Literatura académica sobre el tema "Fast size exclusion liquid chromatography (FastSEC)"

Marine bioactive peptides, as a source of unique bioactive compounds, are the focus of current research. They exert various biological roles, some of the most crucial of which are antioxidant activity, antimicrobial activity, anticancer activity, antihypertensive activity, anti-inflammatory activity, and so forth, and specific characteristics of the bioactivities are described. This review also describes various manufacturing techniques for marine bioactive peptides using organic synthesis, microwave assisted extraction, chemical hydrolysis, and enzymes hydrolysis. Finally, purification of marine bioactive peptides is described, including gel or size exclusion chromatography, ion-exchange column chromatography, and reversed-phase high-performance liquid chromatography, which are aimed at finding a fast, simple, and effective method to obtain the target peptides.

Abstract We determined the zinc binding stoichiometry of peroxisomal RING finger proteins by measuring sulfur/metal ratios using inductively coupled plasma-mass spectrometry coupled to size exclusion chromatography, a strategy that provides a fast and quantitative overview on the binding of metals in proteins. As a quality control, liquid chromatography-electrospray ionisation-time of flight-mass spectrometry was used to measure the molar masses of the intact proteins. The RING fingers of Pex2p, Pex10p, and Pex12p showed a stoichiometry of 2.0, 2.1, and 1.2 mol zinc/mol protein, respectively. Thus, Pex2p and Pex10p possess a typical RING domain with two coordinated zinc atoms, whereas that of Pex12p coordinates only a single zinc atom.

The camel (camelus dromedarius) milk proteose peptone 3 (PP3) was purified successively by size exclusion fast protein liquid chromatography and reversed phase high performance liquid chromatography and then characterized by amino acid residue composition determination and chemical microsequencing after CNBr or trypsin cleavages. In comparison with the previously reported structure of camel milk whey protein, the camel PP3 contains an insertion in the N-terminal region which has approximately 24 residues, whereas the remaining C-terminal regions of these two homologous proteins are essentially identical. The camel PP3 seems to contain a potential O-glycosylation site localized in this insertion and 2 or 3 phosphorylated serine residues. PP3 belongs to the glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1) family and could therefore play an immunological role in the camel or its suckling young.

A NAD-dependent aldehyde dehydrogenase (EC 1.2.1.3) which catalyzes the oxidation of retinal to retinoic acid was purified to homogeneity from rat kidney by using Affi-Gel blue affinity chromatography and chromatofocusing, followed by Mono-Q anion-exchange chromatography. The apparent molecular weight of the native enzyme determined by size-exclusion fast protein liquid chromatography was 140 000. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis gave a subunit molecular weight of 53 000. The isoelectric point as measured by chromatofocusing was 8.5. The enzyme also catalyzed the oxidation of acetaldehyde, but showed much lower Km value for the retinal substrate. We suggest that aldehyde dehydrogenase found in the kidney may be a specific retinal dehydrogenase, involved in vitamin A metabolism.Key words: aldehyde dehydrogenase, vitamin A, retinal, retinoic acid, kidney.

Scorpion venoms are a rich source of bioactive molecules, but characterisation of toxin peptides affecting cytosolic Ca2+, central to cell signalling and cell death, is limited. We undertook a functional screening of the venom of the Australian scorpion Hormurus waigiensis to determine the breadth of Ca2+ mobilisation. A human embryonic kidney (HEK293) cell line stably expressing the genetically encoded Ca2+ reporter GCaMP5G and the rabbit type 1 ryanodine receptor (RyR1) was developed as a biosensor. Size-exclusion Fast Protein Liquid Chromatography separated the venom into 53 fractions, constituting 12 chromatographic peaks. Liquid chromatography mass spectroscopy identified 182 distinct molecules with 3 to 63 components per peak. The molecular weights varied from 258 Da—13.6 kDa, with 53% under 1 kDa. The majority of the venom chromatographic peaks (tested as six venom pools) were found to reversibly modulate cell monolayer bioimpedance, detected using the xCELLigence platform (ACEA Biosciences). Confocal Ca2+ imaging showed 9/14 peak samples, with molecules spanning the molecular size range, increased cytosolic Ca2+ mobilization. H. waigiensis venom Ca2+ activity was correlated with changes in bio-impedance, reflecting multi-modal toxin actions on cell physiology across the venom proteome.

The γ-aminobutyric acid (GABA) transporter 1 (GAT1) belongs to a family of Na+ and Cl−-coupled transport proteins and possesses 12 putative transmembrane domains. To perform structural analyses of the GAT1 protein, the GAT1/green fluorescent protein (GFP) fusion protein was functionally expressed in insect Sf9 cells by the BAC-TO-BAC® baculovirus expression system. A two-step procedure to purify the GAT1/GFP fusion protein from insect Sf9 cells has been established and involves immunoaffinity chromatography using self-prepared anti-GFP antibodies and size-exclusion fast protein liquid chromatography (SE-FPLC). A yield of 200–300 μg of the GAT1/GFP protein could be purified from 400–600 mL of infected Sf9 cells. The purified protein was analyzed by transmission electron microscopy (TEM), which revealed that the GAT1/GFP fusion protein was isolated in its monomeric form.

The low molecular weight peptide composition of virgin olive oil (VOO) is mostly unknown. We hypothesised that unfiltered VOO could possess low molecular weight peptides with antihypertensive activity. We produced unfiltered VOO and obtained a water-soluble peptide extract from it. The peptides were separated by size-exclusion using fast protein liquid chromatography, and the low molecular weight fraction was analysed by nanoscale liquid chromatography-Orbitrap coupled with tandem mass spectrometry and de novo sequencing. We selected 23 peptide sequences containing between 6 and 9 amino acids and molecular masses ranging 698–1017 Da. Those peptides were chemically synthesised and their angiotensin-converting enzyme (ACE) inhibitory activity was studied in vitro. Seven peptides showed a strong activity, with half maximal inhibitory concentration (IC50) <10 µm. The antihypertensive effects of the four most active synthesised ACE inhibitor peptides were studied in spontaneously hypertensive rats (SHR). Acute oral administration of synthetic peptides RDGGYCC and CCGNAVPQ showed antihypertensive activity in SHR. We conclude that unfiltered VOO naturally contains low molecular weight peptides with specific ACE inhibitory activity and antihypertensive effects in SHR.

L'objectif principal de cette recherche est le développement de méthodes analytiques utilisant une technique de séparation couplée à la spectrométrie de masse pour l'analyse de complexes de fer de faible poids moléculaire et une technique de single-particle ICP MS pour la détection de nanoparticules bimétalliques.Dans la première partie, une méthode utilisant la chromatographie liquide avec spectrométrie de masse à double détecteur, spectrométrie de masse (MS) à haute résolution par électrospray (HRAM) et spectrométrie de masse à couplage inductif (ICPMS), a été développée pour les complexes du fer (Fe) de faible poids moléculaire, appelés 'sideophore', dans un échantillon d'un sol. La complexité des échantillons étudiés, les faibles concentrations et la labilité des analytes ont posé un défi dans le développement de méthodes pour leur identification et leur quantification. Pour éliminer la matrice, une extraction en phase solide (SPE) a été développée dans des conditions acides pour purifier la majeure partie des complexes 56Fe-sidérophore et concentrée par évaporation. Les complexes 56Fe-sidérophore ont été identifiés par chromatographie d'exclusion stérique rapide (FastSEC) - Orbitrap MSn sur la base de la masse moléculaire exacte (+ 1 ppm) et de la fragmentation MS2 ou MS3. Leur capacité à échanger facilement le 56Fe naturel contre le 58Fe ajouté a été démontrée par SEC avec détection par l'ICP MS et l'ESI MS. La méthode a été appliquée à l'analyse de tourbe prélevée dans la partie orientale des montagnes pyrénéennes françaises. Dix-neuf sidérophores appartenant à quatre classes différentes ont été identifiés et quantifiés sans avoir besoin d'un standard authentique. Les résultats ont été validés à l'aide de la détection ICP MS du fer en comparant la somme des complexes de fer déterminés par échange isotopique - ESI MS dans chaque pic observé par FastSEC-ICP MS.Dans la deuxième partie du manuscrit, une méthode utilisant la spectrométrie de masse à plasma à couplage inductif -ICP-MS en mode particule unique (SP-ICP-MS) et en mode conventionnel couplée au fractionnement d'écoulement de champ (FlFFF) a été développée. Les conditions de synthèse de nanoparticules bimétalliques (BNP) Ag-Au ont été optimisées pour appliquer celles-ci à la détection colorimétrique basée sur le concept d'agrégation. Les BNP Ag-Au, synthétisés par la réduction par le citrate des ions Ag et Au, ont été utilisées comme capteurs pour la détection du Co2+. Pour mieux comprendre la détection colorimétrique du Co2+ à l'aide de BNP Ag-Au, divers mélanges de solutions ont été étudiés, notamment : (i) uniquement des BNP Ag-Au ; (ii) BNP Ag-Au avec thiosulfate; (iii) BNP Ag-Au avec thiosulfate et éthylènediamine; et (iv) Ag-Au BNPs avec thiosulfate, Co2+ et éthylènediamine. SP-ICP-MS a été utilisé pour déterminer la taille du noyau, la distribution de taille et la concentration en nombre de particules, ainsi que l'hétérogénéité des particules synthétisées en utilisant diverses concentrations de citrate et un rapport de métal. FlFFF-ICP-MS a également été utilisé pour observer la taille hydrodynamique et le rapport d'intensité du signal de Ag et Au dans les BNP et donc pour étayer les informations obtenues à partir de SP-ICP-MS. La combinaison des techniques proposées dans des conditions appropriées a permis de surveiller la réaction de détection colorimétrique. Les informations supplémentaires du fractogramme fournies par FlFFF-ICP-MS ont également été utiles pour comprendre l'agrégation des BNP due au complexe [Co(II)(en)3]2+ autour de la surface des BNP. En outre, par rapport à la détection colorimétrique classique, la limite de détection (LOD) pour la détection des ions Co2+ a été réduite de 20 fois, du niveau ppb au niveau ppt
The research focuses on an analytical method development using chromatography coupled to mass spectrometry for the analysis of low molecular weight iron complexes. In the second part, the study explores the utilization of bimetallic nanoparticles for Co2+ detection.In the first part, a method using liquid chromatography with two detector mass spectrometry, i.e., electrospray high-resolution accurate mass (HRAM) mass spectrometry (MS) and inductively coupled mass spectrometry (ICP-MS), was developed for the analysis of low molecular weight iron (Fe) complexes, called ‘siderophores'. The complexity of the samples, their low concentrations, and the lability of the iron complexe were challenges in the development of methods for their identification and quantification. For the sample clean-up, solid phase extraction (SPE) using acidic conditions was developed to purify the samples, followed by evaporation to dryness. The individual 56Fe-siderophore complexes were identified by fast size-exclusion chromatography (FastSEC) - Orbitrap MSn based on the exact molecular mass (+ 1 ppm) and MS2. Their capability of exchanging the natural 56Fe with the spiked 58Fe was demonstrated by SEC with ICP-MS and ESI-MS detection. The method was applied to the analysis of peat collected in the Eastern part of the French Pyrenean mountains. Nineteen siderophores belonging to four different classes were presumptively identified and quantified. The results were compared with ICP-MS detection of iron and matching of the sum of the moles of iron complexes determined by the isotopic- ESI-MS within each peak as eluted from the fastSEC column.In the second part, a method using inductively coupled plasma mass spectrometry in the single particle mode and the conventional mode coupled to a flow field flow fractionation was developed to select suitable conditions for the synthesis of Ag-Au bimetallic nanoparticles and to monitor the colorimetric changes due to aggregations. Ag-Au BNPs, synthesized by using citrate reduction of Ag and Au ions, were used as sensors for the detection of Co2+. To better understand the colorimetric sensing of Co2+ using the Ag-Au BNPs, various mixtures were studied, viz. (i) only Ag-Au BNPs; (ii) Ag-Au BNPs with thiosulfate; (iii) Ag-Au BNPs with thiosulfate and ethylenediamine; and (iv) Ag-Au BNPs with thiosulfate, Co2+ and ethylenediamine. SP-ICP-MS was used to determine the core size, size distribution, and number concentration, as well as the heterogeneity of the particles synthesized by using various citrate concentrations and metal ratios. Fl-FFF-ICP-MS was also used to observe the hydrodynamic size and the Ag: Au signal intensity ratio of the BNPs to support information obtained from the SP-ICP-MS. The combination of the proposed techniques has been applied to monitor the reaction during colorimetric sensing. Additional information from fractograms provided by Fl-FFF-ICP-MS was also useful for the understanding of the aggregation of BNPs arising from the [Co(II)(en)3]2+ complex surrounding the surface of the BNPs. Furthermore, when compared to colorimetric sensing, the limit of detection for Co2+ ion, using the BNPs and SP-ICP-MS, were 20-fold lower, decreasing from ppb to ppt levels