Expression of recombinant human metallothionein 2A as internal standard for mass spectrometric analysis of metallothioneins

Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2008.The induction of metallothionein (MT) expression in mitochondrial disorders has been well studied on the transcription level by means of RNA measurements in an attempt to understand and confirm the function of this protein in the deficient cells and organs (Olivier, 2004:42; Pretorius, 2006:44; Reinecke, 2004:89). However, MT expression induction still needs to be verified on protein (translation) level in order to confirm previous findings and to gain better perspective on the significance of MT expression induction. Therefore, it is necessary to use a technique that is capable of quantifying MT accurately in biological material. Due to the lack of sensitivity and selectivity of many commonly used techniques (Dabrio et al., 2002:125), it is necessary to develop a mass spectrometric based quantification technique to detect and quantify MT-2A selectively and accurately. For quantification of human MT-2A in biological material using a mass spectrometry-based method, a MT (MT-2A) standard similar to the native form but with a slightly different mass was required. Due to the lack of pure human MT standards and high cost of pure rabbit MT standards, it was decided to create a recombinant human MT-2A with different mass due to additional N-terminal amino acids. In addition, native human MT-2A is also required to develop and optimize an MS quantification technique in a future study. Therefore, pure (98 %) rabbit MT standard, which is highly similar to human MT-2A, was purchased to serve as a positive control for MS detection in this study and which can also be used to develop and optimize an MS quantification technique in a future study. An expression vector for human MT-2A was constructed with the use of recombinant DNA techniques. The correct construct was identified and characterized with PCR and verified by sequencing. This newly created expression vector was transformed into four E.coli BL21(DE3) strains to express a modified human recombinant MT-2A (MT-2AA) using induction with IPTG. This protein comprised of a full length human MT-2A sequence, but excluding the N-terminal Met and including an N-terminal His-tag. MT-2AA expression in the selected strains was extensively optimized and monitored with SDS-PAGE. Ecoli BL21 (DE3) CodonPlus-RIL cells proved to be the strain that expressed MT-2'A at the highest relative levels. Expressed MT-2'A was isolated and purified using a three step purification procedure which included heat treatment, metal chelating chromatography and RP-HPLC. Relative pure (70 %) MT-2'A was successfully obtained as confirmed with SDS-PAGE and mass spectrometry. Removal of the His-tag from MT-2'A with thrombin protease cleavage was, however, unsuccessful. In addition, it was observed that this protein was, compared to native commercially obtained MT-2A, unstable and after extensive purification still had a lower than required purity. It was concluded from this studies' results that, although it was successfully produced, this recombinant MT-2A protein would not be suitable as an internal standard for MS analysis of human MT-2A. On the other hand, rabbit MT-2E (as alternative) holds great promise as internal standard since it is stable and pure.Master

Metabolomics reveals the depletion of intracellular metabolites in HepG2 cells after treatment with gold nanoparticles

Studies on the safety of gold nanoparticles (GNPs) are plentiful due to their successful application in drug delivery and treatment of diseases in trials. Cytotoxicity caused by GNPs has been studied on the physiological and biochemical level; yet, the effect of GNPs (particularly gold nano-spheres) on the metabolome of living organisms remains understudied. In this investigation, metabolomics was used to comprehensively study the metabolic alterations in HepG2 cells caused by GNPs; and to investigate the role of representative GNP coatings. GNPs were synthesized, coated and characterized before use on HepG2 cell cultures. Cells were treated for 3 h with citrate-, poly-(sodiumsterene sulfunate)-, and poly-vinylpyrrolidone (PVP)-capped GNPs, respectively. The internalization of the different GNPs and their effect on mitochondrial respiration and the metabolome were studied. Results indicated that the PVP-capped GNPs internalized more and also caused a more observable effect on the metabolome. Conversely, it was the citrate- and poly-(sodiumsterene sulfunate) coated particles that influenced ATP production in addition to the metabolomic changes. A holistic depletion of intracellular metabolites was observed regardless of GNP coating, which hints to the binding of certain metabolites to the particle

Uncovering the metabolic response of abalone (Haliotis midae) to environmental hypoxia through metabolomics

Introduction Oxygen is essential for metabolic processes and in the absence thereof alternative metabolic pathways are required for energy production, as seen in marine invertebrates like abalone. Even though hypoxia has been responsible for significant losses to the aquaculture industry, the overall metabolic adaptations of abalone in response to environmental hypoxia are as yet, not fully elucidated. Objective To use a multiplatform metabolomics approach to characterize the metabolic changes associated with energy production in abalone (Haliotis midae) when exposed to environmental hypoxia. Methods Metabolomics analysis of abalone adductor and foot muscle, left and right gill, hemolymph, and epipodial tissue samples were conducted using a multiplatform approach, which included untargeted NMR spectroscopy, untargeted and targeted LC–MS spectrometry, and untargeted and semi-targeted GC-MS spectrometric analyses. Results Increased levels of anaerobic end-products specific to marine animals were found which include alanopine, strombine, tauropine and octopine. These were accompanied by elevated lactate, succinate and arginine, of which the latter is a product of phosphoarginine breakdown in abalone. Primarily amino acid metabolism was affected, with carbohydrate and lipid metabolism assisting with anaerobic energy production to a lesser extent. Different tissues showed varied metabolic responses to hypoxia, with the largest metabolic changes in the adductor muscle. Conclusions From this investigation, it becomes evident that abalone have well-developed (yet understudied) metabolic mechanisms for surviving hypoxic periods. Furthermore, metabolomics serves as a powerful tool for investigating the altered metabolic processes in abalone