Development of combined micro -preparation, separation, and mass spectrometry methods and application in the microdialysis study of neuropeptide metabolism

Two sets of mass spectrometry-based methods were developed specifically for the in vivo study of extracellular neuropeptide biochemistry. First, an integrated micro-concentration/desalting/matrix-addition device was constructed for matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) to achieve attomole sensitivity for microdialysis samples. Second, capillary electrophoresis (CE) was incorporated into the above micro-liquid chromatography (LC) and MALDI MS system to provide two-dimensional separation and identification (i.e. electrophoretic mobility and molecular mass) for the analysis of complex mixtures. The latter technique includes two parts of instrumentation: (1) the coupling of a preconcentration LC column to the inlet of a CE capillary, and (2) the utilization of a matrix-precoated membrane target for continuous CE effluent deposition and for automatic MALDI MS analysis (imaging) of the CE track.^ Initial in vivo data reveals a carboxypeptidase A (CPA) activity in rat brain involved in extracellular neurotensin metabolism. Benzylsuccinic acid, a CPA inhibitor, inhibited neurotensin metabolite NT1-12 formation by 70%, while inhibitors of other major extracellular peptide metabolizing enzymes increased NT1-12 formation. CPA activity has not been observed in previous in vitro experiments. Next, the validity of the methodology was demonstrated in the detection and structural elucidation of an endogenous neuropeptide, (L)VV-hemorphin-7, in rat brain upon ATP stimulation. Finally, the combined micro-LC/CE/MALDI MS was used in the in vivo metabolic study of peptide E, a mu-selective opioid peptide with 25 amino acid residues. Profiles of 88 metabolites were obtained, their identity being determined by their mass-to-charge ratio and electrophoretic mobility. The results indicate that there are several primary cleavage sites in vivo for peptide E in the release of its enkephalin-containing fragments. ^

Element concentration and GDGT fractional abundances of sediment core GeoB3910-2

In dieser Arbeit wurde das Zeitintervall zwischen 20 und 10 ka vor heute einschließlich des Heinrichevent 1 und der Younger Dryas am Kern GeoB 3910-2 neu untersucht. An organischen Parametern, basierend auf der Verteilung von bakteriellen GDGTs, und Elementkonzentrationen wurde eine Rekonstruktion der klimatischen Bedingungen und Veränderungen im Hinterland von NO Brasilien durchgeführt. Es zeigt sich, dass sich die durchschnittliche Landtemperatur gleich der Oberflächenwassertemperatur verhält und im Gegensatz zu den Phasen von H6 bis H2 dem antarktischen Erwärmungstrend ab 17 ka vor heute folgt. Weiterhin konnte gezeigt werden, dass durch die südwärts Verlagerung der ITCZ während H1 und der YD die Niederschläge in NO Brasilen intensiviert wurden, was eine Ausbreitung der Flüsse und Änderung der Erosionsgebiete zur Folge hatte.

Biphytane and stable carbon isotopic values of sugars and glycerol from ODP Hole 204-1245D and 201-1229A

The membrane lipids diglycosyl-glycerol dibiphytanyl glycerol tetraethers (2G-GDGTs) in marine subsurface sediments are believed to originate from uncultivated benthic archaea, yet the production of 2G-GDGTs from subseafloor samples has not been demonstrated in vitro. In order to validate sedimentary biosynthesis of 2G-GDGTs, we performed a stable carbon isotope probing experiment on a subseafloor sample with six different 13C-labelled substrates (bicarbonate, methane, acetate, leucine, glucose and Spirulina platensis biomass). After 468 days of anoxic incubation, only glucose and S. platensis resulted in label uptake in lipid moieties of 2G-GDGTs, indicating incorporation of carbon from these organic substrates. The hydrophobic moieties of 2G-GDGTs showed minimal label incorporation, with up to 4 per mil 13C enrichment detected in crenarchaeol-derived tricyclic biphytane from the S. platensis-supplemented slurries. The 2G-GDGT-derived glucose or glycerol moieties also showed 13C incorporation (Dd13C = 18 - 38 per mil) in the incubations with glucose or S. platensis, consistent with a lipid salvage mechanism utilized by marine benthic archaea to produce new 2G-GDGTs. The production rates were nevertheless rather slow, even when labile organic matter was supplied. The 2G-GDGT turnover times of 1700 - 20 500 years were much longer than those estimated for subseafloor microbial communities, implying that sedimentary 2G-GDGTs as biomarkers of benthic archaea are cumulative records of past and present generations.

Chemical characterization and quantification of the brown algal storage compound laminarin

In search of a meaningful stress indicator for Fucus vesiculosus we found that the often used quantitative determination procedures for the polysaccharide laminarin (beta-1,3-glucan) result in different kind of problems, uncertainties and limitations. This chemical long-term storage form of carbon enables perennial brown algae in seasonally fluctuating ecosystems to uncouple growth from photosynthesis. Because of this high ecological relevance a reliable and precise method for determination and quantification of laminarin is needed. Therefore, a simple, cold water extraction method coupled to a new quantitative liquid chromatography-mass spectrometrical method (LC-MS) was developed. Laminarin was determined in nine out of twelve brown algal species, and its expected typical molar mass distribution of 2000-7000 Da was confirmed. Furthermore, laminarin consisted of a complex mixture of different chemical forms, since fifteen chemical laminarin species with distinct molecular weights were measured in nine species of brown algae. Laminarin concentrations in the algal tissues ranged from 0.03 to 0.86% dry weight (DW). The direct chemical characterization and quantification of laminarin by LC-MS represents a powerful method to verify the biochemical and ecological importance of laminarin for brown algae. Single individuals of Laminaria hyperborea, L. digitata, Saccharina latissima, F. serratus, F. vesiculosus, F. spiralis, Himanthalia elongata, Cystoseira tamariscifolia, Pelvetia canaliculata, Ascophyllum nodosum, Halidrys siliquosa and Dictyota dichotoma were collected in fall (18.11.2013) during spring low tide from the shore of Finavarra, Co. Clare, west coast of Ireland (53° 09' 25'' N, 09° 06' 58'' W). After sampling, the different algae were immediately transported to the lab, lyophilized and sent to the University of Rostock. Laminarin was extracted with cold ultrapure water from the algal samples. Before extraction they were ground to < 1 mm grain size with an analytical mill (Ika MF 10 Basic). The algal material (approx. 1.5 g DW) was extracted in ultrapure water (8 mL) on a shaker (250 rpm) for 5 h. After the addition of surplus ultrapure water (4 mL) and shaking manually, 1 mL of the sample was filter centrifuged (45 µm) at 14,000 rpm (Hettich Mikro 22 R). The slightly viscous supernatant was free of suspended material and converted into a microvial (300 µL) for further analysis. The extracts were analyzed using liquid chromatography-mass spectrometry (LC-MS) analysis (LTQ Velos Pro ion trap spectrometer with Accela HPLC, Thermo Scientific). Laminarin species were separated on a KinetexTM column (2.6 µm C18, 150 x 3 mm). The mobile phase was 90 % ultrapure water and 10 % acetonitrile, run isocratically at a flow rate of 0.2 mL min-1. MS was working in ESI negative ion mode in a mass range of 100 - 4000 amu. Glucose contents were determined after extraction using high-performance liquid chromatography (HPLC). Extracted samples were analyzed in an HPLC (SmartLine, Knauer GmbH) equipped with a SUPELCOGELTM Ca column (30 x 7,8 mm without preColumn) and RI-detector (S2300 PDA S2800). Water was used as eluent at a flow rate of 0.8 mL min-1 at 75 °C. Glucose was quantified by comparison of the retention time and peak area with standard solutions using ChromGate software. Mannitol was extracted from three subsamples of 10-20 mg powdered alga material (L. hyperborea, L. digitata, S. latissima, F. serratus, F. vesiculosus, F. spiralis, H. elongata, P. canaliculata, A. nodosum, H. siliquosa) and quantified, following the HPLC method described by Karsten et al. (1991). For analyzing carbon and nitrogen contents, dried algal material was ground to powder and three subsamples of 2 mg from each alga thalli were loaded and packed into tin cartridges (6×6×12 mm). The packages were combusted at 950 °C and the absolute contents of C and N were automatically quantified in an elemental analyzer (Elementar Vario EL III, Germany) using acetanilide as standard according to Verardo et al. (1990).

Molecular dissolved organic matter composition and environmental parameters from a laboratory incubation study

Dissolved organic matter (DOM) in the oceans constitutes a major carbon pool involved in global biogeochemical cycles. More than 96% of the marine DOM resists microbial degradation for thousands of years. The composition of this refractory DOM (RDOM) exhibits a molecular signature which is ubiquitously detected in the deep oceans. Surprisingly efficient microbial transformation of labile into RDOM was shown experimentally, implying that microorganisms produce far more RDOM than needed to sustain the global pool. By assessing the microbial formation and transformation of DOM in unprecedented molecular detail for 3 years, we show that most of the newly formed RDOM is molecularly different from deep sea RDOM. Only <0.4% of the net community production was channeled into RDOM molecularly undistinguishable from deep sea DOM. Our study provides novel experimentally derived molecular evidence and data for global models on the production, turnover and accumulation of marine DOM.

Obtención de un estándar secundario de cuantificación para la síntesis y purificación de allicina.

Desde tiempos ancestrales, el ajo (Allium sativum L.) ha sido utilizado para la prevención y el tratamiento de dolencias. Sus efectos benéficos para la salud han sido atribuidos a los tiosulfinatos, siendo la allicina el más abundante. La determinación rutinaria de la allicina es dificultosa dada su inestabilidad y la naturaleza reactiva de la misma. El objetivo del trabajo fue obtener un estándar secundario para la cuantificación de allicina mediante Cromatografía Líquida de Alta Resolución (HPLC). Fueron ensayadas diferentes metodologías de síntesis y purificación de allicina. En el Laboratorio de Residuos Tóxicos de la Fac. Cs. Agrarias (UNCuyo), bajo condiciones controladas, se preparó polvo de ajo para ser utilizado como estándar secundario de cuantificación. La cuantificación de muestras problema contra ambos estándares (allicina pura y polvo de ajo estandarizado, respectivamente) no presentó diferencias significativas. El empleo de polvo de ajo estandarizado ofrece una alternativa viable simple para la cuantificación de allicina.

Efecto de la interacción genotipo-ambiente sobre la expresión del contenido de allicina y ácido pirúvico en ajo (Allium sativum L.)

La expresión de la intensidad del flavor en los bulbos de ajo (Allium sativum L.), depende tanto de factores genéticos como ambientales. Las características organolépticas se manifiestan por la presencia de compuestos organoazufrados, específicamente tiosulfinatos, siendo el representante mayoritario la allicina (diallil tiosulfinato). El ácido pirúvico constituye un producto secundario de la reacción enzimática generadora del flavor, por lo que su medición se asocia a la intensidad de pungencia en ajo. El objetivo del presente trabajo fue estudiar si la variabilidad en el contenido de allicina y pirúvico está más asociada a las características genéticas o a la influencia de las regiones de cultivo. Se seleccionaron cuatro cultivares (Castaño INTA, Sureño INTA, Lican INTA y Unión) pertenecientes al banco de germoplasma del Instituto Nacional de Tecnología Agropecuaria (INTA) La Consulta, Mendoza, Argentina, cultivados en diferentes zonas geográficas: La Consulta (Dpto. San Carlos, Mendoza), Esquel (Chubut) y Ushuaia (Tierra del Fuego). Se cuantificó la allicina mediante Cromatografía Líquida de Alta Resolución (HPLC) y pungencia mediante espectrofotometría. A partir del análisis estadístico de los valores obtenidos se puede inferir que existen diferencias significativas en el contenido de allicina y pirúvico entre distintas cultivares de una misma zona y en el contenido de allicina y pirúvico para la misma cultivar en distintas zonas.

Total organic carbon content and isorenieratene concentrations from three holes of ODP Leg 160

Carotenoids were analysed in ca. 1-cm thick subsamples of three laterally time-equivalent sapropels from a west-east transect of the eastern Mediterranean Basin to study euxinic periods during Pliocene sapropel formation. The amount of intact isorenieratene (summed all-trans and cis isomers), ranged from non-detectable at the base and top of a sapropel up to 140 µg/g sediment in the central parts. Isorenieratene accumulation rates at the central and western site are remarkably similar and increase sharply to levels of up to 3.0 mg/m**2/ yr in the central part of the sapropel and then drop to low levels. This pattern indicates an expansion of euxinic conditions reaching into the photic zone, followed by deepening of the chemocline during deposition of this Pliocene sapropel. The sapropel from the easternmost site of the basin, which contains less organic carbon, shows much lower isorenieratene accumulation rates and even absence of isorenieratene in the central part of the sapropel. Ba/Al ratios indicate enhanced palaeoproductivity during sapropel formation, supporting previously proposed models, according to which increased productivity is the driving force for the generation of euxinic conditions.

Geochemistry and petrography of organic matter at DSDP Hole 77-535

Organic geochemical and organic petrographic methods were used to study three Lower to middle Cretaceous sediment samples from Hole 535 in the southeastern Gulf of Mexico for organic matter contents and origin and level of maturation. All three samples contain mixed kerogen Type II/III organic matter with a maturity corresponding to about 0.4% vitrinite reflectance. The marine component increases with stratigraphic age, and microbial reworking of the organic matter is significant in each age. The lower two samples of Hauterivian to Valanginian age appear to be impregnated (or contaminated) with soluble polar organic compounds, but there is only a weak indication for the presence of more mature, nonindigenous hydrocarbons.

Temperature proxies of sediment cores from the Sea of Marmara

Reconstructing ocean temperature values is a major target in paleoceanography and climate research. However, most temperature proxies are organism-based and thus suffer from an "ecological bias". Multiproxy approaches can potentially overcome this bias but typically require more investment in time and resources, while being susceptible to errors induced by sample preparation steps necessary before analysis. Three lipid-based temperature proxies are widely used: UK'37 (based on long chain alkenones from phytoplanktonic haptophytes), TEX86 [based on glycerol dialkyl glycerol tetraethers (GDGTs) from pelagic archaea] and LDI (based on long chain diols from phytoplanktonic eustigmatophytes). So far, separate analytical methods, including gas chromatography (GC) and liquid chromatography (LC), have been used to determine these proxies. Here we present a sensitive method for determining all three in a single normal phase high performance LC-atmospheric pressure chemical ionization mass spectrometry (NP-HPLC-APCI-MS) analysis. Each of the long chain alkenones and long chain diols was separated and unambiguously identified from the accurate masses and characteristic fragmentation during multiple stage MS analysis (MS2). Comparison of conventional GC and HPLC-MS methods showed similar results for UK'37 and LDI, respectively, using diverse environmental samples and an Emiliania huxleyi culture. Including the three sea surface temperature (SST) proxies; the NP-HPLC-APCI-MS method in fact allows simultaneous determination of nine paleoenvironmental proxies. The extent to which the ecology of the source organisms (ecological bias) influences lipid composition and thereby the reconstructed temperature values was demonstrated by applying the new method to a sediment core from the Sea of Marmara, covering the last 21 kyr BP. Reconstructed SST values differed considerably between the proxies for the Last Glacial Maximum (LGM) and the period of Sapropel S1 formation at ca. 10 kyr BP, whereas the trends during the late Holocene were similar. Changes in the composition of alkenone-producing species at the transition from the LGM to the Bølling/Allerød (B/A) were inferred from unreasonably high UK'37-derived SST values (ca. 20 °C) during the LGM. We ascribe discrepancies between the reconstructed temperature records during S1 deposition to habitat change, e.g. a different depth due to changes in nutrient availability.