Proteome changes in bovine longissimus thoracis muscle during the first 48 h postmortem: shifts in energy status and myofibrillar stability.

Changes in the insoluble protein fraction of bovine longissimus thoracis muscle from eight Norwegian Red (NRF) dual-purpose young bulls during the first 48 h postmortem were investigated by two-dimensional gel electrophoresis (2DE) and matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry (MALDI-TOF MS/MS). Significant changes were observed in a total of 35 proteins, and of those, 26 were identified and divided into three different groups: metabolic enzymes, cellular defense/stress proteins, and structural proteins, according to their predicted function. The majority of the metabolic enzymes identified are involved in the energy metabolism of the cell, while the cellular defense/stress proteins can be related to regulation and stabilization of the myofibrillar proteins. Both easily soluble proteins as well as structural proteins were identified in the insoluble protein fraction. We have studied the changes in solubility during postmortem storage by comparing the postmortem changes in protein composition between the soluble and insoluble protein fractions. We have identified two metabolic enzymes (2,3-bisphosphoglycerat mutase and NADH dehydrogenase) and one protein involved in the stress responses/apoptosis of the cell (Hsp70) that have not been identified previously in the insoluble protein fraction. The occurrence of these easily soluble proteins in the insoluble protein fraction could be due to precipitation or aggregation, thereby going from a soluble to an insoluble state.

Proteome changes in wheat subjected to different nitrogen and sulfur fertilizations.

Controlling the quality of wheat for breadmaking is a major concern for the milling and baking industry. Wheat flour quality depends on both the genetic background and environmental factors during growth and storage. Amount and timing of application of fertilizer are factors that affect wheat quality. This study investigated the effect of different levels of nitrogen and sulfur on the tris-soluble and glutenin protein fractions by 2D-electrophoresis. Multivariate analysis was performed to study changes in the proteome pattern. In the tris-soluble fraction 20 proteins were changed in abundance due to S fertilization, whereas 16 proteins were changed in the glutenin protein fraction. It was found that induced sulfur deficiency during growth resulted in the most pronounced effect on protein composition. Understanding which proteins are affected by varying levels of fertilizers may help tailor specific traits in various wheat varieties.

Comparison of muscle proteome profiles in pure breeds of Norwegian Landrace and Duroc at three different ages.

We have used proteomics as a tool to unravel the changes in protein composition between two pure pig breeds and three age groups. Forty two female pigs of Norwegian Landrace and Duroc breed slaughtered at 6, 9 and 12 months age were included in the study. Each of the breeds was raised in separate farms and was slaughtered at the same day in a commercial abattoir. A sample from the adductor muscle was collected approximately 45min postmortem. Proteome analyses of the water soluble proteins using 2D electrophoresis showed that of the 1125 analyzed protein spots, 94 and 41 proteins are changed in abundance according to breed and age, respectively. A total of 63 changed proteins were identified by mass spectrometry. The identified proteins were classified as structural proteins, metabolic proteins, stress/defense proteins and other proteins. This demonstrates a difference in metabolism and muscle composition between breeds and age groups and shows that proteomics is a useful tool to uncover the molecular basis for physiological differences in muscles between pig breeds and age groups.

Two-dimensional LC-MS/MS in detection of peptides in hypothalamus of the rat subjected to hypoxic stress.

A capillary 2-D LC method coupled with IT MS has been used for separation and identification of peptides in rat hypothalamus. Animals of two different age groups (8 and 50 wk) were exposed to two different rates of CO(2 )in inhaled air to investigate the influence of different hypoxia/hypercapnia levels and their stress-related factor on the peptide excretion. Peptide compounds were fractionated (strong cation exchange chromatography), trapped, and separated (RP chromatography), and MS/MS mass spectra were used for identification. About 107 peptide compounds were identified and 88 of them were semiquantified. Among the characterized peptides, there were fragments from proteins such as proenkephalin A, proSAAS, prosomatostatin, prooxytocin, vasopressin, etc. Explorative principal component analysis (PCA) combined with hypothesis testing was applied to the obtained data to investigate the impact of age and hypoxic stress factors on the peptide pattern. Twenty-six peptides revealed significant differences in concentrations between the animal groups influenced by age and influx rate.

Pixel-based analysis of multiple images for the identification of changes: a novel approach applied to unravel proteome patterns [corrected] of 2-D electrophoresis gel images.

A novel approach for revealing patterns of proteome variation among series of 2-DE gel images is presented. The approach utilises image alignment to ensure that each pixel represents the same information across all gels. Gel images are normalised, and background corrected, followed by unfolding of the images to 1-D pixel vectors and analysing pixel vectors by multivariate data modelling. Information resulting from the data analysis is refolded back to the image domain for visualisation and interpretation. The method is rapid and suitable for automatic routines applied after the gel alignment. The approach is compared with spot volume analysis to illustrate how this approach can solve persistent problems like mismatch of protein spots, erroneous missing values and failure to detect variation in overlapping proteins. The method may also detect variation in the border area of saturated proteins. The approach is given the name pixel-based analysis of multiple images for the identification of changes (PMC). The method can be used for multiple images in general. Effects of pretreatment of the images are discussed.

Multivariate analysis of 2-DE protein patterns--practical approaches.

Practical approaches to the use of multivariate data analysis of 2-DE protein patterns are demonstrated by three independent strategies for the image analysis and the multivariate analysis on the same set of 2-DE data. Four wheat varieties were selected on the basis of their baking quality. Two of the varieties were of strong baking quality and hard wheat kernel and two were of weak baking quality and soft kernel. Gliadins at different stages of grain development were analyzed by the application of multivariate data analysis on images of 2-DEs. Patterns related to the wheat varieties, harvest times and quality were detected on images of 2-DE protein patterns for all the three strategies. The use of the multivariate methods was evaluated in the alignment and matching procedures of 2-DE gels. All the three strategies were able to discriminate the samples according to quality, harvest time and variety, although different subsets of protein spots were selected. The explorative approach of using multivariate data analysis and variable selection in the analyses of 2-DEs seems to be promising as a fast, reliable and convenient way of screening and transforming many gel images into spot quantities.

Application of proteomics to understand the molecular mechanisms behind meat quality.

The proteome is expressed from the genome, influenced by environmental and processing conditions, and can be seen as the molecular link between the genome and the functional quality characteristics of the meat. In contrast to traditional biochemical methods where one protein is studied at a time, several hundred proteins can be studied simultaneously. Proteomics is a promising and powerful tool in meat science and this is reflected by the increasing number of studies emerging in the literature using proteomics as the key tool to unleash the molecular mechanisms behind different genetic backgrounds or processing techniques of meat. Thus understanding the variations and different components of the proteome with regard to a certain meat quality or process parameter will lead to knowledge that can be used in optimising the conversion of muscles to meat. At present, there has been focus on development of techniques and mapping of proteomes according to genotypes and muscle types. In the future, focus should be more towards understanding and finding markers for meat quality traits. This review will focus on the methods used in the published proteome analyses of meat, with emphasis on the challenges related to statistical analysis of proteome data, and on the different topics of meat science that are investigated.

Challenges related to analysis of protein spot volumes from two-dimensional gel electrophoresis as revealed by replicate gels.

Assumptions that need to be considered prior to statistical analysis of protein spot volumes from two-dimensional gel electrophoresis (2-DE) data are studied using replicate gels of the same sample. The most important observation is that the data tables of protein spot volumes from 2-DE images contain a large number of missing values, which are not consistent with the presence or absence of the proteins. This implies both loss of information and problems for the subsequent statistical analysis. Challenges with 2-DE protein spot volumes are viewed in light of multiple gel comparisons and multivariate data analysis.