1. Préparation des échantillons <ul class="none"><li> 1 mélange de composés: la coumarine, l&#39;ibuprofène et la carbamazépine; 20 mg / ml chacun dans le méthanol </li><li> 2 Mélange composé: l&#39;ibuprofène et la carbamazépine; 20 mg / ml chacun dans le méthanol </li><li> 3 Mélange composé: l&#39;ibuprofène et la carbamazépine; 20 mg / ml chacun dans le DMSO (diméthylsulfoxyde) </li><li> 4 mélange de composés: 10 mg / mL fénoprofène, 15 mg / mL kétoprofène, 20 mg / mL flavone, 20 mg / ml ibuprofène et 20 mg / ml dans le méthanol coumarine </li></ul> 2. Conditions expérimentales <ol><li> Chromatographie analytique a été réalisée sur une méthode Waters système de station de SFC. Colonne: Viridis SFC 2-éthylpyridine colonne, 5 um, 4,6 x 150 mm, Numéro de pièce 186004937. </li><li> Chromatographie préparative a été réalisée sur un Waters Prep 100 Système UV SFC Réalisé. Les Colonnes: <ul><li> Viridis SFC 2-éthylpyridine colonne OBD, 5 pm, 19 x 150 mm, 186 004 945 Référence </li><li> Viridis SFC colonne de silice OBD, 5 pm, 19 x 150 mm, 186 004 918 Référence </li><li> Viridis SFC 2-éthylpyridine colonne OBD, 5 pm, 19 x 100 mm, 186 004 944 Référence </li></ul></li><li> Paramètres Autre méthode: <ul><li> Température de la colonne: 40 ° C </li><li> Cosolvant: Méthanol </li><li> Le volume d&#39;injection, du débit, et Dégradés: Rapporté en chiffres </li></ul></li></ol> 3. Les résultats représentatifs Débit Le débit utilisé dans les séparations LC préparative est limitée par un certain nombre de facteurs, dont la longueur et le diamètre de la colonne, la taille des particules, et la contre-pression du système. Faible viscosité de dioxyde de carbone, le principal composant de la phase mobile dans la SFC, permet l&#39;utilisation de débits plus élevés que ceux généralement utilisés en chromatographie liquide. La contre-pression associé à longueur de colonne est également plus faible, permettant l&#39;utilisation de plus de colonnes pour augmenter la résolution. Des débits plus élevés augmentent le débit en réduisant la quantité de temps pour terminer une séparation. Comme le montre la figure 1, le profil chromatographique est le même à des débits différents, à condition que la pente du gradient exprimé dans le changement pour cent par volume de colonne reste constante .. Ceci est accompli en diminuant le temps d&#39;exécution dans la même proportion que le débit est augmenté. <img src="/files/ftp_upload/2604/2604fig1.jpg" alt="Figure 1" /> Dissolution de l&#39;échantillon et la charge de solvant L&#39;isolement des composés de mélanges exige que l&#39;échantillon soit complètement soluble avant l&#39;injection. Le méthanol, le plus commun de co-solvant dans la SFC, ne solubiliser tous les échantillons à des concentrations élevées habituellement requis pour les séparations préparatives typique. De grands volumes de solvants comme le méthanol peut limiter la capacité de masse de la colonne à cause de forts effets de solvant qui peut fausser la chromatographie. Injection de flux modificateur, la configuration standard brevetée de tous les systèmes des eaux SFC prep, a été conçu pour réduire cet effet de solvant. Diméthylsulfoxyde (DMSO) est utilisé régulièrement dans les laboratoires de la purification de solubiliser de nombreux types de composés. Les figures 2 et 3 illustrent comment des injections de DMSO dans le SFC peut entraîner des charges plus élevées et une meilleure résolution pour des applications achiral par rapport aux échantillons injectés dans le méthanol. Note: Pour les applications chirales, la prudence doit être considéré comme le DMSO peut sévèrement endommager un revêtement traditionnel phase stationnaire chirale. Si le DMSO doit être requis pour la solubilité de l&#39;échantillon, une colonne chirale immobilisé doit être sélectionné. <img src="/files/ftp_upload/2604/2604fig2.jpg" alt="Figure 2" /> <img src="/files/ftp_upload/2604/2604fig3.jpg" alt="Figure 3" /> Sélectivité En SFC, des colonnes multiples sont systématiquement testés afin de déterminer qui offre la meilleure résolution et la forme des pics pour les composants dans un mélange. Bonne résolution entre les composés conduit à des isolements de composés cibles avec une grande pureté. Dans l&#39;exemple ci-dessous, la colonne 2-éthylpyridine montre une excellente séparation des cinq composés dans le mélange de l&#39;échantillon. Le mélange sur un même échantillon sur une colonne de silice montre que trois pics composant car il a une sélectivité différente. Coéluent coumarine et de l&#39;ibuprofène à environ 0,8 minutes et flavones et fénoprofène coéluent un peu plus tard. Flavone et l&#39;ibuprofène également modifier l&#39;ordre d&#39;élution sur la colonne de silice. Le kétoprofène est bien séparé de l&#39;ensemble des autres sommets sur les deux colonnes. <img src="/files/ftp_upload/2604/2604fig4.jpg" alt="Figure 4" /> Mise à l&#39;échelle Mélanges de l&#39;échantillon brut sont généralement analysées avec un gradient de dépistage qui commence à partir d&#39;un faible pourcentage de solvant organique et se termine à un pourcentage plus élevé de solvant organique dans un temps relativement court. Si les composés d&#39;intérêt sont bien réglés, le gradient peut être réduit directement pour la purification. Lorsque tous les composés éluer avant èmee fin de la pente et sont entièrement résolus, une simple réduction de la longueur de la pente est acceptable. Le débit rapide utilisé dans SFC combinée avec des dégradés modifiés considérablement raccourcir le temps total nécessaire pour la purification de composés cibles. Séparations d&#39;échelle nécessite correspondant chimie colonne et la taille des particules ainsi que les volumes d&#39;injection échelle appropriée et dégradés. Eaux préparative colonnes SFC sont emballés avec la technologie OBD, assurant la stabilité du lit et des performances comparables à des eaux analytiques colonnes SFC. OBD colonnes préparatives sont emballés au lit densités qui correspondent étroitement à la colonne équivalente analytique. Cette procédure innovante produit colonnes préparatives avec une excellente stabilité, la reproductibilité et l&#39;efficacité. Une étude de chargement est effectué à la petite échelle pour déterminer combien de masse de l&#39;échantillon peut être chargé sur la colonne. L&#39;objectif est de maximiser la charge sans compromettre la résolution. Amélioration de la résolution de pointe entraîne une hausse des charges et une meilleure pureté des composés isolés. De chargement supérieur colonne de réduire le nombre de pistes nécessaires pour obtenir suffisamment de matériel pour des expériences ultérieures. La chromatographie de l&#39;étude de chargement est représenté dans la figure 5. Le volume d&#39;injection conservatrice 35 uL montre une bonne résolution entre tous les composés dans le mélange et est ensuite mis à l&#39;échelle préparative pour la course sur la grande colonne. Un volume total de 600 uL ont été injectés sur la colonne de 19 x 150 mm préparative SFC. La figure 6 compare la chromatographie en utilisant le gradient à petite échelle modifiées à la charge maximale avec la chromatographie à grande échelle utilisée pour l&#39;isolement. La sélectivité est identique, un facteur critique lors de l&#39;utilisation de chromatographie analytique comme un outil pour l&#39;isolement le scoutisme et les différences dans les purification.The temps de rétention et la résolution entre les analytique et préparative chromatogrammes peuvent être attribués à des facteurs tels que le volume système et le mode d&#39;injection. En particulier, le flux brevetée modificateur de mode d&#39;injection * est employé pour tous les systèmes des eaux SFC préparative, en minimisant l&#39;effet de solvant et donc d&#39;améliorer le débit et l&#39;efficacité. * Brevet américain n ° 6576125 <img src="/files/ftp_upload/2604/2604fig5.jpg" alt="Figure 5" /> <img src="/files/ftp_upload/2604/2604fig6.jpg" alt="Figure 6" />

<ol>
<li>Optimum Bed Density (OBD) Columns: Enabling Technology for Laboratory-Scale Isolation and Purification. Waters White Paper 720001939EN 2006 Nov.</li>
</ol>

<ul><li> Les principes de base de la chromatographie liquide peut être appliqué à la chromatographie fluide supercritique (SFC). </li><li> Chromatographie à fluide supercritique peut être exécuté à des débits plus rapides et isolements faisant augmenter le débit. </li><li> Les échantillons ayant une solubilité limitée peut être dissous dans le diméthylsulfoxyde et purifiée en utilisant la SFC. En utilisant le DMSO pour la dissolution de l&#39;échantillon rend le processus de purification plus facile en réduisant l&#39;incertitude associée à la solubilité de l&#39;échantillon. </li><li> Résolution et le chargement des échantillons dissous dans le DMSO peut être améliorée par rapport aux mêmes échantillons dissous dans du méthanol. Une meilleure résolution de l&#39;échantillon conduit à des produits avec une plus grande pureté. Supérieur de chargement améliore l&#39;efficacité du processus de purification. </li><li> Colonne de sélectivité des impacts de la séparation des composants du mélange. Dépistage des colonnes différentes avec des gradients de vitesse conduit à une décision sur laquelle colonne préparative à utiliser pour isoler les composés dans l&#39;échantillon. </li><li> La capacité d&#39;échelle entre les colonnes analytiques et préparatives OBD conduit à des séparations identiques aux échelles petites et grandes, à condition que le volume d&#39;injection, la pente, et le temps de gradient sont redimensionnées correctement et la chimie et la taille des particules restent constantes. Colonne évolutivité élimine l&#39;incertitude dans l&#39;identification des pics, facilitant ainsi une plus grande efficacité de purification. </li></ul>

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<td>Method Station SFC System</td>
<td>&nbsp;</td>
<td>Waters</td>
<td>More information available at www.waters.com</td>
<td>Analytical SFC system</td>
</tr>
<tr>
<td>SFC-UV Prep 100</td>
<td>&nbsp;</td>
<td>Waters</td>
<td>More information available at www.waters.com</td>
<td>Preparative SFC System</td>
</tr>
<tr>
<td>Viridis&trade; SFC 2-Ethylpyridine, 4.6 x 150 mm, 5 &mu;m Column</td>
<td>&nbsp;</td>
<td>Waters</td>
<td>186004937</td>
<td>Analytical SFC Chromatography Column</td>
</tr>
<tr>
<td>Viridis&trade; SFC 2-Ethylpyridine OBD&trade;, 19 x 150 mm, 5 &mu;m Column</td>
<td>&nbsp;</td>
<td>Waters</td>
<td>186004945</td>
<td>Preparative SFC Chromatography Column</td>
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<td>Viridis&trade; SFC Silica OBD&trade;, 19 x 150 mm, 5 &mu;m Column</td>
<td>&nbsp;</td>
<td>Waters</td>
<td>186004918</td>
<td>Preparative SFC Chromatography Column</td>
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<td>Viridis&trade; SFC 2-Ethylpyridine OBD&trade;, 19 x 100 mm, 5 &mu;m Column</td>
<td>&nbsp;</td>
<td>Waters</td>
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basic principles for purification using supercritical fluid chromatography - advertisement

Bien que la chromatographie fluide supercritique (SFC) n&#39;est pas une technique nouvelle, préparative SFC devient de plus en plus populaire auprès des progrès dans l&#39;instrumentation, de logiciels et de la chimie. Principes de base pour l&#39;isolement des composés avec SFC sont similaires aux règles fondamentales de grande envergure chromatographie liquide préparative. Dans cette étude, nous illustrons l&#39;effet de débit, de la dissolution de l&#39;échantillon de solvant, et la sélectivité de colonne sur l&#39;isolement de composés à partir d&#39;un mélange. Colonnes emballé avec la densité optimale Bed (OBD) 1 et la technologie avec la chimie correspondant d&#39;assurer une évolutivité facile et prévisible à partir de la petite échelle aux grandes séparations préparatives.

Principes de base pour la séparation des composés à partir de mélanges en utilisant la chromatographie fluide supercritique (SFC) sont similaires aux fondements de la chromatographie liquide préparative.

Principes de base pour la purification par chromatographie en phase fluide supercritique - PUBLICITÉ

Although supercritical fluid chromatography (SFC) is not a new technique, preparative SFC is becoming increasingly more popular with advances in ...

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Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. The approach offers an unbiased and in-depth analysis that can enable the development of diagnostic tests, novel therapies, and further our understanding of disease processes. The inherent chemical diversity of the metabolome creates significant analytical challenges and there is no single experimental approach that can detect all metabolites. Additionally, the biological variation in individual metabolism and the dependence of metabolism on environmental factors necessitates large sample numbers to achieve the appropriate statistical power required for meaningful biological interpretation. To address these challenges, this tutorial outlines an analytical workflow for large scale non-targeted metabolite profiling of serum by UPLC-MS. The procedure includes guidelines for sample organization and preparation, data acquisition, quality control, and metabolite identification and will enable reliable acquisition of data for large experiments and provide a starting point for laboratories new to non-targeted metabolite profiling by UPLC-MS.

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry UPLC-MS

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. This article outlines a typical workflow utilized for non-targeted metabolite profiling of serum including sample organization and preparation, data acquisition, data analysis, quality control, and metabolite identification.

À grande échelle non ciblée profilage métabolomique du sérum par Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to ...

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JoVE Journal

Chimie

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

Metabolomics non ciblées de sources biologiques en utilisant UltraPerformance chromatographie liquide haute résolution spectrométrie de masse (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

L&#39;extraction des lipides cellulaires pour ciblées dilution isotopique stable analyse de spectrométrie de masse chromatographie liquide-

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Bio-ingénierie

Biochemical Measurement of Neonatal Hypoxia

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to cause/exacerbate well documented neonatal disorders including neurological impairment 1-3. Decreased adenosine triphosphate production occurs due to a lack of oxidative phosphorylation. To compensate for this energy deprived state molecules containing high energy phosphate bonds are degraded 2. This leads to increased levels of adenosine which is subsequently degraded to inosine, hypoxanthine, xanthine, and finally to uric acid. The final two steps in this degradation process are performed by xanthine oxidoreductase. This enzyme exists in the form of xanthine dehydrogenase under normoxic conditions but is converted to xanthine oxidase (XO) under hypoxia-reperfusion circumstances 4, 5. Unlike xanthine dehydrogenase, XO generates hydrogen peroxide as a byproduct of purine degradation 4, 6. This hydrogen peroxide in combination with other reactive oxygen species (ROS) produced during hypoxia, oxidizes uric acid to form allantoin and reacts with lipid membranes to generate malondialdehyde (MDA) 7-9. Most mammals, humans exempted, possess the enzyme uricase, which converts uric acid to allantoin. In humans, however, allantoin can only be formed by ROS-mediated oxidation of uric acid. Because of this, allantoin is considered to be a marker of oxidative stress in humans, but not in the mammals that have uricase.
We describe methods employing high pressure liquid chromatography (HPLC) and gas chromatography mass spectrometry (GCMS) to measure biochemical markers of neonatal hypoxia ischemia. Human blood is used for most tests. Animal blood may also be used while recognizing the potential for uricase-generated allantoin. Purine metabolites were linked to hypoxia as early as 1963 and the reliability of hypoxanthine, xanthine, and uric acid as biochemical indicators of neonatal hypoxia was validated by several investigators 10-13. The HPLC method used for the quantification of purine compounds is fast, reliable, and reproducible. The GC/MS method used for the quantification of allantoin, a relatively new marker of oxidative stress, was adapted from Gruber et al 7. This method avoids certain artifacts and requires low volumes of sample. Methods used for synthesis of MMDA were described elsewhere 14, 15. GC/MS based quantification of MDA was adapted from Paroni et al. and Cighetti et al. 16, 17. Xanthine oxidase activity was measured by HPLC by quantifying the conversion of pterin to isoxanthopterin 18. This approach proved to be sufficiently sensitive and reproducible.

A method is described to measure biochemical markers of neonatal hypoxia-ischemia. The approach utilizes high pressure liquid chromatography (HPLC) and Gas Chromatography Mass Spectrometry (GC/MS).

Mesure biochimique de l&#39;hypoxie néonatale

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to ...

Médecine

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings (GCMR) in the Insect Antennal Lobe

All organisms inhabit a world full of sensory stimuli that determine their behavioral and physiological response to their environment. Olfaction is especially important in insects, which use their olfactory systems to respond to, and discriminate amongst, complex odor stimuli. These odors elicit behaviors that mediate processes such as reproduction and habitat selection1-3. Additionally, chemical sensing by insects mediates behaviors that are highly significant for agriculture and human health, including pollination4-6, herbivory of food crops7, and transmission of disease8,9. Identification of olfactory signals and their role in insect behavior is thus important for understanding both ecological processes and human food resources and well-being.
	To date, the identification of volatiles that drive insect behavior has been difficult and often tedious. Current techniques include gas chromatography-coupled electroantennogram recording (GC-EAG), and gas chromatography-coupled single sensillum recordings (GC-SSR)10-12. These techniques proved to be vital in the identification of bioactive compounds. We have developed a method that uses gas chromatography coupled to multi-channel electrophysiological recordings (termed 'GCMR') from neurons in the antennal lobe (AL; the insect's primary olfactory center)13,14. This state-of-the-art technique allows us to probe how odor information is represented in the insect brain. Moreover, because neural responses to odors at this level of olfactory processing are highly sensitive owing to the degree of convergence of the antenna's receptor neurons into AL neurons, AL recordings will allow the detection of active constituents of natural odors efficiently and with high sensitivity. Here we describe GCMR and give an example of its use.
	Several general steps are involved in the detection of bioactive volatiles and insect response. Volatiles first need to be collected from sources of interest (in this example we use flowers from the genus Mimulus (Phyrmaceae)) and characterized as needed using standard GC-MS techniques14-16. Insects are prepared for study using minimal dissection, after which a recording electrode is inserted into the antennal lobe and multi-channel neural recording begins. Post-processing of the neural data then reveals which particular odorants cause significant neural responses by the insect nervous system.
	Although the example we present here is specific to pollination studies, GCMR can be expanded to a wide range of study organisms and volatile sources. For instance, this method can be used in the identification of odorants attracting or repelling vector insects and crop pests. Moreover, GCMR can also be used to identify attractants for beneficial insects, such as pollinators. The technique may be expanded to non-insect subjects as well.

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings GCMR in the Insect Antennal Lobe

Olfactory cues mediate many different behaviors in insects, and are often complex mixtures comprised of tens to hundreds of volatile compounds. Using gas chromatography with multi-channel recording in the insect antennal lobe, we describe a method for the identification of bioactive compounds.

L&#39;identification des composés volatils olfactifs chromatographie en phase gazeuse-à logements multiples enregistrements (GCMR) dans le lobe antennaire des insectes

All  organisms inhabit a world full of sensory stimuli that determine their  behavioral and physiological response to their environment. Olfaction is ...

Neurosciences

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography (TLC) Coupled with Gas-Liquid Chromatography (GLC)

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as phosphatidylcholine or phosphatidylethanolamine, form bilayer membranes which act as both boundaries and interfaces for chemical exchange between cells and their surroundings. Unlike animals, plant cells have a special organelle for photosynthesis, the chloroplast. The intricate membrane system of the chloroplast contains unique glycerolipids, namely glycolipids lacking phosphorus: monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG)4. The roles of these lipids are beyond simply structural. These glycolipids and other glycerolipids were found in the crystal structures of photosystem I and II indicating the involvement of glycerolipids in photosynthesis8,11. During phosphate starvation, DGDG is transferred to extraplastidic membranes to compensate the loss of phospholipids9,12.
Much of our knowledge of the biosynthesis and function of these lipids has been derived from a combination of genetic and biochemical studies with Arabidopsis thaliana14. During these studies, a simple procedure for the analysis of polar lipids has been essential for the screening and analysis of lipid mutants and will be outlined in detail. A leaf lipid extract is first separated by thin layer chromatography (TLC) and glycerolipids are stained reversibly with iodine vapor. The individual lipids are scraped from the TLC plate and converted to fatty acyl methylesters (FAMEs), which are analyzed by gas-liquid chromatography coupled with flame ionization detection (FID-GLC) (Figure 1). This method has been proven to be a reliable tool for mutant screening. For example, the tgd1,2,3,4 endoplasmic reticulum-to-plastid lipid trafficking mutants were discovered based on the accumulation of an abnormal galactoglycerolipid: trigalactosyldiacylglycerol (TGDG) and a decrease in the relative amount of 18:3 (carbons : double bonds) fatty acyl groups in membrane lipids 3,13,18,20. This method is also applicable for determining enzymatic activities of proteins using lipids as substrate6.

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC

Composition of polar lipid extracts and the fatty acid composition of individual glycerolipids are determined in a simple and robust lipid profiling experiment. For this purpose, glycerolipids are isolated by thin layer chromatography and subjected to transmethylation of their acyl groups. Fatty acyl methylesters are quantified by gas-liquid chromatography.

Arabidopsis thaliana profilage glycolipides polaires par chromatographie sur couche mince (CCM) couplée à la chromatographie gaz-liquide (GLC)

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as ...

Biologie

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification.
HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4.
The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.

An automated method for identifying suitable hydrophobic interaction chromatography (HIC) media to be used in the process of protein purification is presented. The method utilizes a medium-pressure liquid chromatography system including automated buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection.

Automatisé d&#39;interaction hydrophobe Chromatographie sur colonne de sélection pour l&#39;utilisation dans la purification des protéines

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Purification de Hsp104, une protéine Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

Fruit Volatile Analysis Using an Electronic Nose

Numerous and diverse physiological changes occur during fruit ripening, including the development of a specific volatile blend that characterizes fruit aroma. Maturity at harvest is one of the key factors influencing the flavor quality of fruits and vegetables1. The validation of robust methods that rapidly assess fruit maturity and aroma quality would allow improved management of advanced breeding programs, production practices and postharvest handling. 
Over the last three decades, much research has been conducted to develop so-called electronic noses, which are devices able to rapidly detect odors and flavors2-4. Currently there are several commercially available electronic noses able to perform volatile analysis, based on different technologies. The electronic nose used in our work (zNose, EST, Newbury Park, CA, USA), consists of ultra-fast gas chromatography coupled with a surface acoustic wave sensor (UFGC-SAW). This technology has already been tested for its ability to monitor quality of various commodities, including detection of deterioration in apple5; ripeness and rot evaluation in mango6; aroma profiling of thymus species7; C6 volatile compounds in grape berries8; characterization of vegetable oil9 and detection of adulterants in virgin coconut oil10. 
This system can perform the three major steps of aroma analysis: headspace sampling, separation of volatile compounds, and detection. In about one minute, the output, a chromatogram, is produced and, after a purging cycle, the instrument is ready for further analysis. The results obtained with the zNose can be compared to those of other gas-chromatographic systems by calculation of Kovats Indices (KI). Once the instrument has been tuned with an alkane standard solution, the retention times are automatically converted into KIs. However, slight changes in temperature and flow rate are expected to occur over time, causing retention times to drift. Also, depending on the polarity of the column stationary phase, the reproducibility of KI calculations can vary by several index units11. A series of programs and graphical interfaces were therefore developed to compare calculated KIs among samples in a semi-automated fashion. These programs reduce the time required for chromatogram analysis of large data sets and minimize the potential for misinterpretation of the data when chromatograms are not perfectly aligned.
We present a method for rapid volatile compound analysis in fruit. Sample preparation, data acquisition and handling procedures are also discussed.

A rapid method for volatile compound analysis in fruit is described. The volatile compounds present in the headspace of a homogenate of the sample are rapidly separated and detected with ultra-fast gas chromatography (GC) coupled with a surface acoustic wave (SAW) sensor. A procedure for data handling and analysis is also discussed.

Analyse de fruits instable avec un nez électronique

Numerous and diverse physiological changes occur during fruit ripening, including the development of a specific volatile blend that characterizes fruit ...

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits2. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel&#39;s molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight3. The leading model for DNA movement through an agarose gel is &#34;biased reptation&#34;, whereby the leading edge moves forward and pulls the rest of the molecule along4. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation5; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 
 
1. Understand the mechanism by which DNA fragments are separated within a gel matrix 
2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 
3. Identify an agarose solution of appropriate concentration for their needs 
4. Prepare an agarose gel for electrophoresis of DNA samples 
5. Set up the gel electrophoresis apparatus and power supply 
6. Select an appropriate voltage for the separation of DNA fragments 
7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 
8. Determine the sizes of separated DNA fragments 
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A basic protocol for the separation of DNA fragments using agarose gel electrophoresis is described.

Électrophorèse sur gel d&#39;agarose pour la séparation de fragments d&#39;ADN

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from ...