Contribution of picoplankton to primary production in estuarine,coastal and equatorial waters of Brazil

Quantitative data on primary production of picoplankton are presented for three different Brazilian marine ecosystems: estuarine, coastal and oceanic. The size fraction from 0.45 μm to 1.0 μm was responsible for 3.0% to 28.5% of ¹⁴C uptake in estuarine waters; 18.5% to 40.4% in coastal waters, and 6.7% to 100.0% in oceanic waters, respectively. These results establish the importance of picoplankton as primary producers in different marine environments along the Brazilian waters.     

Robust MS quantification method for phospho-peptides using 18O/16O labeling

Background  

Quantitative measurements of specific protein phosphorylation sites, as presented here, can be used to investigate signal transduction pathways, which is an important aspect of cell dynamics. The presented method quantitatively compares peptide abundances from experiments using ¹⁸O/¹⁶O labeling starting from elaborated MS spectra. It was originally developed to study signaling cascades activated by amyloid-β treatment of neurons used as a cellular model system with relevance to Alzheimer's disease, but is generally applicable.     

Quantitative proteomics using 16O/18O labeling and linear ion trap mass spectrometry

Quantitative proteomics using stable isotopic 16O/18O labeling has emerged as a very powerful tool, since it has a number of advantages over other methods, including the simplicity of chemistry, the constant mass tag at the C termini and its general applicability. However, due to the small mass difference between labeled and unlabeled peptide species, this approach has usually been restricted to high-resolution mass spectrometers. In this study we explored whether the high-resolution scanning mode, together with the extremely high scanning speed of the linear IT allows the 16O/18O-labeling method to be used for accurate, large-scale quantitative analysis of proteomes. A protocol, including digestion, desalting, labeling, MS and quantitative analysis was developed and tested using protein standards and whole proteome extracts. Using this method we were able to identify and quantify 140 proteins from only 10 mug of a proteome extract from mesenchymal stem cells. Relative expression changes larger than twofold can be identified with this method at the 95% confidence level. Our results demonstrate that accurate quantitative analysis using 16O/18O labeling can be performed in the practice using linear IT MS, without compromising large-scale peptide identification efficiency.     

34S/32S and 18O/16O Fractionation During Sulfur Disproportionation by Desulfobulbus propionicus

In the present study, coupled stable sulfur and oxygen isotope fractionation during elemental sulfur disproportionation according to the overall reaction: 4H₂O + 4S^? → 3H₂S + SO₄ ^{2 ?} + 2H⁺, was experimentally investigated for the first time using a pure culture of the sulfate reducer Desulfobulbus propionicus at 35^?C. Bacterial disproportionation of elemental sulfur is an important process in the sulfur cycle of natural surface sediments and leads to the simultaneous formation of sulfide and sulfate. A dual-isotope approach considering both sulfur and oxygen isotope discrimination has been shown to be most effective in evaluating specific microbial reactions. The influence of iron- and manganese bearing-solids (Fe(II)CO₃, Fe(III)OOH, Mn(IV)O₂) acting in natural sediments as scavengers for hydrogen sulfide, was considered, too. Disproportionation of elemental sulfur was observed in the presence of iron solids at a cell-specific sulfur disproportionation rate of about 10^{? 9.5± 0.4} μ mol S^? cell^{? 1} h^{? 1}. No disproportionation, however, was observed with MnO₂. In the presence of iron solids, newly formed sulfate was enriched in ¹⁸ O compared to water by about +21‰ (≡ ? _H2O ), in agreement with a suggested oxygen isotope exchange via traces of intra- or extracellular sulfite that is formed as a disproportionation intermediate. Dissolved sulfate was also enriched in ³⁴S compared to elemental sulfur by up to +35%. Isotope fractionation by Desulfobulbus propionicusis highest for all disproportionating bacteria investigated, so far, and may impact on the development of isotope signals at the redox boundary of surface sediments.     

Regression analysis for comparing protein samples with 16O/18O stable-isotope labeled mass spectrometry

MOTIVATION: Using stable isotopes in global proteome scans, labeled molecules from one sample are pooled with unlabeled molecules from another sample and subsequently subjected to mass-spectral analysis. Stable-isotope methodologies make use of the fact that identical molecules of different stable-isotope compositions are differentiated in a mass spectrometer and are represented in a mass spectrum as distinct isotopic clusters with a known mass shift. We describe two multivariable linear regression models for (16)O/(18)O stable-isotope labeled data that jointly model pairs of resolved isotopic clusters from the same peptide and quantify the abundance present in each of the two biological samples while concurrently accounting for peptide-specific incorporation rates of the heavy isotope. The abundance measure for each peptide from the two biological samples is then used in down-stream statistical analyses, e.g. differential expression analysis. Because the multivariable regression models are able to correct for the abundance of the labeled peptide that appear as an unlabeled peptide due to the inability to exchange the natural C-terminal oxygen for the heavy isotope, they are particularly advantageous for a two-step digestion/labeling procedure. We discuss how estimates from the regression model are used to quantify the variability of the estimated abundance measures for the paired samples. Although discussed in the context of (16)O/(18)O stable-isotope labeled data, the multivariable regression models are generalizable to other stable-isotope labeled technologies.     

Determination of ester carbonyl 18O/16O ratios in phospholipids by gas chromatography-mass spectrometry

A method is described for the quantitation of 18O enrichment in phospholipid acyl groups. Methyl esters are prepared by transesterification with sodium hydroxide in methanol and are hydrogenated prior to gas chromatography-mass spectrometry (GC-MS). Estimation of 18O content of the resulting esters is made using the m/z 74 and m/z 76 fragments, which contain both the ester oxygens. The accuracy of the method is demonstrated with synthetic methyl esters and phosphatidylcholines containing a known amount of 18O.     

Quantitative proteomic analysis of mouse embryonic fibroblasts and induced pluripotent stem cells using 16O/18O labeling

Induced pluripotent stem cells (iPSC) hold great promise for regenerative medicine as well as for investigations into the pathogenesis and treatment of various diseases. Understanding of key intracellular signaling pathways and protein targets that control development of iPSC from somatic cells is essential for designing new approaches to improve reprogramming efficiency. Here, we report the development and application of an integrated quantitative proteomics platform for investigating differences in protein expressions between mouse embryonic fibroblasts (MEF) and MEF-derived iPSC. This platform consists of 16O/18O labeling, multidimensional peptide separation coupled with tandem mass spectrometry, and data analysis with UNiquant software. With this platform, a total of 2481 proteins were identified and quantified from the 16O/18O-labeled MEF-iPSC proteome mixtures with a false discovery rate of 0.01. Among them, 218 proteins were significantly upregulated, while 247 proteins were significantly downregulated in iPSC compared to MEF. Many nuclear proteins, including Hdac1, Dnmt1, Pcna, Ccnd1, Smarcc1, and subunits in DNA replication and RNA polymerase II complex, were found to be enhanced in iPSC. Protein network analysis revealed that Pcna functions as a hub orchestrating complicated mechanisms including DNA replication, epigenetic inheritance (Dnmt1), and chromatin remodeling (Smarcc1) to reprogram MEF and maintain stemness of iPSC.     

Study of bacterial communities in Antarctic coastal waters by a combination of 16S rRNA and 16S rDNA sequencing

An ecological study on distribution of Antarctic bacterial communities was determined by 16S-based phylogenetic analyses of clone libraries derived from RNA and DNA extracted from two different marine areas and compared between each other. Superficial seawater samples were collected from four stations in Ross Sea, three of them located in Rod Bay and one in Evans Cove; for each station two clone libraries (16S rDNA and 16S rRNA) were prepared and evident divergences between DNA and RNA libraries of each site were obtained. Of all phylotypes 93.6% were found in RNA libraries; in contrast, only 31 phylotypes (70.5%) were retrieved from total microbial community (DNA libraries). DNA and RNA sequences related to gamma-Proteobacteria and Bacteroidetes groups, typical for Antarctic sea-ice bacterial communities, were detected in analysed sites. 16S rDNA and rRNA libraries derived from the two different areas were enriched by picophytoplanktonic 16S sequences of plastid and mitochondrion origins, reflecting that the algal blooms occurred during sampling (Antarctic summer 2003). The finding in Rod Bay libraries of high percentage of DNA clones apparently affiliated with beta-Proteobacteria typical for activated sludges and well water could be explained by the presence of a sewage depuration system at this site. Obtained results clearly demonstrate that combination of 16S rDNA and 16S rRNA gene sequencing is preferred approach to have a more reliable vision on the composition of microbial communities.     

Tracing the Sources of Exported Nitrate in the Turkey Lakes Watershed Using 15N/14N and 18O/16O isotopic ratios

Nitrate produced by bacterially mediated nitrification in soils is isotopically distinct from atmospheric nitrate in precipitation. ¹⁵N/¹⁴N and ¹⁸O/¹⁶O isotopic ratios of nitrate can therefore be used to distinguish between these two sources of nitrate in surface waters and groundwaters. Two forested catchments in the Turkey Lakes Watershed (TLW) near Sault Ste. Marie, Ontario, Canada were studied to determine the relative contributions of atmospheric and microbial nitrate to nitrate export. The TLW is reasonably undisturbed and receives a moderate amount of inorganic nitrogen bulk deposition (8.7 kg N · ha⁻¹· yr⁻¹) yet it exhibits unusually low inorganic nitrogen retention (average = 65% of deposition). The measured isotopic ratios for nitrate in precipitation ranged from +35 to +59‰ (VSMOW) for δ¹⁸O and −4 to +0.8‰ (AIR) for δ¹⁵N. Nitrate produced from nitrification at the TLW is expected to have an average isotope value of approximately −1.0‰ for δ¹⁸O and a value of about 0 to +6‰ for δ¹⁵N, thus, the isotopic separation between atmospheric and soil sources of nitrate is substantial. Nitrate produced by nitrification of ammonium appears to be the dominant source of the nitrate exported in both catchments, even during the snowmelt period. These whole catchment results are consistent with the results of small but intensive plot scale studies that have shown that the majority of the nitrate leached from these catchments is microbial in origin. The isotopic composition of stream nitrate provides information about N-cycling in the forested upland and riparian zones on a whole catchment basis. Received 5 October 1999; accepted 18 August 2000     

Incidence of Vibrio parahaemolyticus in U.S. coastal waters and oysters.

Oyster and seawater samples were collected seasonally from May 1984 through April 1985 from shellfish-growing areas in Washington, California, Texas, Louisiana, Alabama, Florida, South Carolina, Virginia, and Rhode Island which had been designated as approved or prohibited by the National Shellfish Sanitation Program. Fecal coliforms counts, aerobic plate counts, and Vibrio parahaemolyticus densities were determined for the samples. Mean V. parahaemolyticus density was more than 100 times greater in oysters than in water, whereas density of fecal coliforms was approximately 10 times higher in oysters. Seasonal and geographical distributions of V. parahaemolyticus were related to water temperature, with highest densities in samples collected in the spring and the summer along the Gulf coast. The synthetic DNA probe for thermostable direct hemolysin hybridized with 2 of 50 isolates, 1 of which was positive by the Kanagawa test.     

Incidence of Vibrio parahaemolyticus in U.S. coastal waters and oysters

Oyster and seawater samples were collected seasonally from May 1984 through April 1985 from shellfish-growing areas in Washington, California, Texas, Louisiana, Alabama, Florida, South Carolina, Virginia, and Rhode Island which had been designated as approved or prohibited by the National Shellfish Sanitation Program. Fecal coliforms counts, aerobic plate counts, and Vibrio parahaemolyticus densities were determined for the samples. Mean V. parahaemolyticus density was more than 100 times greater in oysters than in water, whereas density of fecal coliforms was approximately 10 times higher in oysters. Seasonal and geographical distributions of V. parahaemolyticus were related to water temperature, with highest densities in samples collected in the spring and the summer along the Gulf coast. The synthetic DNA probe for thermostable direct hemolysin hybridized with 2 of 50 isolates, 1 of which was positive by the Kanagawa test.     

16O2/18O2 analysis of oxygen exchange in Dunaliella tertiolecta. Evidence for the inhibition of mitochondrial respiration in the light

A mass spectrometric ¹⁶O₂/¹⁸O₂-isotope technique was used to analyse the rates of gross O₂ evolution, net O₂ evolution and gross O₂ uptake in relation to photon fluence rate by Dunaliella tertiolecta adapted to 0.5, 1.0, 1.5, 2.0 and 2.5 M NaCl at 25°C and pH 7.0.At concentrations of dissolved inorganic carbon saturating for photosynthesis (200 M) gross O₂ evolution and net O₂ evolution increased with increasing salinity as well as with photon fluence rate. Light compensation was also enhanced with increased salinities. Light saturation of net O₂ evolution was reached at about 1000 mol m^-2s^-1 for all salt concentrations tested. Gross O₂ uptake in the light was increased in relation to the NaCl concentration but it was decreased with increasing photon fluence rate for almost all salinities, although an enhanced flow of light generated electrons was simultaneously observed. In addition, a comparison between gross O₂ uptake at 1000 mol photons m^-2s^-1, dark respiration before illumination and immediately after darkening of each experiment showed that gross O₂ uptake in the light paralleled but was lower than mitochondrial O₂ consumption in the dark.From these results it is suggested that O₂ uptake by Dunaliella tertiolecta in the light is mainly influenced by mitochondrial O₂ uptake. Therefore, it appears that the light dependent inhibition of gross O₂ uptake is caused by a reduction in mitochondrial O₂ consumption by light.Abbreviations DCMU 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea - DHAP dihydroxy-acetonephosphate - DIC dissolved inorganic carbon - DR_a rate of dark respiration immediately after illumination - DR_b rate of dark respiration before illumination - E₀ rate of gross oxygen evolution in the light - NET rate of net oxygen evolution in the light - PFR photon fluence rate - RubP rubulose-1,5-bisphosphate - SHAM salicyl hydroxamic acid - U₀ rate of gross oxygen uptake in the light     

Community analysis of high- and low-nucleic acid-containing bacteria in NW Mediterranean coastal waters using 16S rDNA pyrosequencing

The ecological significance of the marine bacterial populations distinguishable by flow cytometry on the basis of the fluorescence (FL) of their nucleic acid (NA) content and proxies of cell size (such as side scatter, SSC) remains largely unknown. Some studies have suggested that cells with high NA (HNA) content and high SSC (HS) represent the active members of the community, while the low NA (LNA) cells are inactive members of the same phylogenetic groups. But group-specific activity measurements and phylogenetic assignment after cell sorting have suggested this is not be the case, particularly in open-ocean communities. To test the extent to which the different NA subgroups are similar, and consequently the extent to which they likely have similar ecological and biogeochemical roles in the environment, we analysed the phylogenetic composition of three populations after cell sorting [high NA-high SC (HNA-HS), high NA-low SC (HNA-LS), low NA (LNA)] by 454 pyrosequencing in two contrasting periods of the year in NW Mediterranean coastal waters (BBMO, Blanes Bay Microbial Observatory) where these three populations have recurrent seasonal patterns. Statistical analyses showed that summer and winter samples were significantly different and, importantly, the sorted populations within a sample were composed of different taxa. The majority of taxa were associated with one NA fraction only, and the degree of overlap (i.e. OTUs present simultaneously in 2 fractions) between HNA and LNA and between summer and winter communities was very small. Rhodobacterales, SAR116 and Bacteroidetes contributed primarily to the HNA fraction, whereas other groups such as SAR11 and SAR86 contributed largely to the LNA fractions. Gammaproteobacteria other than SAR86 showed less preference for one particular NA fraction. An increase in diversity was observed from the LNA to the HNA-HS fraction for both sample dates. Our results suggest that, in Blanes Bay, flow cytometric signatures of natural communities track their phylogenetic composition.     

Inaccuracies in selected ion monitoring determination of isotope ratios obviated by profile acquisition: nucleotide 18O/16O measurements

Precise and accurate measurements of isotopologue distributions (IDs) in biological molecules are needed for determination of isotope effects, quantitation by isotope dilution, and quantification of isotope tracers employed in both metabolic and biophysical studies.While single ion monitoring (SIM) yields significantly greater sensitivity and signal/noise than profile-mode acquisitions, we show that small changes in the SIM window width and/or center can alter experimentally determined isotope ratios by up to 5%, resulting in significant inaccuracies. This inaccuracy is attributed to mass granularity, the differential distribution of digital data points across the m/z ranges sampled by SIM. Acquiring data in the profile mode and fitting the data to an equation describing a series of equally spaced and identically shaped peaks eliminates the inaccuracies associated with mass granularity with minimal loss of precision. Additionally a method of using the complete ID profile data that inherently corrects for “spillover” and for the natural-abundance ID has been used to determine ¹⁸O/¹⁶O ratios for 5′,3′-guanosine bis-[¹⁸O₁]phosphate and TM[¹⁸O₁]P with precisions of ∼0.005. The analysis protocol is also applied to quadrupole time-of-flight tandem mass spectrometry using [2-¹⁸O] arabinouridine and 3′-UM[¹⁸O₁]P which enhances signal/noise and minimizes concerns for background contamination.     

Proteomic analysis of ductal carcinoma of the breast using laser capture microdissection, LC-MS, and 16O/18O isotopic labeling

The goal of this study was the development of a method for quantitative expression proteomics on the limited sample amounts obtained through laser capture microdissection (LCM) of tissues, e.g., approximately 10 000 cells, which typically contain roughly 1-4 microg protein. The 16O/18O labeling method was selected as an approach to measure differential expression. A sample preparation protocol including lysis, digestion and 16O/18O labeling was first developed for LCM cell samples. The selected protocol was examined using two LCM caps of 10 000 cells from invasive ductal carcinoma of the breast and shown to be repeatable. A further test of LC-IT-MS/MS in combination with the 16O/18O post-digestion labeling method for studying low level samples was conducted first on a single protein (BSA) and then on a 5-standard protein mixture digest of different protein amounts, each with a total content approximately 1 microg. Next, protein expression was compared between 10 000 cells, each of microdissected normal ductal epithelium and metastatic ductal carcinoma, using the developed method. The proteins from the microdissected cells were extracted, precipitated, digested with trypsin and then 16O/18O labeled. The normal and metastatic cell samples were analyzed using reversed phase LC-ESI-MS/MS on the ion trap mass spectrometer. A total of 76 proteins were identified. Some, such as mitochondrial isocitrate dehydrogenase, actin and 14-3-3 protein xi/delta were found to be significantly up-regulated in the breast tumor cells.     

Accurate LC Peak Boundary Detection for 16 O/ 18 O Labeled LC-MS Data

In liquid chromatography-mass spectrometry (LC-MS), parts of LC peaks are often corrupted by their co-eluting peptides, which results in increased quantification variance. In this paper, we propose to apply accurate LC peak boundary detection to remove the corrupted part of LC peaks. Accurate LC peak boundary detection is achieved by checking the consistency of intensity patterns within peptide elution time ranges. In addition, we remove peptides with erroneous mass assignment through model fitness check, which compares observed intensity patterns to theoretically constructed ones. The proposed algorithm can significantly improve the accuracy and precision of peptide ratio measurements.     

Quantitative proteome analysis of human plasma following in vivo lipopolysaccharide administration using 16O/18O labeling and the accurate mass and time tag approach

Identification of novel diagnostic or therapeutic biomarkers from human blood plasma would benefit significantly from quantitative measurements of the proteome constituents over a range of physiological conditions. Herein we describe an initial demonstration of proteome-wide quantitative analysis of human plasma. The approach utilizes postdigestion trypsin-catalyzed 16O/18O peptide labeling, two-dimensional LC-FTICR mass spectrometry, and the accurate mass and time (AMT) tag strategy to identify and quantify peptides/proteins from complex samples. A peptide accurate mass and LC elution time AMT tag data base was initially generated using MS/MS following extensive multidimensional LC separations to provide the basis for subsequent peptide identifications. The AMT tag data base contains >8,000 putative identified peptides, providing 938 confident plasma protein identifications. The quantitative approach was applied without depletion of high abundance proteins for comparative analyses of plasma samples from an individual prior to and 9 h after lipopolysaccharide (LPS) administration. Accurate quantification of changes in protein abundance was demonstrated by both 1:1 labeling of control plasma and the comparison between the plasma samples following LPS administration. A total of 429 distinct plasma proteins were quantified from the comparative analyses, and the protein abundances for 25 proteins, including several known inflammatory response mediators, were observed to change significantly following LPS administration.     

Seasonality in the excystment of Alexandrium minutum and Alexandrium tamarense in Irish coastal waters

Excystment experiments were carried out on cysts of Alexandrium minutum and A. tamarense (Group III) taken from Cork Harbour, Ireland. Freshly sampled cysts were isolated into well plates and their excystment was monitored over a 30 day period. A pronounced seasonality was observed in the excystment behaviour in both species when measurements were carried out seasonally. Between 80 and 100% of the isolated cysts excysted within 30 days when samples were taken between March and June. However, between only 0 and 15% excysted in samples taken between August and early February. This seasonal characteristic was observed repeatedly over a 3 year period (2004–2007). The effect was observed in cysts which had been sampled from both inter-tidal and sub-tidal locations. No endogenous clock was evident in the germination of A. tamarense and A. minutum cysts taken from Cork Harbour which had been stored cool and in the dark under anoxic conditions for up to 18 months. The seasonal effect observed was independent of water temperature, although temperature did affect the rate of excystment. Temperature also affected the maturation of cysts which had been freshly formed in the laboratory. The significance of incorporating seasonality in excystment of Alexandrium into models describing its growth is also discussed.     

Occurrence and distribution of halophilic vibrios in subtropical coastal waters of Hong Kong.

The summer occurrence and distribution of halophilic vibrios in the subtropical coastal waters of Hong Kong were investigated. The density of vibrios in six sample sites ranged from 90 to 6,700 per ml, which made up 0.41 to 40% of the total bacterial populations of these sample sites. The sucrose-positive vibrios were found to be much more common (88% of total vibrios) than the sucrose-negative ones. A total of 48 strains belonging to six Vibrio species were fully characterized. Among these, Vibrio alginolyticus was the most frequently isolated, followed by V. parahaemolyticus, V. harveyi, V. vulnificus, V. campbellii, and V. fluvialis. The finding that eight of the nine strains of V. harveyi showed a positive Kanagawa reaction warrants further study.