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.

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.

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.

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.

Differential expression of proteins in the wild type and 7B-1 male-sterile mutant anthers of tomato (Solanum lycopersicum): a proteomic analysis.

In the 7B-1 male-sterile mutant of tomato, pollen development breaks down prior to meiosis in microspore mother cells (MMCs). We have used the proteomic approach to identify differentially expressed proteins in the wild type (WT) and mutant anthers with the objective of analyzing their roles in normal pollen development and in male sterility. By using 2-DE and DIGE technologies, over 1800 spots were detected and of these 215 spots showed 1.5-fold or higher volume ratio in either WT or 7B-1 anthers. Seventy spots, either up-regulated in WT, or in 7B-1, were subjected to mass spectrometry and 59 spots representing 48 distinct proteins were identified. The proteins up-regulated in WT anthers included proteases, e.g., subtilase, proteasome subunits, and 5B-protein with potential roles in tapetum degeneration, FtsZ protein, leucine-rich repeat proteins, translational and transcription factors. In 7B-1 anthers, aspartic protease, superoxide dismutase, ACP reductase, ribonucleoprotein and diphosphate kinase were up-regulated. Also, cystatin inhibitory activity was high in the mutant and correlated with the expression of male sterility. Other proteins including calreticulin, Heat shock protein 70, glucoside hydrolase, and ATPase, were present in both genotypes. The function of identified proteins in tapetum and normal pollen development, and in male sterility is discussed.

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.

The molecular cloning of artemisinic aldehyde Delta11(13) reductase and its role in glandular trichome-dependent biosynthesis of artemisinin in Artemisia annua.

At some point during biosynthesis of the antimalarial artemisinin in glandular trichomes of Artemisia annua, the Delta11(13) double bond originating in amorpha-4,11-diene is reduced. This is thought to occur in artemisinic aldehyde, but other intermediates have been suggested. In an effort to understand double bond reduction in artemisinin biosynthesis, extracts of A. annua flower buds were investigated and found to contain artemisinic aldehyde Delta11(13) double bond reductase activity. Through a combination of partial protein purification, mass spectrometry, and expressed sequence tag analysis, a cDNA clone corresponding to the enzyme was isolated. The corresponding gene Dbr2, encoding a member of the enoate reductase family with similarity to plant 12-oxophytodienoate reductases, was found to be highly expressed in glandular trichomes. Recombinant Dbr2 was subsequently characterized and shown to be relatively specific for artemisinic aldehyde and to have some activity on small alpha,beta-unsaturated carbonyl compounds. Expression in yeast of Dbr2 and genes encoding four other enzymes in the artemisinin pathway resulted in the accumulation of dihydroartemsinic acid. The relevance of Dbr2 to trichome-specific artemisinin biosynthesis is discussed.

Optimization of immobilized gallium (III) ion affinity chromatography for selective binding and recovery of phosphopeptides from protein digests.

Although widely used in proteomics research for the selective enrichment of phosphopeptides from protein digests, immobilized metal-ion affinity chromatography (IMAC) often suffers from low specificity and differential recovery of peptides carrying different numbers of phosphate groups. By systematically evaluating and optimizing different loading, washing, and elution conditions, we have developed an efficient and highly selective procedure for the enrichment of phosphopeptides using a commercially available gallium(III)-IMAC column (PhosphoProfile, Sigma). Phosphopeptide enrichment using the reagents supplied with the column is incomplete and biased toward the recovery and/or detection of smaller, singly phosphorylated peptides. In contrast, elution with base (0.4 M ammonium hydroxide) gives efficient and balanced recovery of both singly and multiply phosphorylated peptides, while loading peptides in a strong acidic solution (1% trifluoracetic acid) further increases selectivity toward phosphopeptides, with minimal carryover of nonphosphorylated peptides. 2,5-Dihydroxybenzoic acid, a matrix commonly used when analyzing phosphopeptides by matrix-assisted laser desorption/ionization mass spectrometry was also evaluated as an additive in loading and eluting solvents. Elution with 50% acetonitrile containing 20 mg/mL dihydroxybenzoic acid and 1% phosphoric acid gave results similar to those obtained using ammonium hydroxide as the eluent, although the latter showed the highest specificity for phosphorylated peptides.

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.

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.

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.

Mass spectrometric determination of the coordination geometry of potential copper(II) surrogates for the mammalian prion protein octarepeat region.

The N-terminal domain of mammalian prion proteins contains several tandem repeats of the octapeptide PHGGGWGQ, each one capable of selectively binding up to 1 equiv of Cu2+. Under saturating conditions Cu2+ is known to coordinate the HGG portion of the repeat sequence via the histidine imidazole side chain, two deprotonated amide N-atoms, and a backbone carbonyl O-atom. Using appropriate selection criteria, we have generated a short list of candidate metal ions (Co3+, Ni2+, Pd2+, Pt2+) that can serve as potential surrogates for Cu2+. The selected metal ions were screened for binding interactions with the OR-derived peptide fragment AcHGGGWNH2 (Ac = acetyl, amino acid residues in italics) using electrospray ionization mass spectrometry. The coordination geometries of these metal ions with the synthetic OR peptide were subsequently determined from fragment analysis using collision-induced dissociation tandem mass spectrometry. Our results indicate that, although Co3+, Pd2+, and Pt2+ all bind to the OR fragment via the peptide backbone to varying extents, each of these metal ions appears to associate with the peptide in a unique manner, which is distinct from the way in which Cu2+ is coordinated. This work illustrates the extremely strong selectivity for Cu2+ of this highly conserved region of the mammalian prion protein.

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.

A comparative proteome and phosphoproteome analysis of differentially regulated proteins during fertilization in the self-incompatible species Solanum chacoense Bitt.

We have used 2-DE for a time-course study of the changes in protein and phosphoprotein expression that occur immediately after fertilization in Solanum chacoense. The phosphorylation status of the detected proteins was determined with three methods: in vivo labeling, immunodetection, and phosphoprotein-specific staining. Using a pI range of 4-7, 262 phosphorylated proteins could be mapped to the 619 proteins detected by Sypro Ruby staining, representing 42% of the total proteins. Among these phosphoproteins, antibodies detected 184 proteins from which 78 were also detected with either of the other two methods (42%). Pro-Q Diamond phosphoprotein stain detected 111 proteins, of which 76 were also detected with either of the other two methods (68%). The 32P in vivo labeling method detected 90 spots from which 78 were also detected with either of other two methods (87%). On comparing before and after fertilization profiles, 38 proteins and phosphoproteins presented a reproducible change in their accumulation profiles. Among these, 24 spots were selected and analyzed by LC-MS/MS using a hybrid quadrupole-TOF (Q-TOF) instrument. Peptide data were searched against publicly available protein and EST databases, and the putative roles of the identified proteins in early fertilization events are discussed.