Effect of temperature and pH on the post-mortem degradation of myofibrillar proteins

Incubation of bovine muscle at 37°C promoted a more drastic proteolytic change in myofibrillar proteins, as determined from sodium dodecyl sulphate polyacrylamide gels of isolated myofibrils, than incubation at 4°C. Degradation of myosin and troponin-T were the most noticeable changes at 37°C. Loss of α-actinin was observed in the 4°C incubated muscle. Ground bovine muscle incubated at pH 5·4 and 7 revealed that alterations in myosin and troponin-T were the most noticeable changes at ph 5·4 while troponin-T and α-actinin were altered at pH7. More troponin-T degradation occurred at pH 5·4 and 37°C than at pH7 and 4°C (similar to the degradation of myosin), indicating that troponin-T degradation in post-mortem muscle may be an indicator of overall myofibrillar proteolysis and not responsible for post-mortem tenderisation per se. Myosin degradation in the ground samples incubated at pH 5·4 and in whole samples incubated at 37°C was compared with the digestion of myofibrillar myosin by papain. Both pyrophosphate and Guba-Straub extracts of the 37°C and pH 5·4 treated samples confirmed that myosin degradation did occur to a much greater extent at this temperature and pH than at 4°C and pH7, and, in addition, at pH 5·4 frequent cleavage occurred near the papain sensitive site of myosin.     

A constrained binary knapsack approximation for shortest path network interdiction

A modified shortest path network interdiction model is approximated in this work by a constrained binary knapsack which uses aggregated arc maximum flow as the objective function coefficient. In the modified shortest path network interdiction problem, an attacker selects a path of highest non-detection probability on a network with multiple origins and multiple available targets. A defender allocates a limited number of resources within the geographic region of the network to reduce the maximum network non-detection probability between all origin-target pairs by reducing arc non-detection probabilities and where path non-detection probability is modeled as a product of all arc non-detection probabilities on that path. Traditional decomposition methods to solve the shortest path network interdiction problem are sensitive to problem size and network/regional complexity. The goal of this paper is to develop a method for approximating the regional allocation of defense resources that maintains accuracy while reducing both computational effort and the sensitivity of computation time to network/regional properties. Statistical and spatial analysis methods are utilized to verify approximation performance of the knapsack method in two real-world networks.     

Probability-based validation of protein identifications using a modified SEQUEST algorithm

Database-searching algorithms compatible with shotgun proteomics match a peptide tandem mass spectrum to a predicted mass spectrum for an amino acid sequence within a database. SEQUEST is one of the most common software algorithms used for the analysis of peptide tandem mass spectra by using a cross-correlation (XCorr) scoring routine to match tandem mass spectra to model spectra derived from peptide sequences. To assess a match, SEQUEST uses the difference between the first- and second-ranked sequences (ACn). This value is dependent on the database size, search parameters, and sequence homologies. In this report, we demonstrate the use of a scoring routine (SEQUEST-NORM) that normalizes XCorr values to be independent of peptide size and the database used to perform the search. This new scoring routine is used to objectively calculate the percent confidence of protein identifications and posttranslational modifications based solely on the XCorr value.     

Automated identification of amino acid sequence variations in proteins by HPLC/microspray tandem mass spectrometry

Amino acid sequence variations resulting from single-nucleotide polymorphisms (SNPs) were identified using a novel mass spectrometric method. This method obtains 99+% protein sequence coverage for human hemoglobin in a single LC-microspray tandem mass spectrometry (microLC-MS/MS) experiment. Tandem mass spectrometry data was analyzed using a modified version of the computer program SEQUEST to identify the sequence variations. Conditions of sample preparation, chromatographic separation, and data collection were optimized to correctly identify amino acid changes in six variants of human hemoglobin (Hb C, Hb E, Hb D-Los Angeles, Hb G-Philadelphia, Hb Hope, and Hb S). Hemoglobin proteins were isolated and purified, dehemed, (S)-carboxyami-domethylated, and then subjected to a combination proteolytic digestion to obtain a complex peptide mixture with multiple overlaps in sequence. Reversed-phase chromatographic separation of peptides was achieved on-line with MS utilizing a robust fritless microelectrospray interface. Tandem mass spectrometry was performed on an ion trap mass spectrometer using automated data-dependent MS/MS procedures. Tandem mass spectra were collected from the five most abundant ions in each scan using dynamic and isotopic exclusion to minimize redundancy. The spectra were analyzed by a version of the SEQUEST algorithm modified to identify amino acid substations resulting from SNPs.     

Proteome profiling-pitfalls and progress

In this review we examine the current state of analytical methods in proteomics. The conventional methodology using two-dimensional electrophoresis gels and mass spectrometry is discussed, with particular reference to the advantages and shortcomings thereof. Two recently published methods which offer an alternative approach are presented and discussed, with emphasis on how they can provide information not available via two-dimensional gel electrophoresis. These two methods are the isotope-coded affinity tags approach of Gygi et al. and the two-dimensional liquid chromatography-tandem mass spectrometry approach as presented by Link et al. We conclude that both of these new techniques represent significant advances in analytical methodology for proteome analysis. Furthermore, we believe that in the future biological research will continue to be enhanced by the continuation of such developments in proteomic analytical technology.     

Sex-and age-related differences in ceramide dihexosides of primary human brain tumors

Neutral glycolipids (NGL) are promising diagnostic markers of human gliomas, but differences in NGL with age and sex have not been examined. Previous work demonstrated that ceramide dihexosides (CDH) levels in mouse kidney are age- and sex-dependent, probably due to levels of sex hormones. We quantitated CDH in 181 human gliomas and found significant differences with sex and age, particularly menopause and male puberty. This emphasizes the importance of assessing results of studies on glycolipids in disease states with respect to age and sex in order to avoid erroneous conclusions concerning the relationship of glycolipid composition with diagnosis and pathogenesis.     

Influence of geotechnical factors on gas flow experienced in a UK longwall coal mine panel

Methane drainage has become an integral part of modern coal mining operations when gas emissions cannot be practically dealt with using conventional ventilation methods alone. Boreholes are often drilled above and below the caving zone and connected to a drainage range located along the return gate. This paper describes the construction and analysis of the results obtained from the two- and three- dimensional geomechanical and gas flow models experienced around an active deep UK longwall coal production panel. The models constructed using the commercial FLAC codes were undertaken to provide information to the ventilation engineers at the mine on the likely gas sources and gas flow paths into the face line areas and gate roads. This information allows for the correct design of the orientation, length and support of the boreholes to maximise gas capture. The paper describes the method adopted to derive the relevant rock mass parameters and the laboratory tests conducted to obtain the stress-dependent permeability of coal measure rock strata. A functional relationship is proposed whereby the intrinsic bulk permeability of a sheared coal measure rock may be predicted from the confining stress. A detailed discussion of the geomechanical modelling methodology and the derivation of the strata permeabilities and gas flow modelling adopted is presented. The output of the models is described and used to interpret the major potential gas sources and pathway into the workings.     

Characterization of ZnS grown by metal-organic chemical vapour deposition on GaAs(100) using t-butyl mercaptan and dimethylzinc

ZnS has been grown on GaAs(100) substrates by atmospheric pressure metal–organic chemical vapour deposition (MOCVD) using dimethylzinc (DMZn) and t-butyl mercaptan (t-BuSH). The effects of the reactant gas phase molar ratio and the growth temperature on the characteristics of the material grown have been investigated. The structural quality of the layer is demonstrated by X-ray rocking curve half-widths of less than 300 arcsec for the epilayers. There is little significant pre-reaction and the layers are of excellent surface morphology and layer uniformity.