Decomposition processes of eelgrass,Zostera marina L.

Summary In Lake Grevelingen decomposition of eelgrass was studied in the field with the litter bag method from July 1977 till February 1978. After 6 months only 6% refractory matter remained in the bags. Under aerobic conditions the decomposition of eelgrass is completed within one year. The organic fraction decreased from 80 to 55%. Chlorophyll a was always present in the detritus, but with this parameter no decomposition stages could be distinguished. Fragmentation was mainly physical, and a particle size spectrum showed a maximum towards the small pieces.The POC content was fairly constant, and the N and P content changed during the decomposition. The C/N and C/P ratios increased the first 10 weeks (leaching) and decreased after 10 weeks (microbial colonization). This did not correspond with the three decomposition stages, based on the dry weight loss per day. The C/N ratio does not seem to be a simple index for the decomposition stage in eelgrass.Communication no. 235 of the Delta Institute for Hydrobiological Research, Yerseke, The Netherlands.     

Sulfur accumulation in eelgrass (Zostera marina) and effect of sulfur on eelgrass growth

Eelgrass (Zostera marina) was grown under exposure to high levels of sediment sulfides to examine their ability to reoxidize sulfides intruding into the plants. The plants were kept under full light (control and high sulfide level) and at 10% of light saturation (high sulfide level) for 3 weeks and growth and accumulation of elemental sulfur (S⁰) in the plants were examined. The growth rate was reduced with ∼75% in the low light treatment, whereas there was no significant difference between the rates at full light saturation. S⁰ was accumulating in the below-ground structures of the plants exposed to high sulfide concentrations with highest concentration in the youngest roots and oldest internodes. There was no accumulation of S⁰ in the leaves, suggesting that the intruding sulfides were reoxidized in the below-ground structures before reaching the leaves. The accumulation of S⁰ was higher in the roots of the low light treatment (up to two times) suggesting a larger intrusion of sulfides. These plants also appeared highly affected by the treatment with rotting meristems and increased mortality after the 3-week growth period. These results are the first to show an accumulation of sulfur compounds internally in seagrasses as a result of reoxidation of sulfides. The reoxidation is facilitated by the internal transport of oxygen and is an example of the advantage of the internal lacunae system in seagrasses.     

Pyrolysis mass spectrometry analysis of free-living and symbiotic cyanobacteria

The potential of pyrolysis mass spectrometry to distinguish closely related cyanobacterial strains was assessed by using the technique to compare symbiotic cyanobacteria isolated from the hornwort Phaeoceros laevis and free-living cyanobacterial strains at the same field site. The same strains had previously been compared using polymerase chain reaction-based DNA fingerprinting techniques (West & Adams 1997, Appl. Environ. Microbiol. 63: 4479–4484). Many of the strains were grouped identically by the two techniques, although there were some differences, possibly resulting from the ability of these cyanobacteria to develop a range of specialised cell types having different chemical compositions to the vegetative cells. Although growth conditions were chosen to suppress cellular differentiation, this may not always have been completely successful. With careful control of growth conditions pyrolysis mass spectrometry has considerable potential as an additional tool for the phenetic comparison of cyanobacterial strains. It has the advantage that analysis is directly derived from whole cells, and hence is simpler and cheaper than DNA-based methods, although it does require the growth of axenic strains. The technique may be particularly useful in the study of some of the more cryptic unicellular and non-heterocystous filamentous cyanobacterial groups, in which the lack of cellular differentiation should minimise any variability in the chemical composition of cells.     

An attempt at taxonomical characterization of some Rhizobial species by intact cell MALDI mass spectrometry

In the present study an attempt was made to exploit the benefit of intact cell MALDI mass spectrometry (ICM-MS) in bringing out similarities and differences among some Rhizobium species and a species of Agrobacterium based on specific mass:charge (m/z) values. Rhizobium species isolated from the root nodules of selected leguminous plants were analysed by ICM-MS. The spectra were acquired in the range of 500–10,000 Da yielding several peaks specific to each species. The peaks obtained corresponded to the respective bacterial cell surface molecules which were desorbed during matrix-assisted laser desorption ionization. The number of peaks were more in the range of 500–1200 Da. Dice similarity coefficient analysis of m/z values indicated that Rhizobium species isolated from Trigonella foenum-graecum and Pisum arvense showed more similarity than any other species. Agrobacterium species did show a few common m/z values in comparison with other Rhizobium species. This clearly shows that Agrobacterium is closely related to Rhizobium. Eventually, ICM-MS technique offers clear, distinct, and consistent results for replicates, in less than an hour’s time, therefore this technique has high potential in molecular taxonomy.     

Analysis of intact protein isoforms by mass spectrometry

The diverse proteome of an organism arises from such events as single nucleotide substitutions at the DNA level, different RNA processing, and dynamic enzymatic post-translational modifications. This minireview focuses on the measurement of intact proteins to describe the diversity found in proteomes. The field of biological mass spectrometry has steadily advanced, enabling improvements in the characterization of single proteins to proteins derived from cells or tissues. In this minireview, we discuss the basic technology for "top-down" intact protein analysis. Furthermore, examples of studies involved with the qualitative and quantitative analysis of full-length polypeptides are provided.     

Reproduction of annual eelgrass: Variation among habitats and comparison with perennial eelgrass (Zostera marina L.)

The variation in reproductive potential of annual eelgrass was examined along a continuous gradient on an extensive mudflat bordering on and sloping down from the shore (Eastern River) and in a habitat mosaic in a salt marsh (Petpeswick Inlet) in Nova Scotia. Spathe and flower production as well as plant density were compared among habitats. Of the four habitats investigated in Petpeswick Inlet, the largest numbers of spathes and flowers per spathe were produced by plants in ponds on raised flats of Spartina alterniflora Loisel. The highest number of seeds per unit area was produced by plants in depressions on these flats which drained with each low tide. Seed production of annual eelgrass in drained depressions (4 889 seeds per 625 cm²) was seven times that of perennial shoots in creeks and higher than any records in the literature for perennial eelgrass. On average, seed production of annual eelgrass in this study was higher than values reported for other locations. Along the gradient, both annual and perennial eelgrass showed peaks in reproductive potential, but the annual peaked further up the gradient where there was greater exposure to air at low tide. Transplanting studies indicated that the among-habitat differences in reproductive potential were largely controlled by environmental as opposed to genetic factors. The possible effect of inter- and intraspecific competition on the reproductive potential of annual eelgrass was investigated experimentally in two habitats where co-existing species were abundant. In creeks the presence of perennial eelgrass significantly reduced the reproductive potential of annual eelgrass, but in a drained depression, the removal of Ruppia maritima L. s.l. had no effect. The upper distribution limit of annual eelgrass is likely determined by desiccation while the lower limit is probably determined by a combination of light availability (to some extent affected by perennial eelgrass) and exposure to spring rains which would significantly enhance seed germination.     

Pyrolysis mass spectrometry characterisation and numerical taxonomy ofAeromonas spp.

Reference strains (2) and 29 isolates ofAeromonas spp. from clinical material and environmental specimens were characterised in traditional biochemical tests, and in pyrolysis mass spectrometry, which gives data reflecting whole-cell composition. Numerical taxonomic analyses of the data sets were compared with conventional identification at species level, and pathogenic potential, as inferred from the origin of the isolates. Clustering with conventional test reaction patterns showed, for each of the species represented, a clearly defined core group of typical isolates, surrounded by a halo of aberrant strains. One further cluster comprised strains intermediate betweenA. caviae andA. hydrophila, and one strain was grossly atypical in both analyses. Clustering from pyrolysis data corresponded less well with species identification. Broadly, the biochemical division between core and halo strains was supported in pyrolysis forA. caviae andA. sobria, but the main group ofA. hydrophila in pyrolysis comprised strains clustering in the core and halo groups of this species, and three strains intermediate betweenA. hydrophila andA. caviae in biochemical tests. Two further pyrolysis clusters comprised core and halo strains ofA. hydrophila. However, pyrolysis clustering correlated well with inferred pathogenicity, showing four clusters of probable pathogens, six clusters of probable nonpathogens, and one two member cluster of doubtful status. Most strains that clustered in the species haloes, or in species-intermediate groups in biochemical tests, were non-human isolates, or were isolated in the absence of symptomatic infection. The correlation of inferred pathogenicity with biochemical clustering was poorer than that with pyrolysis clustering.Abbreviations CTRP conventional test reaction pattern - PyMS pyrolysis mass spectrometry     

Characterisation of intact microorganisms using electrospray ionisation mass spectrometry

We report the first application of electrospray ionisation mass spectrometry (ESI-MS) for the reproducible characterisation of strains of intact Gram-negative and Gram-positive bacteria. Electrospray ionisation was performed in both the positive and negative ion modes and the spectra obtained from Escherichia coli and Bacillus cereus were very information rich. Several of the observed negative mass ion fragments from E. coli could be assigned to specific fragmentation from bacterial phospholipids.Cluster analyses of these spectra showed that ESI-MS could be used to discriminate between these microorganisms to below species level. Therefore we conclude that ESI-MS constitutes a powerful approach to the characterisation and speciation of intact microorganisms.     

Electrospray-ionization mass spectrometry of intact intrinsic membrane proteins.

Membrane proteins drive and mediate many essential cellular processes making them a vital section of the proteome. However, the amphipathic nature of these molecules ensures their detailed structural analysis remains challenging. A versatile procedure for effective electrospray-ionization mass spectrometry (ESI-MS) of intact intrinsic membrane proteins purified using reverse-phase chromatography in aqueous formic acid/isopropanol is presented. The spectra of four examples, bacteriorhodopsin and its apoprotein from Halobacterium and the D1 and D2 reaction-center subunits from spinach thylakoids, achieve mass measurements that are within 0.01% of calculated theoretical values. All of the spectra reveal lesser quantities of other molecular species that can usually be equated with covalently modified subpopulations of these proteins. Our analysis of bovine rhodopsin, the first ESI-MS study of a G-protein coupled receptor, yielded a complex spectrum indicative of extensive molecular heterogeneity. The range of masses measured for the native molecule agrees well with the range calculated based upon variable glycosylation and reveals further heterogeneity arising from other covalent modifications. The technique described represents the most precise way to catalogue membrane proteins and their post-translational modifications. Resolution of the components of protein complexes provides insights into native protein/protein interactions. The apparent retention of structure by bacteriorhodopsin during the analysis raises the potential of obtaining tertiary structure information using more developed ESI-MS experiments.     

The application of hybrid mass spectrometry/mass spectrometry and high-resolution mass spectrometry to the analysis of fish samples for polychlorinated dibenzo-p-dioxins and dibenzofurans

A hybrid mass spectrometer operated in low-resolution selected decomposition monitoring (SDM) mode was used for the analysis of whole fish samples for the 2,3,7,8-substituted polychlorinated dibenzo-p-dioxins and poly-chlorinated dibenzofurans (PCDD/Fs). These fish samples were previously analyzed for PCDD/Fs using a high-resolution mass spectrometer following EPA protocols. The hybrid tandem mass spectrometric method using loss of COCl gave similar quantification results to those obtained by high-resolution mass spectrometry and eliminated the interferences attributed to polychlorinated biphenyls that were encountered in the high-resolution mass spectrometric analysis. Comparison of the two methods shows that the high-resolution mass spectrometric method surpasses the tandem mass spectrometric method in other analytical figures of merit, such as detection limit, linearity and reproducibility.     

Lipid fingerprints of intact viruses by MALDI-TOF/mass spectrometry

A number of viruses contain lipid membranes, which are in close contact with capsid proteins and/or nucleic acids and have an important role in the viral infection process. In this study membrane lipids of intact viruses have been analysed by MALDI-TOF/MS with a novel methodology avoiding lipid extraction and separation steps. To validate the novel method, a wide screening of viral lipids has been performed analysing highly purified intact bacterial and archaeal viruses displaying different virion architectures. Lipid profiles reported here contain all lipids previously detected by mass spectrometry analyses of virus lipid extracts. Novel details on the membrane lipid composition of selected viruses have also been obtained. In addition we show that this technique allows the study of lipid distribution easily in subviral particles during virus fractionation. The possibility to reliably analyse minute amounts of intact viruses by mass spectrometry opens new perspectives in analytical and functional lipid studies on a wider range of viruses including pathogenic human ones, which are difficult to purify in large amounts.     

Ultra-performance liquid chromatography-tandem mass spectrometry for the determination of atypical antipsychotics and some metabolites in in vitro samples

The ultra-performance liquid chromatography-electrospray tandem mass spectrometry (UPLC-ESI-MS/MS) method has been developed to perform the determination of quetiapine, perospirone, aripiprazole and quetiapine sulfoxide in in vitro samples in less than 3 min. The UPLC separation was carried out using an Acquity UPLC BEH C18 column (100 mm x 2.1mm i.d., 1.7 microm particle size) that provided high efficiency and resolution in combination with high linear velocities. The UPLC system was coupled to a Waters Micromass Quattro Premier XE tandem quadrupole mass spectrometer. This system permits high-speed data acquisition without peak intensity degradation, and produces sharp and narrow chromatographic peaks (w(h) about 2.5s) of compounds. The determination was performed in multiple reaction monitoring (MRM) mode. The quantification parameters of the developed method were established, obtaining instrumental LODs lower than 0.005 microg/l and a repeatability at a low concentration level lower than 10% CV (n=10). Finally, the method was successfully applied to the analysis of atypical antipsychotics and some metabolites in in vitro samples.     

Production and decomposition of eelgrass (Zostera marina L.) in saline Lake Grevelingen

Summary During five 28-hours measurements in 1981, the oxygen production and consumption in an eelgrass community in saline Lake Grevelingen were investigated using light plexiglass enclosures. Applying a conversion factor of 0.29 the amount of carbon fixed and the amount of organic carbon mineralized were estimated. Gross and net production were estimated over 24-hours periods.There appeared to be a good correlation between production and insolation on the water surface. For every measurement period the production as a function of light and aboveground eelgrass biomass in the enclosure were calculated. This showed a maximum of 5.10^–6 mg C.J.^–1 g dry weight^–1 in April and minimum of 1.4.10^–6 mg C.J.^–1 g^–1 in August.Using the calculated production coefficients, the insolation and the eelgrass biomass the gross production, net production and consumption during the growing season of 1976 were calculated. Gross production amounted to 340 gC.m^–2, and net production came to 130 g C.m^–2. Approximately 60 gC.m^–2 was respired by the eelgrass plants while the remaining 150 gC.m^–2 was consumed or mineralized by other organisms on the sampling spot. Approximately 120 g C.m^–2.yr^–1 was transported by wind and wave action towards the eastern part of the lake where it became anaerobically degraded. This resulted in the formation of sulfide and methane.Communication no. 236 of the Delta Institute for Hydrobiological Research, Yerseke, The Netherlands.     

Hamamelitol purification,identification by electrospray ionization mass spectrometry,and quantitation in plant leaves

Branched-chain sugars and sugar alcohols are unusual, but perhaps widespread, plant constituents whose associated biochemistry and function are poorly understood. Herein we show that one such sugar alcohol, hamamelitol (2-C-hydroxymethyl-D-ribitol), does occur in leaves of many different species often in very high amounts. Hamamelitol levels were quantitated by an isotope dilution assay we developed with a detection limit of about 15 nmol per g fresh weight, and its identity was verified using electrospray ionization mass spectrometry. The taxonomic distribution of hamamelitol was disjunct: hamamelitol was present in species of distantly related orders such as Laurales, Fabales, and Primulales, but was not necessarily present in different genera of the same family. Species with high leaf levels of carboxyarabinitol (2-C-hydroxymethyl-D-ribonic acid) generally have low hamamelitol levels. Leaves of Hedera helix L. contain the most hamamelitol of any species examined, with levels comparable to those of sucrose. The youngest leaves of H. helix accumulated the most hamamelitol, about 11 mol per g fresh weight. Growth of H. helix with periodic sub-freezing temperatures did not induce further accumulation of leaf hamamelitol. Hamamelitol levels were also high in leaf petioles of H. helix, which indicates that this sugar alcohol may be translocatable. Further, the mass spectrometry analysis indicates that a non-covalent dimer of hamamelitol may be more prevalent in vivo than is the monomeric form.Abbreviations CA1P 2-carboxyarabinitol 1-phosphate - CAD collisionally activated dissociation - ESI electrospray ionization - FW fresh weight This work was supported by US Department of Agriculture-National Research Institute grant 93-37306-9240 to J.R.S. and B.d.M. Mass spectra were obtained with an instrument purchased under NSF grant DIR-9102839. We appreciate general technical support from Kitty Spreeman, and useful discussions with Professor David Schooley. We thank Dr. Lewis Carey for carrying out the NMR analysis, Dr. Houle Wang for general help with the mass spectrome try, and Dr. Guo Lin for doing the chemical structure drawing.     

Enumeration and characterization of nitrogen-fixing bacteria in an eelgrass (Zostera marina) bed

Marine nitrogen-fixing bacteria distributed in the eelgrass bed and seawater of Aburatsubo Inlet, Kanagawa, Japan were investigated using anaerobic and microaerobic enrichment culture methods. The present enrichment culture methods are simple and efficient for enumeration and isolation of nitrogen-fixing bacteria from marine environments. Mostprobable-number (MPN) values obtained for nitrogen-fixing bacteria ranged from 1.1×10² to 4.6×10²/ml for seawater, 4.0×10⁴ to 4.3×10⁵/g wet wt for eelgrass-bed sediment, and 2.1 × 10⁵ to 1.2 × 10⁷/g wet wt for eelgrass-root samples. More than 100 strains of halophilic, nitrogen-fixing bacteria belonging to the family Vibrionaceae were isolated from the MPN tubes. These isolates were roughly classified into seven groups on the basis of their physiological and biochemical characteristics. The majority of the isolates were assigned to the genusVibrio and one group to the genusPhotobacterium. However, there was also a group that could not be identified to the generic level. All isolates expressed nitrogen fixation activities under anaerobic conditions, and no organic growth factors were required for their activities.     

Charge variant native mass spectrometry benefits mass precision and dynamic range of monoclonal antibody intact mass analysis

ABSTRACT

The preponderance and diversity of charge variants in therapeutic monoclonal antibodies has implications for antibody efficacy and degradation. Understanding the extent and impact of minor antibody variants is of great interest, and it is also a critical regulatory requirement. Traditionally, a combination of approaches is used to characterize antibody charge heterogeneity, including ion exchange chromatography and independent mass spectrometric variant site mapping after proteolytic digestion. Here, we describe charge variant native mass spectrometry (CVMS), an integrated native ion exchange mass spectrometry-based charge variant analytical approach that delivers detailed molecular information in a single, semi-automated analysis. We utilized pure volatile salt mobile phases over a pH gradient that effectively separated variants based on minimal differences in isoelectric point. Characterization of variants such as deamidation, which are traditionally unattainable by intact mass due to their minimal molecular weight differences, were measured unambiguously by mass and retention time to allow confident MS1 identification. We demonstrate that efficient chromatographic separation allows introduction of the purified forms of the charge variant isoforms into the Orbitrap mass spectrometer. Our CVMS method allows confident assignment of intact monoclonal antibody isoforms of similar mass and relative abundance measurements across three orders of magnitude dynamic range.     

Twentyfive years of changes in the distribution and biomass of eelgrass,Zostera marina,in Grevelingen lagoon,The Netherlands

The wax and wane of the eelgrass (Zostera marina L.) population in Grevelingen lagoon (East Atlantic; The Netherlands) has been documented for over 25 years, together with quantitative and semi-quantitative data on environmental variables. The population expanded after the closure of the Grevelingen estuary in 1971, but declined from 4600 ha surface area in 1978 to less than 100 ha in 1993. There is little causal evidence which factors are responsible for the observed dynamics of the population. The incomplete picture emerging from the data is that of an extremely impoverished eelgrass population, living under constant oligo-mesotrophic marine conditions. Both the sexual and the vegetative modes of reproduction are severely stressed by environmental variables, most likely a combination of low temperatures, high salinity, low dissolved silicate and low ammonium concentrations. Survival of the population asks for the restoration of moderate estuarine conditions.Contribution No. 2180 of the Netherlands Institute of Ecology, Nieuwersluis, The Netherlands.     

Pyrolysis mass spectrometry as a method for inter-strain discrimination of Candida albicans

A claim that Candida albicans strains NCPF 3153 and B311 were identical was investigated. Authentic strains were shown to be distinct (P less than 0.1%) by pyrolysis mass spectrometry (PyMS). Of twelve strains, provided as clones of NCPF 3153, seven were authenticated, one yielded an equivocal result and four were distinct from both NCPF 3153 and B311. Of eight B311 clones, six were authenticated and two yielded equivocal results. Although five non-C. albicans yeast strains were identified as distinct from B311 and NCPF 3153, Torulopsis glabrata NCPF 3240 was identified as B311, and one clinical isolate of C. albicans as NCPF 3153. This could be explained by the specificity of the mathematical analysis for discrimination between the authentic strains.