Metabolomics and systems biology require the acquisition of reproducible, robust, reliable, and homogeneous biological data sets. Therefore, we developed and validated standard operating procedures (SOPs) for quenching and efficient extraction of metabolites from Escherichia coli to determine the best methods to approach global analysis of the metabolome. E. coli was grown in chemostat culture so that cellular metabolism could be held in reproducible, steady-state conditions under a range of precisely defined growth conditions, thus enabling sufficient replication of samples. The metabolome profiles were generated using gas chromatography/time-of-flight mass spectrometry (GC/TOF-MS). We employed univariate and multivariate statistical analyses to determine the most suitable method. This investigation indicates that 60% cold (-48 degrees C) methanol solution is the most appropriate method to quench metabolism, and we recommend 100% methanol, also at -48 degrees C, with multiple freeze-thaw cycles for the extraction of metabolites. However, complementary extractions would be necessary for coverage of the entire complement of metabolites as detected by GC/TOF-MS. Finally, the observation that metabolite leakage was significant and measurable whichever quenching method is used indicates that methods should be incorporated into the experiment to facilitate the accurate quantification of intracellular metabolites.

We show that pentaerythritol tetranitrate reductase (PETNR), a member of the 'ene' reductase old yellow enzyme family, catalyses the asymmetric reduction of a variety of industrially relevant activated alpha,beta-unsaturated alkenes including enones, enals, maleimides and nitroalkenes. We have rationalised the broad substrate specificity and stereochemical outcome of these reductions by reference to molecular models of enzyme-substrate complexes based on the crystal complex of the PETNR with 2-cyclohexenone 4a. The optical purity of products is variable (49-99% ee), depending on the substrate type and nature of substituents. Generally, high enantioselectivity was observed for reaction products with stereogenic centres at Cbeta (>99% ee). However, for the substrates existing in two isomeric forms (e.g., citral 11a or nitroalkenes 18-19a), an enantiodivergent course of the reduction of E/Z-forms may lead to lower enantiopurities of the products. We also demonstrate that the poor optical purity obtained for products with stereogenic centres at Calpha is due to non-enzymatic racemisation. In reactions with ketoisophorone 3a we show that product racemisation is prevented through reaction optimisation, specifically by shortening reaction time and through control of solution pH. We suggest this as a general strategy for improved recovery of optically pure products with other biocatalytic conversions where there is potential for product racemisation.

Expression of the gene encoding the S100 calcium-modulated protein family member MRP-14 (also known as S100A9) is elevated in platelets from patients presenting with acute myocardial infarction (MI) compared with those from patients with stable coronary artery disease; however, a causal role for MRP-14 in acute coronary syndromes has not been established. Here, using multiple models of vascular injury, we found that time to arterial thrombotic occlusion was markedly prolonged in Mrp14 -/-mice. We observed that MRP-14 and MRP-8/ MRP-14 heterodimers (S100A8/A9) are expressed in and secreted by platelets from WT mice and that thrombus formation was reduced in whole blood from Mrp14 -/-mice. Infusion of WT platelets, purified MRP-14, or purified MRP-8/MRP-14 heterodimers into Mrp14 -/-mice decreased the time to carotid artery occlusion after injury, indicating that platelet-derived MRP-14 directly regulates thrombosis. In contrast, infusion of purified MRP-14 into mice deficient for both MRP-14 and CD36 failed to reduce carotid occlusion times, indicating that CD36 is required for MRP-14-dependent thrombosis. Our data identify a molecular pathway of thrombosis that involves platelet MRP-14 and CD36 and suggest that targeting MRP-14 has potential for treating atherothrombotic disorders, including MI and stroke.

Chinese hamster ovary (CHO) cells are the primary platform for commercial expression of recombinant therapeutic proteins. Obtaining maximum production from the expression platform requires optimal cell culture medium (and associated nutrient feeds). We have used metabolite profiling to define the balance of intracellular and extracellular metabolites during the production process of a CHO cell line expressing a recombinant IgG4 antibody. Using this metabolite profiling approach, it was possible to identify nutrient limitations, which acted as bottlenecks for antibody production, and subsequently develop a simple feeding regime to relieve these metabolic bottlenecks. This metabolite profiling-based strategy was used to design a targeted, low cost nutrient feed that increased cell biomass by 35% and doubled the antibody titer. This approach, with the potential for utilization in non-specialized laboratories, can be applied universally to the optimization of production of commercially important biopharmaceuticals.

Global metabolite analysis approaches, coupled with sophisticated data analysis and modeling procedures (metabolomics), permit a dynamic read-out of how cellular proteins interact with cellular and environmental conditions to determine cell status. This type of approach has profound potential for understanding, and subsequently manipulating, the regulation of cell function. As part of our study to define the regulatory events that may be used to maximize production of commercially valuable recombinant proteins from cultured mammalian cells, we have optimized the quenching process to allow retention of physiologically relevant intracellular metabolite profiles in samples from recombinant Chinese hamster ovary (CHO) cells. In a comparison of a series of candidate quenching procedures, we have shown that quenching in 60% methanol supplemented with 0.85% ammonium bicarbonate (AMBIC) at -40 degrees C generates a profile of metabolites that is representative of a physiological status based upon examination of key labile cellular metabolites. This represents a key feature for any metabolomic study with suspension cultured mammalian cells and provides confidence in the validity of subsequent data analysis and modeling procedures.