Development of multidimensional liquid chromatographic methods hyphenated to mass spectrometry, preparation and analysis of complex biological samples

Immunoadsorbers based on monolithic epoxy-activated CIM disks have been developed in order to target biomarkers of heart diseases. The developed immunoadsorbers permitted to selectively isolate myoglobin and NT-proBNP from human serum. Anti-NT-proBNP-CIM disks permitted a quantitative isolation of NT-proBNP at concentrations down to 750 amol/µL in serum (R2 = 0.998). Six different restricted access materials have been evaluated with respect to their ability to remove hemoglobin from hemolysates. Experiments at different pH revealed that the retention of hemoglobin can be drastically diminished at pH 10.7. Because of better chemical stability at high pH, the polymeric Biotrap 500 MS RAM column was optimized for the analysis of hemolysates. The setup permits to quantitatively extract antibiotics from whole blood hemolysates at biologically relevant concentrations (200 pg/µL), and without carry-over of hemoglobin. A new 2D-HPLC-ESI-MS/MS setup for proteome analysis was developed. It consisted of a peptide separation by RP-HPLC at pH 10.0, followed by IP-RP-HPLC at pH 2.1. This new setup was compared with a classical SCX x IP-RP-HPLC setup. Separation repeatability is similar with both setups. The orthogonality between methods of separation is higher in the SCX x IP-RP-HPLC approach than in the RP x IP-RP-HPLC scheme. However, the better peptide distribution and separation efficiency achieved with the RP x IP-RP-HPLC setup permitted to identify significantly more peptides than with the classical SCX x IP-RP-HPLC setup. Both approaches are complementary and a combination of both setups permits to identify more peptides than replicate injections performed with a single setup.Immunoadsorber, die auf monolithischen Epoxy-aktivierten CIM-Disks basieren, wurden entwickelt, um Biomarker für Herzkrankheiten nachweisen zu können. Die entwickelten Immunoadsorber erlaubten eine selektive Isolierung von Myoglobin und NT-proBNP aus menschlichen Serum. Anti-NT-proBNP-CIM-Disks ermöglichten die quantitative Isolierung von NT-proBNP mit Konzentrationen bis zu 750 amol/µL im Serum (R2 = 0,998). Sechs verschiedene "Restricted Access Materials" wurden im Hinblick auf ihre Fähigkeit, bzgl. der Entfernung von Hämoglobin aus Hämolysaten untersucht. Experimente bei unterschiedlichen pH-Werten ergaben, dass die Retention von Hämoglobin bei einem pH Wert von 10,7 deutlich verkleinert werden kann. Aufgrund ihrer höheren chemischen Stabilität bei höheren pH-Werten wurde die polymere "Biotrap 500 MS RAM" für die Analyse von Hämolysaten optimiert. Die Methode ermöglicht die quantitative Extraktion von Antibiotika aus Gesamtblut Hämolysaten mit biologisch relevanten Konzentrationen (200 pg/µL), ohne die Verschleppung des Hämoglobins. Eine neue 2D-HPLC-ESI-MS/MS-Methode wurde für die Proteomanalyse entwickelt. Sie bestand aus einer Peptid-Trennung mittels RP-HPLC bei einem pH-Wert von 10,0 und anschließender IP-RP-HPLC bei einem pH-Wert von 2,1. Anschließend wurde diese neue Methode mit einer klassischen SCX x IP-RP-HPLC-Methode verglichen. Die Orthogonalität zwischen den beiden Trennmethoden bei der SCX x IP-RP-HPLC ist hierbei höher als bei der entsprechenden RP x IP-RP-HPLC-Methode. Allerdings erlaubt die bessere Verteilung der Peptide und die bessere Trenneffizienz der SCX x IP-RP-HPLC-Methode die Identifizierung einer höheren Anzahl an Peptiden. Beide Methoden sind komplementär, und eine Kombination beider Methoden erlaubt die Identifizierung einer größeren Anzahl an Peptiden als wiederholte Injektionen bei einer eindimensionalen Methode

Bacterial RuBisCO Is Required for Efficient Bradyrhizobium/Aeschynomene Symbiosis

Rhizobia and legume plants establish symbiotic associations resulting in the formation of organs specialized in nitrogen fixation. In such organs, termed nodules, bacteria differentiate into bacteroids which convert atmospheric nitrogen and supply the plant with organic nitrogen. As a counterpart, bacteroids receive carbon substrates from the plant. This rather simple model of metabolite exchange underlies symbiosis but does not describe the complexity of bacteroids' central metabolism. A previous study using the tropical symbiotic model Aeschynomene indica/photosynthetic Bradyrhizobium sp. ORS278 suggested a role of the bacterial Calvin cycle during the symbiotic process. Herein we investigated the role of two RuBisCO gene clusters of Bradyrhizobium sp. ORS278 during symbiosis. Using gene reporter fusion strains, we showed that cbbL1 but not the paralogous cbbL2 is expressed during symbiosis. Congruently, CbbL1 was detected in bacteroids by proteome analysis. The importance of CbbL1 for symbiotic nitrogen fixation was proven by a reverse genetic approach. Interestingly, despite its symbiotic nitrogen fixation defect, the cbbL1 mutant was not affected in nitrogen fixation activity under free living state. This study demonstrates a critical role for bacterial RuBisCO during a rhizobia/legume symbiotic interaction

Entwicklung von mehrdimensionalen, flüssigkeitschromatographischen Methoden gekoppelt mit Massenspektrometrie, Vorbereitung und Analyse von komplexen biologischen Proben

Immunoadsorbers based on monolithic epoxy-activated CIM disks have been developed in order to target biomarkers of heart diseases. The developed immunoadsorbers permitted to selectively isolate myoglobin and NT-proBNP from human serum. Anti-NT-proBNP-CIM disks permitted a quantitative isolation of NT-proBNP at concentrations down to 750 amol/µL in serum (R2 = 0.998). Six different restricted access materials have been evaluated with respect to their ability to remove hemoglobin from hemolysates. Experiments at different pH revealed that the retention of hemoglobin can be drastically diminished at pH 10.7. Because of better chemical stability at high pH, the polymeric Biotrap 500 MS RAM column was optimized for the analysis of hemolysates. The setup permits to quantitatively extract antibiotics from whole blood hemolysates at biologically relevant concentrations (200 pg/µL), and without carry-over of hemoglobin. A new 2D-HPLC-ESI-MS/MS setup for proteome analysis was developed. It consisted of a peptide separation by RP-HPLC at pH 10.0, followed by IP-RP-HPLC at pH 2.1. This new setup was compared with a classical SCX x IP-RP-HPLC setup. Separation repeatability is similar with both setups. The orthogonality between methods of separation is higher in the SCX x IP-RP-HPLC approach than in the RP x IP-RP-HPLC scheme. However, the better peptide distribution and separation efficiency achieved with the RP x IP-RP-HPLC setup permitted to identify significantly more peptides than with the classical SCX x IP-RP-HPLC setup. Both approaches are complementary and a combination of both setups permits to identify more peptides than replicate injections performed with a single setup.Immunoadsorber, die auf monolithischen Epoxy-aktivierten CIM-Disks basieren, wurden entwickelt, um Biomarker für Herzkrankheiten nachweisen zu können. Die entwickelten Immunoadsorber erlaubten eine selektive Isolierung von Myoglobin und NT-proBNP aus menschlichen Serum. Anti-NT-proBNP-CIM-Disks ermöglichten die quantitative Isolierung von NT-proBNP mit Konzentrationen bis zu 750 amol/µL im Serum (R2 = 0,998). Sechs verschiedene "Restricted Access Materials" wurden im Hinblick auf ihre Fähigkeit, bzgl. der Entfernung von Hämoglobin aus Hämolysaten untersucht. Experimente bei unterschiedlichen pH-Werten ergaben, dass die Retention von Hämoglobin bei einem pH Wert von 10,7 deutlich verkleinert werden kann. Aufgrund ihrer höheren chemischen Stabilität bei höheren pH-Werten wurde die polymere "Biotrap 500 MS RAM" für die Analyse von Hämolysaten optimiert. Die Methode ermöglicht die quantitative Extraktion von Antibiotika aus Gesamtblut Hämolysaten mit biologisch relevanten Konzentrationen (200 pg/µL), ohne die Verschleppung des Hämoglobins. Eine neue 2D-HPLC-ESI-MS/MS-Methode wurde für die Proteomanalyse entwickelt. Sie bestand aus einer Peptid-Trennung mittels RP-HPLC bei einem pH-Wert von 10,0 und anschließender IP-RP-HPLC bei einem pH-Wert von 2,1. Anschließend wurde diese neue Methode mit einer klassischen SCX x IP-RP-HPLC-Methode verglichen. Die Orthogonalität zwischen den beiden Trennmethoden bei der SCX x IP-RP-HPLC ist hierbei höher als bei der entsprechenden RP x IP-RP-HPLC-Methode. Allerdings erlaubt die bessere Verteilung der Peptide und die bessere Trenneffizienz der SCX x IP-RP-HPLC-Methode die Identifizierung einer höheren Anzahl an Peptiden. Beide Methoden sind komplementär, und eine Kombination beider Methoden erlaubt die Identifizierung einer größeren Anzahl an Peptiden als wiederholte Injektionen bei einer eindimensionalen Methode

Développement de méthodes chromatographiques liquides multidimensionnelles couplées à la spectrométrie de masse, préparation et analyse d'échantillons biologiques complexes

Des immunoadsorbeurs ont été développés à partir de disques CIM monolithiques pour l'analyse de biomarqueurs impliqués dans des maladies cardio-vasculaires. Les colonnes développées ont permis d'isoler sélectivement la myoglobine et le NT-proBNP du sérumImmunoadsorbers based on monolithic epoxy-activated CIM disks have been developed in order to target biomarkers of heart diseases. The developed immunoadsorbers permitted to selectively isolate myoglobin and NT-proBNP from human serum. Anti-NT-proBNP-CI

Développement de méthodes chromatographiques liquides multidimensionnelles couplées à la spectrométrie de masse (Préparation et analyse d échantillons biologiques complexes)

Des immunoadsorbeurs ont été développés à partir de disques CIM monolithiques pour l analyse de biomarqueurs impliqués dans des maladies cardio-vasculaires. Les colonnes développées ont permis d isoler sélectivement la myoglobine et le NT-proBNP du sérum humain. Les colonnes anti-NT-proBNP ont permis l isolation quantitative du NT-proBNP (R2 = 0,998) à des concentrations jusqu à 750 amol/ L de sérum. Six matériaux à accès restreints ont été évalués en fonction de leur aptitude à exclure l hémoglobine d hémolysats sanguins. Des injections à différents pH ont montré que la rétention de l hémoglobine est drastiquement restreinte à pH 10,7. En raison d une bonne stabilité à pH basique, la colonne polymérique Biotrap 500 MS RAM a été retenue pour l extraction d antibiotiques d hémolysats sanguins. Des extractions quantitatives d analytes à faibles concentrations (200 pg/ L) ont été réalisées sans effet mémoire d hémoglobine sur la colonne. Un nouveau système 2D-HPLC-ESI-MS/MS pour l analyse protéomique a été développé. Le système est composé d une séparation par RP-HPLC à pH 10,0, suivie d une séparation par IP-RP-HPLC à pH 2,1. Ce nouveau système a été comparé à un système conventionnel SCX x IP-RP-HPLC. L orthogonalité des méthodes de séparation est plus élevée dans l approche SCX x IP-RP-HPLC que dans le schéma RP x IP-RP-HPLC. Cependant, en raison d une meilleure distribution des peptides et d une meilleure efficacité de séparation, le système RP x IP-RP-HPLC permet d identifier significativement plus de peptides. Les deux approches sont complémentaires et une combinaison des deux systèmes permet d identifier plus de peptides que des analyses répétées par un système unique.Immunoadsorbers based on monolithic epoxy-activated CIM disks have been developed in order to target biomarkers of heart diseases. The developed immunoadsorbers permitted to selectively isolate myoglobin and NT-proBNP from human serum. Anti-NT-proBNP-CIM disks permitted a quantitative isolation of NT-proBNP at concentrations down to 750 amol/ L in serum (R2 = 0.998). Six different restricted access materials have been evaluated with respect to their ability to remove hemoglobin from hemolysates. Experiments at different pH revealed that the retention of hemoglobin can be drastically diminished at pH 10.7. Because of better chemical stability at high pH, the polymeric Biotrap 500 MS RAM column was optimized for the analysis of hemolysates. The setup permits to quantitatively extract antibiotics from whole blood hemolysates at biologically relevant concentrations (200 pg/ L), and without carry-over of hemoglobin. A new 2D-HPLC-ESI-MS/MS setup for proteome analysis was developed. It consisted of a peptide separation by RP-HPLC at pH 10.0, followed by IP-RP-HPLC at pH 2.1. This new setup was compared with a classical SCX x IP-RP-HPLC setup. Separation repeatability is similar with both setups. The orthogonality between methods of separation is higher in the SCX x IP-RP-HPLC approach than in the RP x IP-RP-HPLC scheme. However, the better peptide distribution and separation efficiency achieved with the RP x IP-RP-HPLC setup permitted to identify significantly more peptides than with the classical SCX x IP-RP-HPLC setup. Both approaches are complementary and a combination of both setups permits to identify more peptides than replicate injections performed with a single setup.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Fast sampling method for mammalian cell metabolic analyses using liquid chromatography-mass spectrometry

Metabolomics has emerged as a powerful tool for addressing biological questions. Liquid chromatography coupled with mass spectrometry (LC-MS) is widely used for metabolic characterization, including targeted and untargeted approaches. Despite recent innovations, a crucial aspect of this technique is the sample preparation for accurate data analyses. In this protocol, we present a robust and adaptable workflow for metabolic analyses of mammalian cells from adherent cell cultures, which is particularly suited for qualitative and quantitative central metabolite characterization by LC-MS. Each sample consists of 600,000 mammalian cells grown on cover glasses, allowing for fast and complete transfer of the cells for metabolite extraction or medium exchange, e.g., for labeling experiments. The sampling procedure includes a fast and efficient washing step in liquid flow in water, which reduces cross-contamination and matrix effects while minimizing perturbation of the metabolic steady state of the cells; it is followed by quenching cell metabolism. The latter is achieved by using a -20 °C cold methanol acetonitrile mixture acidified with formic acid, followed by freeze drying, metabolite extraction and LC-MS. The protocol requires 2 s for cell sampling until quenching, and the entire protocol takes a total of 1.5 h per sample when the provided nanoscale LC-MS method is applied

Identification of CbbL1 (BRADO1659) peptides in bacteroids protein extract by tandem mass spectrometry and database search.

<p>Identification of CbbL1 (BRADO1659) peptides in bacteroids protein extract by tandem mass spectrometry and database search.</p

Evolutionary relationships of RuBisCO large subunit sequences.

<p>Neighbour joining method was used. Bootstrap support values (10000 replicates with Mega4) are provided as percentage at the corresponding nodes. CbbL1 and CbbL2 proteins from ORS278 strain are highlighted in bold characters. For RuBisCO IA members, the RuBisCO subclasses IAc and IAq is indicated before bacterial name.</p

RuBisCO 1 mutant displays typical traits of Fix^- mutant on <i>A. indica</i> but is able to reduce acetylene <i>in vitro</i>.

<p>A. Symbiotic acetylene reduction assays: <i>A. indica</i> plants were inoculated with the indicated <i>Bradyrhizobium</i> sp. ORS278 strain one week after germination. Acetylene reduction activities were measured on individual plants two weeks after inoculation. B. Nodule numbers induced by the indicated strain on <i>A. indica</i> were determined two weeks after infection. C. Acetylene reduction activities were measured during growth on BNM medium supplemented with oxo-glutarate. Error bars represent standard deviations.</p

Hypothetical model of ORS278 central metabolism during the symbiotic process.

<p>Upper part of the figure represents the WT and WT-like situation: Plant supplied organic carbon to bacteria which use it via the tricarboxylic acid cycle (TCA) resulting in the production of reduced cofactors (Red Cof). Reduced cofactors are totally oxidized by the respiratory chain at the early stage of nodule formation, then oxygen concentration drops and Calvin cycle drives the electron excess to CO₂. Once nitrogenase synthesis has started, electron excess is consumed by the nitrogen fixation process. Lower part of the figure represents the situation with a <i>cbbL1</i> mutant leading to a Fix–like nodule: electron excess cannot be drive to CO₂ therefore, reduced cofactors accumulate and disturbed bacterial metabolism preventing normal nodule development.</p