Simultaneous determination of steroid hormones and pharmaceuticals in killer whale (Orcinus orca) faecal samples by liquid chromatography tandem mass spectrometry.

We describe a non-invasive method for profiling selected hormones, pharmaceuticals and personal care products (PPCPs) in killer whales (Orcinus orca) based on analysis of faecal samples by liquid chromatography tandem mass spectrometry (LC-MS/MS). The method targets 21 compounds of interest including glucocorticoids, mineralocorticoids, androgens, estrogens, progestogens, selective serotonin uptake inhibitors and an antibacterial/antifungal agent. This method is suitable for routine simultaneous determination of target compounds in killer whale faecal samples as well as validation of immunoassays for the detection and measurement of steroid hormones in faeces. The optimized method involves extraction of freeze-dried faecal material with reagent alcohol and water followed by isolation of the analytes using solid phase extraction with hydrophilic-lipophilic balance cartridges and liquid-liquid extraction with methyl tertiary-butyl ether. Reconstituted extracts were analysed by LC-MS/MS using an electrospray ionization interface. Method limit of quantification ranged from 0.06 to 45.2 ng/g in freeze-dried faecal samples. Except for sertraline, triclosan and estradiol (which was not recovered at the lowest spiked concentration), average intra- and inter-day precisions were within 10%, and average recoveries were between 89.3% and 129.3%, for faecal samples spiked with 5.3, 26.7 or 133 ng/g of each analyte. The method was applied successfully to the analysis of hormones and PPCPs in whale faeces during which 17α-hydroxyprogesterone, a common intermediate in steroid biosynthesis that cross-reacts with precursors and sulphated conjugates in immunoassays, was identified and quantified in all samples.

Dissolved Algal Toxins along the Southern Coast of British Columbia Canada.

Harmful algal blooms (HABs) in coastal British Columbia (BC), Canada, negatively impact the salmon aquaculture industry. One disease of interest to salmon aquaculture is Net Pen Liver Disease (NPLD), which induces severe liver damage and is believed to be caused by the exposure to microcystins (MCs). To address the lack of information about algal toxins in BC marine environments and the risk they pose, this study investigated the presence of MCs and other toxins at aquaculture sites. Sampling was carried out using discrete water samples and Solid Phase Adsorption Toxin Tracking (SPATT) samplers from 2017-2019. All 283 SPATT samples and all 81 water samples tested positive for MCs. Testing for okadaic acid (OA) and domoic acid (DA) occurred in 66 and 43 samples, respectively, and all samples were positive for the toxin tested. Testing for dinophysistoxin-1 (DTX-1) (20 samples), pectenotoxin-2 (PTX-2) (20 samples), and yessotoxin (YTX) (17 samples) revealed that all samples were positive for the tested toxins. This study revealed the presence of multiple co-occurring toxins in BC's coastal waters and the levels detected in this study were below the regulatory limits for health and recreational use. This study expands our limited knowledge of algal toxins in coastal BC and shows that further studies are needed to understand the risks they pose to marine fisheries and ecosystems.

Acute Toxicity of 6PPD-Quinone to Early Life Stage Juvenile Chinook (Oncorhynchus tshawytscha) and Coho (Oncorhynchus kisutch) Salmon.

The breakdown product of the rubber tire antioxidant N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD)-6-PPD-quinone has been strongly implicated in toxic injury and death in coho salmon (Oncorhynchus kisutch) in urban waterways. Whereas recent studies have reported a wide range of sensitivity to 6PPD-quinone in several fish species, little is known about the risks to Chinook salmon (Oncorhynchus tshawytscha), the primary prey of endangered Southern Resident killer whales (Orcinus orca) and the subject of much concern. Chinook face numerous conservation threats in Canada and the United States, with many populations assessed as either endangered or threatened. We evaluated the acute toxicity of 6PPD-quinone to newly feeding (~3 weeks post swim-up) juvenile Chinook and coho. Juvenile Chinook and coho were exposed for 24 h under static conditions to five concentrations of 6PPD-quinone. Juvenile coho were 3 orders of magnitude more sensitive to 6PPD-quinone compared with juvenile Chinook, with 24-h median lethal concentration (LC50) estimates of 41.0 and more than 67 307 ng/L, respectively. The coho LC50 was 2.3-fold lower than what was previously reported for 1+-year-old coho (95 ng/L), highlighting the value of evaluating age-related differences in sensitivity to this toxic tire-related chemical. Both fish species exhibited typical 6PPD-quinone symptomology (gasping, increased ventilation, loss of equilibrium, erratic swimming), with fish that were symptomatic generally exhibiting mortality. The LC50 values derived from our study for coho are below concentrations that have been measured in salmon-bearing waterways, suggesting the potential for population-level consequences in urban waters. The higher relative LC50 values for Chinook compared with coho merits further investigation, including for the potential for population-relevant sublethal effects. Environ Toxicol Chem 2023;42:815-822. © 2023 His Majesty the King in Right of Canada and The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. Reproduced with the permission of the Minister of Fisheries and Oceans Canada.

Domoic acid in Canadian Pacific waters, from 2016 to 2021, and relationships with physical and chemical conditions.

Domoic acid, a phycotoxin produced by species of the marine diatom Pseudo-nitzschia, can cause deleterious impacts to marine food webs and human health. Domoic acid and Pseudo-nitzschia spp. were surveyed from 2016 to 2021 in the Pacific waters of Canada to assess their occurrences, concentrations, and relationships with physical and chemical conditions. Domoic acid was common, occurring in measurable concentrations in 73 % of the 454 samples. It occurred in all regions (west coast of Vancouver Island, Salish Sea, Queen Charlotte Sound / Hecate Strait, deep oceanic NE Pacific), in all years and all seasons. Median concentrations were highest along the west coast of Vancouver Island, and lowest in the oceanic waters of the NE Pacific. Winter had the lowest concentrations; no significant differences occurred between spring, summer, and autumn. High domoic acid concentrations equal to or above 100 ng/L were not common, occurring in about 5 % of samples, but in all seasons and all years except 2019. All six Pseudo-nitzschia taxa identified had similar median concentrations, but different frequencies of occurrence. P. cf. australis appeared to be the major contributor to high concentrations of domoic acid. Physico-chemical conditions were described by ten variables: temperature, salinity, density difference between 30 m and the surface (a proxy for vertical stability), chlorophyll a, nitrate, phosphate, silicate, and the ratios nitrate:phosphate, nitrate:silicate, and silicate:phosphate. Statistical analyses, using general linear models, of their relationships with the absence/presence of Pseudo-nitzschia spp. found silicate (negative) to be the most influential variable common in both the west coast of Vancouver Island and Salish Sea regions. Temperature and chlorophyll a were the most influential variables which determined the log10 abundance of Pseudo-nitzschia spp. in both regions. Analyses of the absence/presence of particulate domoic acid per Pseudo-nitzschia cell (excluding P. americana) found chlorophyll a to be the most influential variable common in both regions, whereas no common influential variable determined the log10 concentration of particulate domoic acid per Pseudo-nitzschia cell (excluding P. americana). These results were generally similar to those of other studies from this area, although this study extends these findings to all seasons and all regions of Canada's Pacific waters. The results provide important background information against which major outbreaks and unusual events can be compared. A domoic acid surveillance program during synoptic oceanographic surveys can help to understand where and when it reaches high concentrations at sea and the potential impacts to the marine ecosystem.

Identification of phosphoproteins in Arabidopsis thaliana leaves using polyethylene glycol fractionation, immobilized metal-ion affinity chromatography, two-dimensional gel electrophoresis and mass spectrometry.

Reversible protein phosphorylation is a key regulatory mechanism in cells. Identification and characterization of phosphoproteins requires specialized enrichment methods, due to the relatively low abundance of these proteins, and is further complicated in plants by the high abundance of Rubisco in green tissues. We present a novel method for plant phosphoproteome analysis that depletes Rubisco using polyethylene glycol fractionation and utilizes immobilized metal-ion affinity chromatography to enrich phosphoproteins. Subsequent protein separation by one- and two-dimensional gel electrophoresis is further improved by extracting the PEG-fractionated protein samples with SDS/phenol and methanol/chloroform to remove interfering compounds. Using this approach, we identified 132 phosphorylated proteins in a partial Arabidopsis leaf extract. These proteins are involved in a range of biological processes, including CO(2) fixation, protein assembly and folding, stress response, redox regulation, and cellular metabolism. Both large and small subunits of Rubisco were phosphorylated at multiple sites, and depletion of Rubisco enhanced detection of less abundant phosphoproteins, including those associated with state transitions between photosystems I and II. The discovery of a phosphorylated form of AtGRP7, a self-regulating RNA-binding protein that affects floral transition, as well as several previously uncharacterized ribosomal proteins confirm the utility of this approach for phosphoproteome analysis and its potential to increase our understanding of growth and development in plants.

Modification of Akt1 by methylglyoxal promotes the proliferation of vascular smooth muscle cells.

Methylglyoxal (MG), a reactive dicarbonyl molecule, can modify protein to form advanced glycation endproducts. Increased MG level has been implicated in proliferative vascular diseases, but the underlying mechanisms are not clear yet. The serine/threonine kinase, Akt, regulates multiple signaling pathways that control cell proliferation. Using mass spectrometric analysis, we have detected the modification of Akt1 by MG at Cys(77). This structural modification increased Akt1 phosphorylation at Ser(473) and Thr(308). Akt1 phosphorylation and activity were also increased by MG treatment (<50 µM) in cultured vascular smooth muscle cells (VSMCs). MG treatment of VSMCs led to increased DNA synthesis (EC(50)=5.8 µM), cell proliferation, phosphorylation of p21 and glycogen synthase kinase-3α/ß (GSK-3α/ß), and increased cyclin-dependent kinase 2 (CDK2) activity. These effects of MG were significantly inhibited by silencing Akt1 or by an Akt inhibitor. Overexpression of Akt1 Cys(77)Ser mutant in HEK-293 cells increased cell proliferation and DNA synthesis, concurrent with an increase in Akt1 activity, which could not be further augmented by MG treatment. It is concluded that MG-induced VSMC proliferation is mediated by the activation of Akt1 via the modification of Akt1 at Cys(77).

Enrichment and analysis of phosphopeptides under different experimental conditions using titanium dioxide affinity chromatography and mass spectrometry.

Titanium dioxide metal oxide affinity chromatography (TiO(2)-MOAC) is widely regarded as being more selective than immobilized metal-ion affinity chromatography (IMAC) for phosphopeptide enrichment. However, the widespread application of TiO(2)-MOAC to biological samples is hampered by conflicting reports as to which experimental conditions are optimal. We have evaluated the performance of TiO(2)-MOAC under a wide range of loading and elution conditions. Loading and stringent washing of peptides with strongly acidic solutions ensured highly selective enrichment for phosphopeptides, with minimal carryover of non-phosphorylated peptides. Contrary to previous reports, the addition of glycolic acid to the loading solution was found to reduce specificity towards phosphopeptides. Base elution in ammonium hydroxide or ammonium phosphate provided optimal specificity and recovery of phosphorylated peptides. In contrast, elution with phosphoric acid gave incomplete recovery of phosphopeptides, whereas inclusion of 2,5-dihydroxybenzoic acid in the eluant introduced a bias against the recovery of multiply phosphorylated peptides. TiO(2)-MOAC was also found to be intolerant of many reagents commonly used as phosphatase inhibitors during protein purification. However, TiO(2)-MOAC showed higher specificity than immobilized gallium (Ga(3+)), immobilized iron (Fe(3+)), or zirconium dioxide (ZrO(2)) affinity chromatography for phosphopeptide enrichment. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was more effective in detecting larger, multiply phosphorylated peptides than liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS), which was more efficient for smaller, singly phosphorylated peptides.

Dynamics of protein expression during pollen germination in canola (Brassica napus).

The proteome of mature (MP) and in vitro germinating pollen (GP) of canola (Brassica napus) were analyzed using the DIGE technology with the objective of identifying proteins and their function in pollen germination. Of the 2,238 protein spots detected in gel images, 344 were differentially expressed in MP and GP samples of which 165 were subjected to MALDI-TOF/TOF and 130 were successfully identified using the NCBInr and Brassica EST databases. The major proteins up-regulated in GP, relative to MP, have roles in carbohydrate metabolism, protein metabolism, and cell wall remodeling. Others with roles in cytoskeleton dynamics, nucleotide and amino acid metabolism, signal transduction, and stress response also showed higher expression in GP. Proteins concerned with transcriptional regulation and ion transport were similar in MP and GP, and some catalases and LEA proteins were down-regulated in GP. A number of proteins including, oleosin, cruciferin, and enolase, were released into the pollen germination medium indicating their potential role in pollen-stigma interaction. Glycosylated proteins were also identified in MP and GP, but their protein profiles were not different. This study has documented the dynamics of protein expression during pollen germination and early tube growth in B. napus and provides insights into the fundamental mechanisms involved in these processes, and in cell growth, cell-cell communication, and cell signaling.

Hormonal regulation of oil accumulation in Brassica seeds: metabolism and biological activity of ABA, 7'-, 8'- and 9'-hydroxy ABA in microspore derived embryos of B. napus.

Developing seeds of Brassica napus contain significant levels of ABA and products of oxidation at the 7'- and 9'-methyl groups of ABA, 7'- and 9'-hydroxy ABA, as well stable products of oxidation of the 8'-methyl group, phaseic acid and dihydrophaseic acid. To probe the biological roles of the initially formed hydroxylated compounds, we have compared the effects of supplied ABA and the hydroxylated metabolites in regulating oil synthesis in microspore-derived embryos of B. napus, cv Hero that accumulate long chain fatty acids. Uptake into the embryos and metabolism of each of the hormone metabolites was studied by using deuterium labeled analogs. Supplied ABA, which was rapidly metabolized, induced expression of oleosin and fatty acid elongase genes and increased the accumulation of triacylglycerols and very long chain fatty acids. The metabolites 7'- and 9'-hydroxy ABA had similar effects, with the 9'-hydroxy ABA having even greater activity than ABA. The principal catabolite of ABA, 8'-hydroxy ABA, also had hormonal activity and led to increased oil synthesis but induced the genes weakly. These results indicate that all compounds tested could be involved in lipid synthesis in B. napus, and may have hormonal roles in other ABA-regulated processes.

Phosphorylation of the 12 S globulin cruciferin in wild-type and abi1-1 mutant Arabidopsis thaliana (thale cress) seeds.

Cruciferin (a 12 S globulin) is the most abundant storage protein in the seeds of Arabidopsis thaliana (thale cress) and other crucifers, sharing structural similarity with the cupin superfamily of proteins. Cruciferin is synthesized as a precursor in the rough endoplasmic reticulum. Subunit assembly is accompanied by structural rearrangements involving proteolysis and disulfide-bond formation prior to deposition in protein storage vacuoles. The A. thaliana cv. Columbia genome contains four cruciferin loci, two of which, on the basis of cDNA analysis, give rise to three alternatively spliced variants. Using MS, we confirmed the presence of four variants encoded by genes At4g28520.1, At5g44120.3, At1g03880.1 and At1g3890.1 in A. thaliana seeds. Two-dimensional gel electrophoresis, along with immunological detection using anti-cruciferin antiserum and antibodies against phosphorylated amino acid residues, revealed that cruciferin was the major phosphorylated protein in Arabidopsis seeds and that polymorphism far exceeded that predicted on the basis of known isoforms. The latter may be attributed, at least in part, to phosphorylation site heterogeneity. A total of 20 phosphorylation sites, comprising nine serine, eight threonine and three tyrosine residues, were identified by MS. Most of these are located on the IE (interchain disulfide-containing) face of the globulin trimer, which is involved in hexamer formation. The implications of these findings for cruciferin processing, assembly and mobilization are discussed. In addition, the protein phosphatase 2C-impaired mutant, abi1-1, was found to exhibit increased levels of cruciferin phosphorylation, suggesting either that cruciferin may be an in vivo target for this enzyme or that abi1-1 regulates the protein kinase/phosphatase system required for cruciferin phosphorylation.

Comparative approaches to the investigation of responses to stress and viral infection in cattle.

Fatal bovine respiratory disease (BRD) is a major cause of financial losses in the cattle industry. A variety of stressors have been implicated as contributing to disease severity. However, it has proven difficult to determine the role these individual factors may play in the final outcome of this disease complex. The objective of the present investigation was to obtain proteomic, metabonomic, and elemental profiles of bovine serum samples from stressed and control animals before and after a primary viral infection to determine if these profiles could distinguish between responses to stressors and viral infection. Multivariate analysis revealed distinct differential trends in the distribution profile of proteins, metabolites, and elements following a stress response both before and after primary viral infection. A group of acute phase proteins, metabolites, and elements could be specifically linked to either a stress response (decreased serum amyloid A and Cu, increased apolipoprotein CIII, amino acids, LDL, P, and Mo) or a primary viral respiratory infection (increased apolipoprotein A1, haptoglobin, glucose, amino acids, LDL and Cu, decreased Lipid, and P). Thus, combined OMICS analysis of serum samples revealed that multimethod analysis could be used to discriminate between the complex biological responses to stress and viral infection.

Identification of histidine phosphorylations in proteins using mass spectrometry and affinity-based techniques.

Histidine phosphorylation plays a key role in prokaryotic signaling and accounts for approximately 6% of the protein phosphorylation events in eukaryotics. Phosphohistidines generally act as intermediates in the transfer of phosphate groups from donor to acceptor molecules. Examples include the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) and the histidine kinases found in two-component signal transduction pathways. The latter are utilized by bacteria and plants to sense and adapt to changing environmental conditions. Despite the importance of histidine phosphorylation in two-component signaling systems, relatively few proteins have so far been identified as containing phosphorylated histidine residues. This is largely due to the instability of phosphohistidines, which, unlike the phosphoesters formed by serine, threonine, and tyrosine, are labile and susceptible to acid hydrolysis. Nevertheless, it is possible to preserve and identify phosphorylated histidine residues in target proteins using appropriate sample preparation, affinity purification, and mass spectrometric techniques. This chapter provides a brief overview of such techniques, describes their use in confirming histidine phosphorylation of a known PTS protein (HPr), and suggests how this approach might be adapted for large-scale identification of histidine-phosphorylated proteins in two-component systems.

Proteomic analysis of tomato (Lycopersicon esculentum) pollen.

In flowering plants, pollen grains are produced in the anther and released to the external environment with the primary function of delivering sperm cells to the female gametophyte. This study was conducted to identify proteins in tomato pollen and to analyse their roles in relation to pollen function. Tomato is an important crop which is grown worldwide and is an excellent experimental system. Proteins were extracted from pollen, separated by two-dimensional gel electrophoresis (2-DE), and identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and peptide mass fingerprinting. Of the 960 spots observed on Colloidal Coomassie Blue (CCB)-stained 2-DE gels, 190 were selected for analysis. Of these, 158 spots, representing 133 distinct proteins, were identified by searching the NCBInr and Expressed Sequence Tag databases. The identified proteins were classified based on designated functions and the majority included those involved in defence mechanisms, energy conversions, protein synthesis and processing, cytoskeleton formation, Ca(2+) signalling, and as allergens. A number of proteins in tomato pollen were similar to those reported in the pollen of other species; however, several additional proteins with roles in defence mechanisms, metabolic processes, and hormone signalling were identified. The potential roles of the identified proteins in the survival strategy of the small, independent, two-celled pollen grain of tomato, and subsequently in pollen germination and tube growth are discussed.

Structural and functional changes in human insulin induced by methylglyoxal.

Elevated methylglyoxal (MG) levels have been reported in insulin-resistance syndrome. The present study investigated whether MG, a highly reactive metabolite of glucose, induced structural and functional changes of insulin. Incubation of human insulin with MG in vitro yielded MG-insulin adducts, as evidenced by additional peaks observed on mass spectrometric (MS) analysis of the incubates. Tandem MS analysis of insulin B-chain adducts confirmed attachment of MG at an arginine residue. [3H]-2-deoxyglucose uptake by 3T3-L1 adipocytes was significantly and concentration-dependently decreased after the treatment with MG-insulin adducts, in comparison with the effect of native insulin at the same concentrations. A significant decrease of glucose uptake induced by MG-insulin adducts was also observed in L8 skeletal muscle cells. MG alone had no effect on glucose uptake or the transcriptional expression of insulin receptor. Unlike native insulin, MG-insulin adducts did not inhibit insulin release from pancreatic beta-cells. The degradation of MG-insulin through liver cells was also decreased. In conclusion, MG modifies insulin by attaching to internal arginine residue in beta-chain of insulin. The formation of this MG-insulin adduct decreases insulin-mediated glucose uptake, impairs autocrine control of insulin secretion, and decreases insulin clearance. These structural and functional abnormalities of insulin molecule may contribute to the pathogenesis of insulin resistance.

Identification of the SRC pyrimidine-binding protein (SPy) as hnRNP K: implications in the regulation of SRC1A transcription.

The human SRC gene encodes pp60(c-src), a non-receptor tyrosine kinase involved in numerous signaling pathways. Activation or overexpression of c-Src has also been linked to a number of important human cancers. Transcription of the SRC gene is complex and regulated by two closely linked but highly dissimilar promoters, each associated with its own distinct non-coding exon. In many tissues SRC expression is regulated by the housekeeping-like SRC1A promoter. In addition to other regulatory elements, three substantial polypurine:polypyrimidine (TC) tracts within this promoter are required for full transcriptional activity. Previously, we described an unusual factor called SRC pyrimidine-binding protein (SPy) that could bind to two of these TC tracts in their double-stranded form, but was also capable of interacting with higher affinity to all three pyrimidine tracts in their single-stranded form. Mutations in the TC tracts, which abolished the ability of SPy to interact with its double-stranded DNA target, significantly reduced SRC1A promoter activity, especially in concert with mutations in critical Sp1 binding sites. Here we expand upon our characterization of this interesting factor and describe the purification of SPy from human SW620 colon cancer cells using a DNA affinity-based approach. Subsequent in-gel tryptic digestion of purified SPy followed by MALDI-TOF mass spectrometric analysis identified SPy as heterogeneous nuclear ribonucleoprotein K (hnRNP K), a known nucleic-acid binding protein implicated in various aspects of gene expression including transcription. These data provide new insights into the double- and single-stranded DNA-binding specificity, as well as functional properties of hnRNP K, and suggest that hnRNP K is a critical component of SRC1A transcriptional processes.

Potent inhibitor of N-myristoylation: a novel molecular target for cancer.

N-myristoyltransferase (NMT) is an essential eukaryotic enzyme that catalyzes the cotranslational and/or posttranslational transfer of myristate to the NH(2) terminus of the glycine residue of a number of important proteins that have diverse biological functions and thus have been proposed as potential targets for chemotherapeutic drug design. Earlier, we demonstrated that NMT is more active in colonic epithelial neoplasms than in corresponding normal-appearing colonic tissue. Furthermore, an increased expression of NMT was also observed in gallbladder carcinoma. In the present study, we report a novel protein inhibitor of NMT. This protein caused a potent concentration-dependent inhibition of human NMT with half-maximal inhibition at 4.5 +/- 0.35 nM. This study will serve as a template for further investigations in the area of protein myristoylation.

Speciation of cyclo(Pro-Gly)3 and its divalent metal-ion complexes by electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) was used to study the binding of selected group II and divalent transition-metal ions by cyclo(Pro-Gly)3 (CPG3), a model ion carrier peptide. Metal salts (CatXn) were combined with the peptide (M) at a molar ratio of 1:10 M/Cat in aqueous solvents containing 50% vol/vol acetonitrile or methanol and 1 or 10 mM ammonium acetate (NH4Ac). Species detected include [M+H]+, [M+Cat-H]+, [M2+Cat]2+, [M+Cat+Ac]+, and [M+Cat+X]+. The relative stabilities of complexes formed with different cations (Mg2+, Ca2+, Sr2+, Mn2+, Co2+, Ni2+, Cu2+, and Zn2+) were determined from the abundance of 1:1 and 2:1 M/Cat species relative to that of the unbound peptide. The largest metal ions (Ca2+, Sr2+, and Mn2+) formed the most stable complexes. Reducing the buffer concentration increased the overall extent of metal binding. Results show that the binding specificity of CPG3 depends upon the size of the metal ion and its propensity for electrostatic interaction with oxygen atoms. Product ion tandem mass spectrometry of [M+H]+ and [M+Cu-H]+ confirmed the cyclic structure of the peptide, although the initial site(s) of metal attachment could not be determined.

A novel method for the rapid determination of polyethoxylated tallow amine surfactants in water and sediment using large volume injection with high performance liquid chromatography and tandem mass spectrometry.

Polyethoxylated tallow amine (POEA) surfactants have been used in many glyphosate-based herbicide formulations for agricultural, industrial and residential weed control. The potential for release of these compounds into the environment is of increasing concern due to their toxicity towards aquatic organisms. Current methods for analysis of POEA surfactants require significant time and effort to achieve limits of quantification that are often higher than the concentrations at which biological effects have been observed (as low as 2 ng mL(-1)). We have developed a rapid and robust method for quantifying the POEA surfactant mixture MON 0818 at biologically relevant concentrations in fresh water, sea water and lake sediment using reversed phase high-performance liquid chromatography and electrospray ionization-tandem mass spectrometry. Water samples preserved by 1:1 v/v dilution with methanol are analyzed directly following centrifugation. Sediment samples undergo accelerated solvent extraction in aqueous methanol prior to analysis. Large volume (100 µL) sample injection and multiple reaction monitoring of a subset of the most abundant POEA homologs provide limits of quantification of 0.5 and 2.9 ng mL(-1) for MON 0818 in fresh water and sea water, respectively, and 2.5 ng g(-1) for total MON 0818 in lake sediment. Average recoveries of 93 and 75% were achieved for samples of water and sediment, respectively spiked with known amounts of MON 0818. Precision and accuracy for the analysis of water and sediment samples were within 10 and 16%, respectively based upon replicate analyses of calibration standards and representative samples. Results demonstrate the utility of the method for quantifying undegraded MON 0818 in water and sediment, although a more comprehensive method may be needed to identify and determine other POEA mixtures and degradation profiles that might occur in the environment.

Enrichment and Analysis of Intact Phosphoproteins in Arabidopsis Seedlings.

Protein phosphorylation regulates diverse cellular functions and plays a key role in the early development of plants. To complement and expand upon previous investigations of protein phosphorylation in Arabidopsis seedlings we used an alternative approach that combines protein extraction under non-denaturing conditions with immobilized metal-ion affinity chromatography (IMAC) enrichment of intact phosphoproteins in Rubisco-depleted extracts, followed by identification using two-dimensional gel electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). In-gel trypsin digestion and analysis of selected gel spots identified 144 phosphorylated peptides and residues, of which only 18 phosphopeptides and 8 phosphosites were found in the PhosPhAt 4.0 and P3DB Arabidopsis thaliana phosphorylation site databases. More than half of the 82 identified phosphoproteins were involved in carbohydrate metabolism, photosynthesis/respiration or oxidative stress response mechanisms. Enrichment of intact phosphoproteins prior to 2-DE and LC-MS/MS appears to enhance detection of phosphorylated threonine and tyrosine residues compared with methods that utilize peptide-level enrichment, suggesting that the two approaches are somewhat complementary in terms of phosphorylation site coverage. Comparing results for young seedlings with those obtained previously for mature Arabidopsis leaves identified five proteins that are differentially phosphorylated in these tissues, demonstrating the potential of this technique for investigating the dynamics of protein phosphorylation during plant development.

Rapid extraction of abscisic acid and its metabolites for liquid chromatography-tandem mass spectrometry.

We have described a simple, reliable and rapid method of extracting and partially purifying the phytohormone (+)-abscisic acid and its catabolites for liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS) analysis. Lyophilized tissue samples were powdered by high-speed agitation with ceramic beads for 5 s. Metabolites were extracted from the tissue powder using acetone-water-acetic acid (80:19:1, v/v) with the addition of deuterated internal standards for quantification. Essentially all endogenous hormones were recovered by three successive tissue extractions. However we demonstrated that, with the use of internal standards, one extraction with vigorous vortexing was sufficient to obtain accurate results (recovery 65-90%). Solvents were optimized for partial purification of abscisic acid and related compounds by solid-phase extraction using Oasis HLB cartridges. The eluted metabolites were then analyzed by LC-MS-MS. To illustrate the applicability of these techniques, we analyzed the levels of abscisic acid and metabolites in seeds and valves of Brassica napus siliques at two stages of development. We detected abscisic acid, phaseic acid, 7'-hydroxyabscisic acid, dihydrophaseic acid and abscisic acid glucose ester. In both tissues, dihydrophaseic acid was the major accumulating product, reaching 97300 pmol/g dry mass in valves at 24 days after anthesis. The amount of abscisic acid in seeds was high at 24 days after anthesis (23300 pmol/g dry mass), but low in the other samples (292-447 pmol/g dry mass).