) was performed on a C(18) column using an eluent containing 0.1% (v/v) acetic acid within 10 min, where the acidic eluent not only suppressed the ionization of the carboxylic acids to be retained on the column, but was also compatible with ESI-MS detection. In addition, an additional standard was used to overcome the matrix effect. The results showed that peak areas correlated linearly with the concentration of carboxylic acids over the range 0.05-10 mg/L. The detection limits of target acids (signal-to-noise S/N ratio of 3) ranged from 20 to 30 microg/L. Finally, the proposed method was used for the confirmation and determination of low-molecular-weight carboxylic acids in plant root exudates, and provided a simple analytical procedure, including sample processing, fast separation, and high specificity and sensitivity.     

Comparing a selenium accumulator plant (Brassica juncea) to a nonaccumulator plant (Helianthus annuus) to investigate selenium-containing proteins

Selenoproteins have been identified in a diverse range of organisms, including bacteria and animals. Their occurrence and role in the plant kingdom are, however, less well-understood. This work investigated the water-soluble selenium-containing proteins extracted from a selenium-accumulating plant species (Brassica juncea) and a nonaccumulator species (Helianthus annuus) exposed to varying forms and concentrations of selenium. Firstly, protein extracts were analyzed by size exclusion chromatography coupled to inductively coupled plasma mass spectrometry; specific detection was achieved by monitoring characteristic isotopes. Then, proteolytic digests of the plant extracts were analyzed by reversed phase chromatography coupled to ICP–MS in order to investigate selenoamino acid and selenopeptide content. Selenomethionine was observed to be the primary constituent of the proteins of the nonaccumulator plant, while selenocystine and selenomethionine were found in the same proportion in the accumulator extract. One main selenium-containing species was present at higher levels in the root digests than in the leaf digests; levels were greater in the nonaccumulator than in the accumulator plant.     

Investigation of the binding properties of heavy-metal-peptide complexes in plant cell cultures using HPLC-ICP-MS

Metal-induced, sulfhydryl-rich peptides (phytochelatins) found in plants, algae, yeasts and fungi have been described as sequesters and detoxifiers of heavy metal ions. High-performance liquid chromatography (HPLC) coupled on-line with inductively coupled plasma mass spectrometry (ICP-MS) has been used for the determination of heavy metal binding properties of phytochelatins in Silene vulgaris cell cultures. The induction of phytochelatins and the binding of heavy metals to these complexes were investigated by exposure of cell cultures with different concentrations of Cd, Cu, Pb and Zn. An in vitro heavy metal saturation assay and in vivo stress experiments with those elements able to bind to phytochelatins were carried out in order to characterize the binding affinity and binding stability of these compounds. It is shown that of the metals investigated, Cu binds most stably to phytochelatins under in vitro and in vivo conditions.     

Identification and characterization of Se-methyl selenomethionine in Brassica juncea roots

The present work shows the identification and characterization of Se-methyl selenomethionine (SeMMet) as an important Se species in Brassica juncea roots when grown in the presence of Se-methionine (SeMet) as the Se source. SeMMet was isolated by liquid chromatography employing two different liquid chromatographic mechanisms: reversed-phase ion-pairing using heptafluorobutyric acid as counter ion and cation exchange using a pyridinium formate gradient (pH 3). Inductively coupled plasma mass spectrometry was used for the detection of Se. SeMMet was characterized by electrospray quadrupole time-of-flight MS in both a synthesized standard and in the roots extract using collision-induced dissociation of the selected ion. Preliminary evidence suggests that Brassica juncea may also produce dimethylselenonium propionate, although to a much lesser extent.     

Silver Nanoparticle-Mediated Enhancement in Growth and Antioxidant Status of Brassica juncea

Metal nanoparticles can potentially be used as tools for engineering biological redox reactions. Present study underlines the effect of silver metal nanoparticles (at 0, 25, 50, 100, 200 and 400?ppm) on the growth and antioxidant status of 7-day-old Brassica juncea seedlings. Fresh weight, root and shoot length, and vigor index of seedlings is positively affected by silver nanoparticle treatment. It induced a 326?% increase in root length and 133?% increase in vigor index of the treated seedlings. Improved photosynthetic quantum efficiency and higher chlorophyll contents were recorded in leaves of treated seedlings, as compared to the control seedlings. Levels of malondialdehyde and hydrogen peroxide decreased in the treated seedlings. Nanoparticle treatment induced the activities of specific antioxidant enzymes, resulting in reduced reactive oxygen species levels. Decrease in proline content confirmed the improvement in antioxidant status of the treated seedlings. The observed stimulatory affects of silver nanoparticles are found to be dose dependent, with 50?ppm treatment being optimum for eliciting growth response. Present findings, for the first time indicate that silver nanoparticles promote the growth of B. juncea seedlings by modulating their antioxidant status.     

Arsenic speciation in chinese seaweeds using HPLC-ICP-MS and HPLC-ES-MS

Three common Chinese edible seaweeds, one brown (Laminaria japonica) and two red (Porphyra crispata and Eucheuma denticulatum), were examined for their total arsenic content. The As species were extracted with yields of 76.4, 69.8 and 25.0%, respectively. Anion-exchange and cation-exchange high-performance liquid chromatography (HPLC) in combination with inductively coupled plasma mass spectrometry (ICP-MS) were used for the separation of the different arsenic species in two of the three seaweed extracts (Laminaria and Porphyra). The main arsenic species in the algal extracts are arseno sugars, although it has been shown that the Laminaria seaweed contains significant amounts of dimethylarsinic acid (DMA). HPLC was coupled with electrospray mass spectrometry (ES-MS) for structural confirmation of the arsenic species. The mass spectrometer settings for the arseno sugars were optimised using standards. The conclusions drawn on the basis of HPLC-ICP-MS were confirmed by the HPLC-ES-MS data. The HPLC-ES-MS method is capable of determining both arseno sugars and DMA in the seaweeds. The unknown compounds seen in the HPLC-ICP-MS chromatogram of Laminaria could not be ascribed to trimethylarsenic oxide or tetramethylarsonium ion.     

The analysis of antimony species by using ESI-MS and HPLC-ICP-MS

A new method for the separation of organic antimony as trimethylantimony dichloride (TMSbCl₂) and inorganic Sb(V) and Sb(III) by using anion exchange high-performance liquid chromatography coupled with inductively-coupled plasma mass spectrometry (ICP-MS) is presented. In comparison with previous work the detection limits for both species were significantly decreased, down to 5 ngL^–1, mainly by avoiding any contamination from the chromatographic device. Using an ultrasonic nebulizer (USN) improved the detection limits for inorganic Sb species, but was useless for the HPLC method due to problems in the recovery of the TMSbCl₂. Matrix interferences of the chromatographic determination were studied in detail and the method was applied to environmental samples assumed to contain organic antimony species. Additionally, the molecular structure of the TMSbCl₂ in solution was studied by using electrospray-ionization mass spectrometry (ESI-MS) showing that this species occurs most probably as [TMSbOH]⁺ in aqueous solutions. Received: 22 September 1997 / Revised: 21 November 1997 / Accepted: 27 November 1997     

Analytical challenges and solutions for performing metabolomic analysis of root exudates

Plant stress responses are mediated by the release of chemical compounds called exudates into the rhizosphere. These chemical substances include primary and secondary plant metabolites and play an important role in the plant defense mechanism. The identification, characterization and study of these compounds can open the door to numerous applications, from greener agriculture to enhanced phytoremediation. This paper critically reviews the most relevant sampling strategies, analytical methodologies, and data-mining approaches to study root exudates.Common analytical techniques are grounded in mass spectrometry or nuclear mass spectrometry, but less common biospectroscopy techniques could offer a new perspective in plant metabolomics due to the minimal sample processing they require. Finally, after analysis, the collected raw data must then be analyzed by means of different multivariate and univariate statistical approaches to test biological-response hypotheses. All in all, the assessment of root exudates calls for the development of hyphenated analytical methodologies, as well as efforts to consolidate data-preprocessing workflows.     

Modified QuEChERS method for phenolic compounds determination in mustard greens (Brassica juncea) using UHPLC-MS/MS

A modified QuEChERS method (Quick, Easy, Cheap, Effective, Rugged, and Safe) for the determination of fifteen phenolic compounds in mustard greens (Brassica juncea) using ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) analysis was developed. The QuEChERS partitioning step and dispersive solid phase extraction (d-SPE) clean-up sorbents were investigated, aimed at phenolic compound extraction and pigment removal, respectively. QuEChERS acetate version combined with 25 mg of diatomaceous earth (DE) and 5.0 mg of graphitized carbon black (GCB) provided the best conditions for sample preparation of the target compounds. Under the optimized conditions, all phenolic compounds showed good linearity (r ≥ 0.99) over the concentration range of 0.1 to 8000 μg kg⁻¹, and the quantification limits were in the range of 0.06–230 μg kg⁻¹. The spectrophotometric analysis showed that the clean-up step promoted a significant removal of chlorophyll, which is the major pigment present in the sample. Furthermore, antioxidant activity analysis was also carried out after the clean-up step and, together with chromatographic data, showed that no significant retention of the phenolic compounds occurs in the clean-up step. Two mustard greens varieties – Southern Giant Curled (SGC) and Florida Broadleaf (FB) - were analyzed with the proposed method. Seven phenolic compounds (4-hydroxybenzoic, p-coumaric, ferulic and sinapic acids, naringenin, apigenin and kaempferol) were found in both varieties, the greatest abundance being for sinapic acid (1261.5 ± 23 μg kg⁻¹ in SGC and 1235.5 ± 26 μg kg⁻¹ in FB) and ferulic acid (2861 ± 24 μg kg⁻¹ in SGC and 3204.5 ± 45 μg kg⁻¹ in FB).     

高效液相色谱法测定铬超富集植物李氏禾根系分泌物中的有机酸

建立了高效液相色谱（HPLC）测定Cr超富集植物李氏禾根系分泌物中低相对分子质量有机酸的分析方法。采用XSelect HSS T3色谱柱（250 mm×4.6 mm,5 μm,Waters）,以40 mmol/L磷酸二氢钾-磷酸缓冲溶液（pH=2.40）作流动相,流速1.0 mL/min,柱温25℃,在波长205 nm处检测。该方法在13 min内简便快速地分离出8种有机酸（草酸、酒石酸、苹果酸、乳酸、甲酸、乙酸、马来酸和柠檬酸）,且峰形良好。有机酸的检出限（LOD）为0.12~12.32 mg/L;草酸的加标回收率为73.15%,其他有机酸的加标回收率为94.54%~109.98%。李氏禾的根系分泌物中各有机酸含量分别为酒石酸（130.90±1.44）μg/g（根干重）、苹果酸（1031.34±4.38）μg/g（根干重）、乳酸（65.54±1.01）μg/g（根干重）、马来酸（0.96000±0.00367）μg/g（根干重）和柠檬酸（201.50±1.13）μg/g（根干重）。该方法简便快速,灵敏可靠,适用于植物根系分泌物样品中有机酸的测定。     

HPLC-ICP-MS测定中药中砷的形态

报道了高效液相色谱-电感耦合等离子体质谱（HPLC—ICP-MS）联用技术测定中药中砷的形态，采用阴离子交换柱，以0．2mmol／L EDTA和2mmol／L NaH2PO4的溶液为流动相，pH6．0，流速为1．0mL／min，成功分离了亚砷酸（AsⅢ）、砷酸（AsV）、甲基砷（MMA）和二甲基砷（DMA）。检出限分别为0．67μg／L（AsⅢ），0．85μg／L（DMA），0．43μg／L（MMA），0．70μg／L（AsV）。中药样品经过（1＋1）甲醇和水的溶液超声提取，离心、过滤、氮气吹干甲醇，超纯水定容。样品加标平均萃取回收率分别为：92．8％（AsⅢ），108％（DMA），104％（MMA），101％（AsV），相对标准偏差（RsD，n=7）均小于10％。     

HPLC-ICP-MS测定中药中砷的形态

报道了高效液相色谱-电感耦合等离子体质谱(HPLC-ICP-MS)联用技术测定中药中砷的形态.采用阴离子交换柱,以含0.2 mmol/L乙二胺四乙酸(EDTA)和2 mmol/L NaH2PO4的水溶液为流动相,pH 6.0,流速为1.0 mL/min,成功分离了亚砷酸(AsⅢ)、砷酸(AsⅤ)、甲基砷(MMA)和二甲基砷(DMA).检出限分别为0.67 μg/L (AsⅢ),0.85 μg/L (DMA),0.43 μg/L (MMA),0.70 μg/L (AsⅤ).中药样品经过(1 1)甲醇水溶液超声提取,离心、过滤、氮气吹干甲醇,超纯水定容.样品加标平均萃取回收率分别为: 92.8% (AsⅢ),108% (DMA),104% (MMA),101% (AsⅤ),RSD (n=7)均小于10%.     

Physico-chemical characterization of metal binding proteins using HPLC-ICP-MS, HPLC-MA-AAS and electrospray-MS

A size exclusion HPLC method was developed and interfaced with ICP-MS detection for determining the metal profiles of commercially available rabbit liver metallothioneins (MT) and metallothionein-like proteins (MLP) extracted from fresh water mussels and hemolyzed osprey blood. The redox state of the cysteine residues was indirectly evaluated via a cadmium saturation approach in the presence or absence of a reducing agent, followed by HPLC-microatomization (MA)-AAS and HPLC-ICP-MS analyses. An electrospray-MS protocol was also developed to accurately measure the molecular weight of rabbit MT isoform II. Nanogram quantities of Cd-MT/MLP were poorly chromatographed on silica based supports. A copolymeric styrene-divinylbenzene size exclusion support provided a symmetrical peak (rabbit MT standard) and linear HPLC-MA-AAS calibration curves [r=0.9988; from the LOD (27 ng, as protein) to about 300×LOD], indicating negligible losses of Cd during the chromatography of trace quantities. Co-injection of Cd²⁺ saturated samples with beta-mercaptoethanol (BMSH) was essential to repress Cd²⁺-support interactions which otherwise induced an undesirable metalaffinity retention mechanism. In the presence of added Cd²⁺, 22 mmol/L BMSH did not significantly compete for Cd²⁺ specifically bound to MT, while preventing non-specific binding to non-thiolic complexing sites. Crude mussel and osprey blood MLP extracts (in cold, deoxygenated Tris-HCl buffer) were obtained by ultracentrifugation (145,000 g) and thermocoagulation/centrifugation, respectively. Incubation with BMSH was prerequisite to obtain a maximum saturation of mussel and osprey blood MLP by Cd²⁺, even for samples conserved (–80° C) in the presence of BMSH (22 mmol/L). These observations indicated that a major proportion of the cysteine residues present in these MLP were oxidized. The assumption of a fully reduced MT/MLP pool binding metals in a definite stoichiometry has been the basis of several quantitative metal binding assays involving the saturation of the thiolic complexing sites with a metallic marker (Ag+, Cd²⁺, or Hg²⁺). Since thiolic agents may interfere, the metal saturation protocols do not include a reducing step to ensure that all cysteines in a MT/MLP extract are available for co-ordination. Given that variations in the redox state of crude MT/MLP extracts may compromise the accuracy of metal saturation assays, it is concluded that the preparation of reference samples certified for total metallothionein content would be desirable.     

Peagol and peagoldione, two new strigolactone-like metabolites isolated from pea root exudates

Two new strigolactone-like metabolites, named peagol and peagoldione, with germinative activity for root parasitic plants, were isolated from pea root exudates and were characterized by spectroscopic methods. Peagol was more active on Orobanche foetida and Phelipanche aegyptiaca seeds, while peagoldione was active on P. aegyptiaca only. Low activity was found on Orobanche crenata and Orobanche minor. Stimulatory activity of peagol on O. foetida seeds is most relevant as this species does not respond to the synthetic strigolactone analogue GR24, usually used as Orobanche germination standard.     

Flavonoids and strigolactones in root exudates as signals in symbiotic and pathogenic plant-fungus interactions

Secondary plant compounds are important signals in several symbiotic and pathogenic plant-microbe interactions. The present review is limited to two groups of secondary plant compounds, flavonoids and strigolactones, which have been reported in root exudates. Data on flavonoids as signaling compounds are available from several symbiotic and pathogenic plant-microbe interactions, whereas only recently initial data on the role of strigolactones as plant signals in the arbuscular mycorrhizal symbiosis have been reported. Data from other plant-microbe interactions and strigolactones are not available yet. In the present article we are focusing on flavonoids in plant-fungal interactions such as the arbuscular mycorrhizal (AM) association and the signaling between different Fusarium species and plants. Moreover the role of strigolactones in the AM association is discussed and new data on the effect of strigolactones on fungi, apart from arbuscular mycorrhizal fungi (AMF), are provided.     

Possible interferences of mercury sulfur compounds with ethylated and methylated mercury species using HPLC-ICP-MS

The HPLC-ICP-MS coupling technique is able to separate and detect methyl, ethyl and inorganic mercury isotopes specifically. An identification of ethyl mercury(+) is not possible when the widely used sodium tetraethylborate derivatisation method in combination with GC-AFS/AAS or ICP-MS techniques is performed because it contains ethyl groups.An unidentified compound with the same retention time as ethyl mercury was found in the HPLC chromatograms of industrial sewage samples and humic-rich soils of microcosm experiments after applying water vapour distillation. We also observed such unidentified peaks in samples of heavily contaminated sites in Eastern Germany, separated by HPLC fractionation only. In the experiments described, different mercury sulfur adducts were synthesised and tested for their retention times in the HPLC-ICP-MS system. It was found that the compound CH₃–S–Hg⁺ showed the same retention time as the ethyl mercury standard. It is therefore possible that ethyl mercury detected in chromatography by comparison of the retention time could also be due to an adduct of a sulfur compound and a mercury species. CH₃–S–Hg⁺ should be tested in other chromatographic mercury speciation methods for this effect.This work can also be regarded as a contribution to the discussion of artificially occurring methyl mercury in sediments during sample preparation.     

Chemical Characterization of Three Accessions of Brassica juncea L. Extracts from Different Plant Tissues

Indian mustard or Brassica juncea (B. juncea) is an oilseed plant used in many types of food (as mustard or IV range salad). It also has non-food uses (e.g., as green manure), and is a good model for phytoremediation of metals and pesticides. In recent years, it gained special attention due to its biological compounds and potential beneficial effects on human health. In this study, different tissues, namely leaves, stems, roots, and flowers of three accessions of B. juncea: ISCI 99 (Sample A), ISCI Top (Sample B), and “Broad-leaf” (Sample C) were analyzed by HPLC-PDA/ESI-MS/MS. Most polyphenols identified were bound to sugars and phenolic acids. Among the three cultivars, Sample A flowers turned were the richest ones, and the most abundant bioactive identified was represented by Isorhamnetin 3,7-diglucoside (683.62 µg/100 mg dry weight (DW) in Sample A, 433.65 µg/100 mg DW in Sample B, and 644.43 µg/100 mg DW in Sample C). In addition, the most complex samples, viz. leaves were analyzed by GC-FID/MS. The major volatile constituents of B. juncea L. leaves extract in the three cultivars were benzenepropanenitrile (34.94% in Sample B, 8.16% in Sample A, 6.24% in Sample C), followed by benzofuranone (8.54% in Sample A, 6.32% in Sample C, 3.64% in Sample B), and phytone (3.77% in Sample B, 2.85% in Sample A, 1.01% in Sample C). The overall evaluation of different tissues from three B. juncea accessions, through chemical analysis of the volatile and non-volatile compounds, can be advantageously taken into consideration for future use as dietary supplements and nutraceuticals in food matrices.     

Determination of inorganic arsenic in white fish using microwave-assisted alkaline alcoholic sample dissolution and HPLC-ICP-MS

An analytical method for the determination of inorganic arsenic in fish samples using HPLC-ICP-MS has been developed. The fresh homogenised sample was subjected to microwave-assisted dissolution by sodium hydroxide in ethanol, which dissolved the sample and quantitatively oxidised arsenite (As(III)) to arsenate (As(V)). This allowed for the determination of inorganic arsenic as a single species, i.e. As(V), by anion-exchange HPLC-ICP-MS. The completeness of the oxidation was verified by recovery of As(V) which was added to the samples as As(III) prior to the dissolution procedure. The full recovery of As(V) at 104±7% (n=5) indicated good analytical accuracy. The uncertified inorganic arsenic content in the certified reference material TORT-2 was 0.186±0.014 ng g^–1 (n=6). The method was employed for the determination of total arsenic and inorganic arsenic in 60 fish samples including salmon from fresh and saline waters and in plaice. The majority of the results for inorganic arsenic were lower than the LOD of 3 ng g^–1, which corresponded to less than one per thousand of the total arsenic content in the fish samples. For mackerel, however, the recovery of As(III) was incomplete and the method was not suited for this fat-rich fish.