Differential recovery of peptides from sample tubes and the reproducibility of quantitative proteomic data

Differential recovery of peptides due to nonspecific adsorption can seriously compromise reproducibility and quality of proteomic data for peptide analyses by liquid chromatography-mass spectrometry (LC-MS). This study demonstrates large variations in reproducibility and quantitation of LC-MS data for peptides derived from tryptic digests of BSA upon storage in different sample tubes. Notably, we show that highly improved consistency and lower errors in quantitation of BSA tryptic peptides in replicate measurements is achieved with low-retention tubes compared to regular eppendorf tubes. Furthermore, qualitative differences in peptides detected by LC-MS were observed in the two types of storage tubes. These results illustrate the necessity for careful evaluation of storage vessels and conditions to minimize variability in sample quality for LC-MS experiments.     

Experimental investigation of the success of stocked, domestic brown trout and their effects on wild brown trout in upland and lowland rivers

The stocking of adult, domestic brown trout (DBT)( Salmo trutta L.) to supplement angling catches is a common practice. There is increasing concern, however, over the effects of such stockings on indigenous wild brown trout (WBT) populations. This paper describes an experimental field trial to explore the success of stocked DBT and their effects on WBT growth and abundance. DBT adults were stocked into upland rain‐fed and lowland spring‐fed streams. Three stocking treatments and a control were replicated six times for each of the two stream categories. In spring, baseline adult (defined as >200 mm LF) WBT densities were increased by 25, 50 and 100% using equal quantities of two different strains of DBT from two large suppliers. All DBT were measured, weighed and tagged before being stocked. The stocking procedure approximated to current stocking practice. A total of 48 500 m experimental sites were used. The abundance of WBT was estimated at each survey site in the summer prior to stocking via electrofishing and the depletion method. All WBT captured were measured and weighed. In addition, all WBT >100 mm were tagged. Habitat was assessed using the HABSCORE model. Post‐treatment monitoring was undertaken in the summer following stocking. Data will be presented regarding the effects of the stocking treatments on the growth and abundance of WBT as well as the strain‐dependent performance of DBT across all sites and on a river type‐specific basis. The suitability of using HABSCORE to formulate appropriate stocking densities will be discussed.     

Comparative genome analysis of potential regulatory elements in the ABCG5-ABCG8 gene cluster

The excretion of sterols from the liver and intestine is regulated by the ABCG5 and ABCG8 transporters. To identify potential regulatory elements, 152 kb of the human ABCG5-ABCG8 gene cluster was sequenced and comparative genome analysis was performed. The two genes are oriented in a head-to-head configuration and are separated by a 374-bp intergenic region, which is highly conserved among several species. Using a reporter construct, the intergenic region was found to act as a bidirectional promoter. A conserved GATA site in the intergenic region was shown by site-directed mutagenesis to act as a repressor for the ABCG5 promoter. The intergenic region was also shown to be partially responsive to treatment by LXR agonists. In summary, several potential regulatory elements were found for the ABCG5 and ABCG8 genes, and the intergenic region was found to act as a bidirectional promoter.     

Detecting low-abundance vasoactive peptides in plasma: Progress toward absolute quantitation using nano liquid chromatography-mass spectrometry

Profiling changes in the concentration of functionally related peptide hormones is critical to understanding the etiology of many diseases and therapies. We present novel data using nano liquid chromatography-mass spectrometry (LC-MS) to simultaneously measure a select group of vasoactive peptides (angiotensin, bradykinin, and related hormones) in 50-μl plasma samples, enabling repeated sampling in rodent models. By chromatographically resolving target peptides and using multiple reaction monitoring to enhance MS sensitivity, linear responses down to 10⁻¹⁷ mol were achieved. Purification of plasma peptides by either methanol precipitation or off-line high-performance liquid chromatography (HPLC) fractionation enabled the detection of endogenous peptides and revealed approaches for enhancing recovery. As proof of principle, seven vasoactive peptides were profiled before, during, and after acute angiotensin-converting enzyme (ACE) inhibition in an anesthetized rat. Of note was an apparent 10-fold increase in vasodilatory bradykinin that reversed after drug infusion but relatively minor changes in angiotensin II levels. Targeted MS analysis used to profile functionally related peptides or other analytes will greatly enhance our ability to define the sequence of events regulating complex and dynamic physiological processes.     

Studies on non-steroidal inhibitors of aromatase enzyme; 4-(aryl/heteroaryl)-2-(pyrimidin-2-yl)thiazole derivatives

Steroidal and non-steroidal aromatase inhibitors target the suppression of estrogen biosynthesis in the treatment of breast cancer. Researchers have increasingly focused on developing non-steroidal derivatives for their potential clinical use avoiding steroidal side-effects.Non-steroidal derivatives generally have planar aromatic structures attached to the azole ring system. One part of this ring system comprises functional groups that inhibit aromatization through the coordination of the haem group of the aromatase enzyme. Replacement of the triazole ring system and development of aromatic/cyclic structures of the side chain can increase selectivity over aromatase enzyme inhibition.In this study, 4-(aryl/heteroaryl)-2-(pyrimidin-2-yl)thiazole derivatives were synthesized and physical analyses and structural determination studies were performed. The IC₅₀ values were determined by a fluorescence-based aromatase inhibition assay and compound 1 (4-(2-hydroxyphenyl)-2-(pyrimidine-2-yl)thiazole) were found potent inhibitor of enzyme (IC₅₀:0.42?nM). Then, their antiproliferative activity over MCF-7 and HEK-293 cell lines was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Compounds 1, 7, 8, 13, 15, 18, 21 were active against MCF-7 breast cancer cells. Lastly, a series of docking experiments were undertaken to analyze the crystal structure of human placental aromatase and identify the possible interactions between the most active structure and the active site.     

Preparation of soybean seed samples for FT-IR microspectroscopy

Typical preparation of seed samples for infrared (IR) microspectroscopy involves imbibition of the seed for varying time periods followed by cryosectioning. Imbibition, however, may initiate germination even at 4° C with associated changes in the chemistry of the sample. We have found that it is possible to section seeds that are sufficiently hard, such as soybeans, on a standard laboratory microtome without imbibition. The use of dry sectioning of unimbibed seeds is reported here, as well as a comparison of different mounting media and modes of analysis. Glycerol, Tissue-Tek, and ethanol were used as mounting media, and the quality of the resulting spectra was assessed. Ethanol was the preferred mountant, because it dried quickly with no residue and thus did not interfere with the spectrum of interest. Analysis in transmission mode using barium fluoride windows to hold the samples was compared with transmission-reflection analysis with sections mounted on special infrared-reflecting slides. The two modes of analysis performed well in different regions of the spectrum. The mode of analysis (transmission vs. transmission-reflection) should be based on the components of greatest interest in the sample.     

The incipient stage in thrombin-induced fibrin polymerization detected by FCS at the single molecule level.

We used fluorescence correlation spectroscopy (FCS) to study the activation of fibrinogen by thrombin and the subsequent aggregation of fibrin monomers into fibrin polymers at a very low and at physiological fibrinogen concentrations. In the labeling procedure used the fibrinogen was randomly labeled and the label was bound to the fibrinopeptide A and/or to the part of fibrinogen which after activation takes part in fibrin formation. We measured a diffusion coefficient for fibrinogen of 2.48 x 10(-7) +/- 0.10 x 10(-7) cm2/s. After activation with thrombin both fibrinopeptide A and fibrin polymerization products could be demonstrated. From our findings we suggest a model for the formation of a three-dimensional network as two parallel processes, elongation and branching and that fibrin oligomers are not only intermediates in the polymerization process but also are substrates for branching.     

A comparison of clustering methods for river benthic community analysis

Four commonly used clustering methods (UPGMA, Ward Linkage,Complete Linkage and TWINSPAN) were compared in their abilitytorecognise the structure of three river macroinvertebratesdatasetswhich were pre-determined based on habitat and biologicalcharacteristics or chemical water quality of sampling sites.DCA,NMDS and ANOSIM were applied to the same datasets to providefurther information about data structure, and nonparametrictestswere also undertaken on major chemical variables to justifythepredeterminations. The modified Rand Index was used to measuretheagreement between a particular solution and the pre-determinedclassification. The results showed that Ward Linkage performedbestwhen its use was broadened and used with the CY DissimilarityMeasure, followed by TWINSPAN and Complete Linkage with UPGMAbeingleast successful. There was evidence to suggest that theeffectiveness of some clustering methods (e.g. UPGMA) may varyatdifferent clustering levels, and simulation techniques whichhavebeen used to assess clustering methods could leave somepropertiesof clustering methods unexamined.