Sensitive quantification of omeprazole and its metabolites in human plasma by liquid chromatography-mass spectrometry

A sensitive method was developed for the simultaneous determination of omeprazole and its major metabolites 5-hydroxyomeprazole and omeprazole sulfone in human plasma by HPLC-electrospray mass spectrometry. Following liquid-liquid extraction HPLC separation was achieved on a ProntoSil AQ, C18 column using a gradient with 10 mM ammonium acetate in water (pH 7.25) and acetonitrile. The mass spectrometer was operated in the selected ion monitoring mode using the respective MH(+) ions, m/z 346 for omeprazole, m/z 362 for 5-hydroxy-omeprazole and omeprazol-sulfone and m/z 300 for the internal standard (2-{[(3,5-dimethylpyridine-2-yl)methyl]thio}-1H-benzimidazole-5-yl)methanol. The limit of quantification (LOQ) achieved with this method was 5 ng/ml for 5-hydroxyomeprazole and 10 ng/ml for omeprazole and omeprazole-sulfone using 0.25 ml of plasma. Intra- and inter-assay variability was below 11% over the whole concentration range from 5 to 250 ng/ml for 5-hydroxyomeprazol and from 10 to 750 ng/ml for omeprazole and omeprazole-sulfone. The method was successfully applied to the determination of pharmacokinetic parameters of esomeprazole and the two major metabolites after a single dose and under steady state conditions.     

Multiplex amplification of ancient DNA

This method is designed to assemble long, continuous DNA sequences using minimal amounts of fragmented ancient DNA as template. This is achieved by a two-step approach. In the first step, multiple fragments are simultaneously amplified in a single multiplex reaction. Subsequently, each of the generated fragments is amplified individually using a single primer pair, in a standard simplex (monoplex) PCR. The ability to amplify multiple fragments simultaneously in the first step allows the generation of large amounts of sequence from rare template DNA, whereas the second nested step increases specificity and decreases amplification of contaminating DNA. In contrast to current protocols using many template-consuming simplex PCRs, the method described allows amplification of several kilobases of sequence in just one reaction. It thus combines optimal template usage with a high specificity and can be performed within a day.     

Cysteine engineering of polyproteins for single-molecule force spectroscopy

Single-molecule methods such as force spectroscopy give experimental access to the mechanical properties of protein molecules. So far, owing to the limitations of recombinant construction of polyproteins, experimental access has been limited to mostly the N-to-C terminal direction of force application. This protocol gives a fast and simple alternative to current recombinant strategies for preparing polyproteins. We describe in detail the method to construct polyproteins with precisely controlled linkage topologies, based on the pairwise introduction of cysteines into protein structure and subsequent polymerization in solution. Stretching such constructed polyproteins in an atomic force microscope allows mechanical force application to a single protein structure via two precisely controlled amino acid positions in the functional three-dimensional protein structure. The capability for site-directed force application can provide valuable information about both protein structure and directional protein mechanics. This protocol should be applicable to almost any protein that can be point mutated. Given correct setup of all necessary reagents, this protocol can be accomplished in fewer than 10 d.     

An anthocyanin/polyphenolic-rich fruit juice reduces oxidative DNA damage and increases glutathione level in healthy probands

Oxidative cell damage is involved in the pathogenesis of atherosclerosis, cancer, diabetes and other diseases. Uptake of fruit juice with especially high content of antioxidant flavonoids/polyphenols, might reduce oxidative cell damage. Therefore, an intervention study was performed with a red mixed berry juice [trolox equivalent antioxidative capacity (TEAC): 19.1 mmol/L trolox] and a corresponding polyphenol-depleted juice (polyphenols largely removed, TEAC 2.4 mmol/L trolox), serving as control. After a 3-week run-in period, 18 male probands daily consumed 700 mL juice, and 9 consumed control juice, in a 4-week intervention, followed by a 3-week wash-out. Samples were collected weekly to analyze DNA damage (comet assay), lipid peroxidation (plasma malondialdehyde: HPLC/fluorescence; urinary isoprostanes: GC-MS), blood glutathione (photometrically), DNA-binding activity of nuclear factor-kappaB (ELISA) and plasma carotenoid/alpha-tocopherol levels (HPLC-DAD). During intervention with the fruit juice, a decrease of oxidative DNA damage (p<5x10(-4)) and an increase of reduced glutathione (p<5x10(-4)) and of glutathione status (p<0.05) were observed, which returned to the run-in levels in the subsequent wash-out phase. The other biomarkers were not significantly modulated by the juice supplement. Intervention with the control juice did not result in reduction of oxidative damage. In conclusion, the fruit juice clearly reduces oxidative cell damage in healthy probands.     

Dietary patterns and changes in body weight in women

Objective: Our objective was to examine the association between adherence to dietary patterns and weight change in women. Research Methods and Procedures: Women (51,670, 26 to 46 years old) in the Nurses’ Health Study II were followed from 1991 to 1999. Dietary intake and body weight were ascertained in 1991, 1995, and 1999. A Western pattern, characterized by high intakes of red and processed meats, refined grains, sweets and desserts, and potatoes, and a prudent pattern, characterized by high intakes of fruits, vegetables, whole grains, fish, poultry, and salad dressing, were identified with principal component analysis, and associations between patterns and change in body weight were estimated. Results: Women who increased their Western pattern score had greater weight gain (multivariate adjusted means, 4.55 kg for 1991 to 1995 and 2.86 kg for 1995 to 1999) than women who decreased their Western pattern score (2.70 and 1.37 kg for the two time periods), adjusting for baseline lifestyle and dietary confounders and changes in confounders over time (p < 0.001 for both time periods). Furthermore, among women who increased their prudent pattern score, weight gain was smaller (multivariate‐adjusted means, 1.93 kg for 1991 to 1995 and 0.66 kg for 1995 to 1999) than among women who decreased their prudent pattern score (4.83 and 3.35 kg for the two time periods) (p < 0.001). The largest weight gain between 1991 and 1995 and between 1995 and 1999 was observed among women who decreased their prudent pattern score while increasing their Western pattern score (multivariate adjusted means, 6.80 and 4.99 kg), whereas it was smallest for the opposite change in patterns (0.87 and ?0.64 kg) (p < 0.001). Discussion: Adoption of a Western dietary pattern is associated with larger weight gain in women, whereas a prudent dietary pattern may facilitate weight maintenance.     

Modular stop and go extraction tips with stacked disks for parallel and multidimensional Peptide fractionation in proteomics

Proteome complexity necessitates protein or peptide separation prior to analysis. We previously described a pipet-tip based peptide micropurification system named StageTips (STop and Go Extraction Tips), which consists of a very small disk of membrane-embedded separation material. Here, we extend this approach in several dimensions by stacking disks containing reversed phase (C(18)) and strong cation exchange (SCX) materials. Multidimensional fractionation as well as desalting, filtration, and concentration prior to mass spectrometry in single or tandem columns is described. C(18)-SCX-C(18) stacked disks significantly improved protein identification by LC-MS/MS for an E. coli protein digest and by MALDI-MS for a 12 standard protein digest. Sequential fractionation based on C(18)- followed by SCX material was also developed. This multidimensional fractionation approach was expanded to parallel sample preparation by incorporating C(18)-SCX-StageTips into a 96-well plate (StagePlate). Fractions were collected into other C(18)-StagePlates and desalted and eluted in parallel to sample well plates or MALDI targets. This approach is suitable for high throughput protein identification for moderately complex, low abundance samples using automated nanoelectrospray-MS/MS or MALDI-MS.     

Mild protease treatment as a small-scale biochemical method for mitochondria purification and proteomic mapping of cytoplasm-exposed mitochondrial proteins

Because of its importance in basic biology and medicine, great efforts are being devoted to unraveling of the genuine mitochondrial proteome, which is the dynamic protein complement that the organelle uses to maintain its structure and functionality. Several proteomic investigations have now clearly shown that all the purification approaches we have at our disposal suffer from the problem of co-purification; therefore, it is very difficult to distinguish novel mitochondrial proteins from those that are just contaminants of the preparation. The question is further complicated by the fact that the mitochondrial proteome depends on the tissue source. Density gradient centrifugation is the most widespread purification method for obtaining highly pure mitochondrial fractions. The main disadvantage of these methods is the low yield of purified mitochondria that precludes their use in low-scale purifications. Here, we have treated small aliquots of crude mitochondria from mouse liver and from cultured hepatocytes (HEPA1-6) with trypsin under mild proteolysis conditions and have evaluated the suitability of this reaction as a small-scale purification approach. The protease removed several cytoplasmic and endoplasmic reticulum proteins, together with a fraction of mitochondrial proteins that we hypothesize to be associated with the cytosolic face of the outer mitochondrial membrane. The peculiar topology of these mitochondrial proteins could be indicative of their functional roles. Finally, our study represents an application of advanced mass spectrometry technology to the evaluation of biochemical approaches for the treatment of mitochondria.     

Gel-free and gel-based proteomics in Bacillus subtilis: a comparative study

The proteome of exponentially growing Bacillus subtilis cells was dissected by the implementation of shotgun proteomics and a semigel-based approach for a particular exploration of membrane proteins. The current number of 745 protein identifications that was gained by the use of two-dimensional gel electrophoresis could be increased by 473 additional proteins. Therefore, almost 50% of the 2500 genes expressed in growing B. subtilis cells have been demonstrated at the protein level. In terms of exploring cellular physiology and adaptation to environmental changes or stress, proteins showing an alteration in expression level are of primary interest. The large number of vegetative proteins identified by gel-based and gel-free approaches is a good starting point for comparative physiological investigations. For this reason a gel-free quantitation with the recently introduced iTRAQ (isobaric tagging for relative and absolute quantitation) reagent technique was performed to investigate the heat shock response in B. subtilis. A comparison with gel-based data showed that both techniques revealed a similar level of up-regulation for proteins belonging to well studied heat hock regulons (SigB, HrcA, and CtsR). However, additional datasets have been obtained by the gel-free approach indicating a strong heat sensitivity of specific enzymes involved in amino acid synthesis.