| Skeletal muscle represents the most abundant tissue in the adult mammalian body and plays a central role in motility and the control of whole-body metabolism. So understanding of growth and development of skeletal muscle is one of the most important goals in meat production science and is also related to particular aspects of human medicine. In this study, the platform for porcine skeletal muscle proteomics research was established using two-dimensional electrophoresis (2-DE) and mass spectrometry (MS). The skeletal muscle proteome of different pig breeds and different development stages was analyzed. Screening and molecular biological analysis of differentially expressed proteins were carried. The aim is to understand the molecular mechanism of meat quality formation. The results are as follws:1. The experimental conditions of two-dimensional gel electrophoresis in porcine skeletal muscle was optimized by different experimental conditions, including different sample processing, different loading quantities, different pH IPG strips, different concentration in gels, different methods for gel staining, et al. The results show that the method of grinding in liquid nitrogen and ultrasonication is more appropriate than the method of grinding in liquid nitrogen for porcine skeletal muscle. The better map of two-dimensional gel electrophoresis can be obtained; low nucleic acid contamination and more protein spots can be separated from the method of grinding in liquid nitrogen and ultrasonication. Compared with previous studies, the 2-DE maps from this work have better resolution and repeatability.2. Western and indigenous Chinese pig breeds show obvious differences in muscle growth and meat quality, however, the underlying molecular mechanism remains unclear. In this study, proteome analysis of longissimus dorsi muscle between purebred Meishan and Large White pigs was performed by two-dimensional gel electrophoresis and mass spectrometry. A total of 25 protein spots were differentially expressed in the two breeds. The 14 identified proteins could be divided into 4 groups:energy metabolism, defense and stress, myofibrillar filaments, and other unclassified proteins. Quantitative real-time PCR was used to analyze the partly differentially expressed proteins in mRNA level, which revealed a positive correlation between the content of the proteins and their mRNA levels. We also analyzed the mRNA levels of myosin heavy chain isoforms using quantitative real-time PCR. The results indicated that IIA and IIX fibers were elevated in Meishan pigs, whereas the IIB fiber was more highly expressed in Large White pigs. To the best of our knowledge, this was the first proteomics-based investigation of total skeletal muscle protein in different pig breeds, and these results may provide valuable information for understanding the molecular mechanism responsible for breed specific differences in growth performance and meat quality.3. We used 2-DE method in combination with mass spectrometric.2-DE gels were generated in triplicate at four stages from embryo to mature. Using Image Master 2D Platinum software Version 6.0, protein spots were quantified on every gel at stages 65 dpc, 3d,60d and 120d. Only spots present in all three animals per group and under at least 2 of 4 conditions were taken into consideration. Statistical analysis (one-way ANOVA, P<0.01) revealed 78 proteins significantly altered in expression in 65 dpc compared to 3d (51 up-and 27 down-regulated); 70 altered proteins in 3d compared to 60d (49 up- and 21 down-regulated); and 40 alterations in 60d compared to 120d (18 up- and 22 down-regulated). In total,99 protein spots were altered in expression during development, which was consistently found between neighbouring stages points investigated. Therefore, most differential spots characterized 65dpc animals, whereas 60-day and 120-day adults exhibited similar patterns of expression. For subsequent MALDI-TOF-MS analysis, a total of 90 spots were successfully identified, and matched to 66 different proteins. To compare the functions of differential proteins, we collected the functional annotations (biological process and molecular function) of all identified proteins. It is evident that a large portion (25%) of the proteins has roles in metabolism. The second largest group of proteins (19%) was identified as myofibrillar regulatory protein, whereas 11% could not be classified on the basis of available information. Other major categories comprised of proteins involved in celluar defense and stress (14%), cytoskeleton (11%), signal transduction (6%), antioxidant and detoxification (8%) and transport (6%). The 66 differential proteins in control term placenta localized to the cytoplasm (43%), cytoskeleton (26%), membrane (6%), mitochondria (11%), nucleus (9%), and other (5%). Four proteins (desmin, AFP, vimentin and HSP90) were confirmed by immunoblotting. It is highly likely that these proteins are closely related with the 2-DE and MS results. Comparing with the 2-DE experiments, the results obtained with microarrays were significantly more comprehensive. To demonstrate whether protein expression levels changed during skeletal muscle development can be correlated with their mRNA expression levels, we assessed the differential expression at the RNA level of those proteins that were identified as relatively abundant in either stage. The expression patterns of most differential proteins showed a positive correlation with their gene expression at the transcript level during skeletal muscle development.4. Three differentially expressed proteins, ATP5B, ATP5A and 14-3-3ζwere further analyzed in mRNA expression levels of spatio-temporal, polymorphism detection and association analysis with meat quality traits.
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