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.

Shigella reroutes host cell central metabolism to obtain high-flux nutrient supply for vigorous intracellular growth.

Shigella flexneri proliferate in infected human epithelial cells at exceptionally high rates. This vigorous growth has important consequences for rapid progression to life-threatening bloody diarrhea, but the underlying metabolic mechanisms remain poorly understood. Here, we used metabolomics, proteomics, and genetic experiments to determine host and Shigella metabolism during infection in a cell culture model.

A proteomic approach of bradyrhizobium/aeschynomene root and stem symbioses reveals the importance of the fixA locus for symbiosis.

Rhizobia are soil bacteria that are able to form symbiosis with plant hosts of the legume family. These associations result in the formation of organs, called nodules in which bacteria fix atmospheric nitrogen to the benefit of the plant. Most of our knowledge on the metabolism and the physiology of the bacteria during symbiosis derives from studying roots nodules of terrestrial plants.

A link between arabinose utilization and oxalotrophy in Bradyrhizobium japonicum.

Rhizobia have a versatile catabolism that allows them to compete successfully with other microorganisms for nutrients in the soil and in the rhizosphere of their respective host plants. In this study, Bradyrhizobium japonicum USDA 110 was found to be able to utilize oxalate as the sole carbon source. A proteome analysis of cells grown in minimal medium containing arabinose suggested that oxalate oxidation extends the arabinose degradation branch via glycolaldehyde.

On-line solid-phase extraction high-performance liquid chromatography-tandem mass spectrometry for the quantitative analysis of tacrolimus in whole blood hemolyzate.

Tacrolimus is an immunosuppressive drug essential for preventing organ rejection after transplantation. Since tacrolimus strongly binds to erythrocytes, therapeutic monitoring requires its quantification in whole blood lyzate, representing one of the most difficult to analyze biological fluids due to its high protein load. In this communication, we report on the successful combination of whole blood hemolysis employing ionic liquids, followed by sample preparation by means of on-line solid phase extraction (SPE) using restricted access materials (RAM), which permitted the efficient removal of hemoglobin and other large biomolecules.

Metaproteogenomic analysis of microbial communities in the phyllosphere and rhizosphere of rice.

The above- and below-ground parts of rice plants create specific habitats for various microorganisms. In this study, we characterized the phyllosphere and rhizosphere microbiota of rice cultivars using a metaproteogenomic approach to get insight into the physiology of the bacteria and archaea that live in association with rice. The metaproteomic datasets gave rise to a total of about 4600 identified proteins and indicated the presence of one-carbon conversion processes in the rhizosphere as well as in the phyllosphere.

The ethylmalonyl-CoA pathway is used in place of the glyoxylate cycle by Methylobacterium extorquens AM1 during growth on acetate.

Acetyl-CoA assimilation was extensively studied in organisms harboring the glyoxylate cycle. In this study, we analyzed the metabolism of the facultative methylotroph Methylobacterium extorquens AM1, which lacks isocitrate lyase, the key enzyme in the glyoxylate cycle, during growth on acetate. MS/MS-based proteomic analysis revealed that the protein repertoire of M.

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.

Bacterial adaptation to life in association with plants - A proteomic perspective from culture to in situ conditions.

Diverse bacterial taxa that live in association with plants affect plant health and development. This is most evident for those bacteria that undergo a symbiotic association with plants or infect the plants as pathogens. Proteome analyses have contributed significantly toward a deeper understanding of the molecular mechanisms underlying the development of these associations.

Nanoscale ion-pair reversed-phase HPLC-MS for sensitive metabolome analysis.

In this article, we introduce a method using nanoscale ion-pair reversed-phase high-performance liquid chromatography (nano-IP-RP-HPLC) hyphenated to nanoelectrospray ionization high-resolution mass spectrometry (nano-ESI-HRMS) to separate and identify metabolites in cell extracts. Separation of metabolites was performed on a 100 μm i.d.

Rhizobial adaptation to hosts, a new facet in the legume root-nodule symbiosis.

Rhizobia are able to infect legume roots, elicit root nodules, and live therein as endosymbiotic, nitrogen-fixing bacteroids. Host recognition and specificity are the results of early programming events in bacteria and plants, in which important signal molecules play key roles. Here, we introduce a new aspect of this symbiosis: the adaptive response to hosts.

An integrated proteomics and transcriptomics reference data set provides new insights into the Bradyrhizobium japonicum bacteroid metabolism in soybean root nodules.

Bradyrhizobium japonicum, a gram-negative soil bacterium that establishes an N(2)-fixing symbiosis with its legume host soybean (Glycine max), has been used as a symbiosis model system. Using a sensitive geLC-MS/MS proteomics approach, we report the identification of 2315 B. japonicum strain USDA110 proteins (27.

Community proteogenomics reveals insights into the physiology of phyllosphere bacteria.

Aerial plant surfaces represent the largest biological interface on Earth and provide essential services as sites of carbon dioxide fixation, molecular oxygen release, and primary biomass production. Rather than existing as axenic organisms, plants are colonized by microorganisms that affect both their health and growth. To gain insight into the physiology of phyllosphere bacteria under in situ conditions, we performed a culture-independent analysis of the microbiota associated with leaves of soybean, clover, and Arabidopsis thaliana plants using a metaproteogenomic approach.

Rapid selection of peptide containing fractions in off-line 2-D HPLC in shotgun proteome analysis by screening with MALDI TOF MS.

A simple and fast screening method for the selection of fractions of first dimension separation to be analyzed in second dimension-MS/MS experiments in offline multidimensional liquid chromatographic separation schemes for shotgun proteome analysis was developed. The method is based on the measurement of total peptide content of the first dimension fractions by MALDI MS and was established using a tryptic digest of a bacterial proteome. The results of the screening process were in good agreement with those obtained in a detailed proteome analysis performed by RPxion-pair RP-MALDI TOF/TOF MS analysis.

Repeatability of peptide identifications in shotgun proteome analysis employing off-line two-dimensional chromatographic separations and ion-trap MS.

The repeatability of peptide identifications in shotgun proteome analyses employing strong cation-exchange-xion-pair RP HPLC hyphenated to ESI MS/MS was compared to an alternative scheme, comprising high-pH RP chromatography combined with low-pH ion-pair RP chromatography. Equivalent results were obtained with both methods in proteome analysis of Corynebacterium glutamicum. From a total number of 1350 to 1850 peptides identified in triplicate analyses of five consecutive fractions chosen from the first-dimension separation, 41-45% of the peptides were identified three times, whereas 16-22 and 37-39% of the peptides were identified only twice or once, respectively.

Shotgun proteomic analysis of the microsomal fraction of eukaryotic cells using a two-dimensional reversed-phase x ion-pair reversed-phase HPLC setup.

A RPxIP-RP HPLC separation scheme was combined with on-line ESI-IT tandem MS or off-line MALDI tandem TOF MS and applied to the analysis of eukaryotic subcellular proteomes. Previous proteomic studies [1] were complemented by the approval of the approach to eukaryotic proteomes using the fission yeast Schizosaccharomyces pombe. The major focus was set to the analysis of primary human hepatocyte microsomes, representing a compartment of high interest due to its involvement in xenobiotic detoxification and cholesterol homeostasis.

A 2D reversed-phase x ion-pair reversed-phase HPLC-MALDI TOF/TOF-MS approach for shotgun proteome analysis.

The separation of complex peptide mixtures in shotgun proteome analysis using a 2D separation scheme encompassing reversed-phase x ion-pair reversed-phase (IP-RP) liquid chromatography coupled online to electrospray ion trap mass spectrometry (MS) has been shown earlier to be superior in terms of separation efficiency and technical robustness compared to the classically used separation scheme encompassing strong cation exchange x IP-RP-chromatography in shotgun proteome analysis. In the present study, this novel separation scheme was coupled offline to matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF)/TOF-MS for the analysis of the same sample, a tryptic digest of the cytosolic proteome of the bacterium Corynebacterium glutamicum. Compared to the earlier study, the MALDI-based platform led to a significantly increased number of peptides (7,416 vs.

Two-dimensional reversed-phase x ion-pair reversed-phase HPLC: an alternative approach to high-resolution peptide separation for shotgun proteome analysis.

A two-dimensional separation scheme for shotgun proteome analysis employing high-pH reversed-phase HPLC in the first and low-pH ion-pair reversed-phase HPLC in the second dimension (RP x IP-RP-HPLC) has been developed and evaluated. Compared to the classical strong cation exchange x ion-pair reversed-phase (SCX x IP-RP-HPLC) approach, the RP x IP-RP-HPLC system was characterized by a lower degree of orthogonality, which was, however, more than counterbalanced by higher separation efficiency, more homogeneous distribution of peptide elution, and easier experimental handling. Peptide fragment fingerprinting by electrospray-ionization tandem mass spectrometry (ESI-MS/MS) was employed for peptide detection and identification.

Monolithic column HPLC separation of intact proteins analyzed by LC-MALDI using on-plate digestion: An approach to integrate protein separation and identification.

A method is developed to integrate a protein separation by monolithic capillary reversed-phase high-performance liquid chromatography to on-probe tryptic digestion for subsequent analyses by MALDI-TOF MS and MALDI-TOF/TOF MS. The method provides a means of directly interfacing separations to MALDI-MS, reducing the amount of time required for traditional procedures involving in-solution enzymatic digestion and sample cleanup prior to MALDI-MS analysis. When used with pI-based fractionation as a first dimension, it provides a means of analyzing complex mixtures of proteins with minimal sample handling and cleanup.