Proteome Analysis of Subsarcolemmal Cardiomyocyte Mitochondria: A Comparison of Different Analytical Platforms

Mitochondria are complex organelles that play critical roles in diverse aspects of cellular function. Heart disease and a number of other pathologies are associated with perturbations in the molecular machinery of the mitochondria. Therefore, comprehensive, unbiased examination of the mitochondrial proteome represents a powerful approach toward system-level insights into disease mechanisms. A crucial aspect in proteomics studies is design of bioanalytical strategies that maximize coverage of the complex repertoire of mitochondrial proteins. In this study, we evaluated the performance of gel-based and gel-free multidimensional platforms for profiling of the proteome in subsarcolemmal mitochondria harvested from rat heart. We compared three different multidimensional proteome fractionation platforms: polymeric reversed-phase liquid chromatography at high pH (PLRP), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and isoelectric focusing (IEF) separations combined with liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), and bioinformatics for protein identification. Across all three platforms, a total of 1043 proteins were identified. Among the three bioanalytical strategies, SDS-PAGE followed by LC-MS/MS provided the best coverage of the mitochondrial proteome. With this platform, 890 proteins with diverse physicochemical characteristics were identified; the mitochondrial protein panel encompassed proteins with various functional roles including bioenergetics, protein import, and mitochondrial fusion. Taken together, results of this study provide a large-scale view of the proteome in subsarcolemmal mitochondria from the rat heart, and aid in the selection of optimal bioanalytical platforms for differential protein expression profiling of mitochondria in health and disease.     

Mass spectrometry of peptides and proteins from human blood

It is difficult to convey the accelerating rate and growing importance of mass spectrometry applications to human blood proteins and peptides. Mass spectrometry can rapidly detect and identify the ionizable peptides from the proteins in a simple mixture and reveal many of their post‐translational modifications. However, blood is a complex mixture that may contain many proteins first expressed in cells and tissues. The complete analysis of blood proteins is a daunting task that will rely on a wide range of disciplines from physics, chemistry, biochemistry, genetics, electromagnetic instrumentation, mathematics and computation. Therefore the comprehensive discovery and analysis of blood proteins will rank among the great technical challenges and require the cumulative sum of many of mankind's scientific achievements together. A variety of methods have been used to fractionate, analyze and identify proteins from blood, each yielding a small piece of the whole and throwing the great size of the task into sharp relief. The approaches attempted to date clearly indicate that enumerating the proteins and peptides of blood can be accomplished. There is no doubt that the mass spectrometry of blood will be crucial to the discovery and analysis of proteins, enzyme activities, and post‐translational processes that underlay the mechanisms of disease. At present both discovery and quantification of proteins from blood are commonly reaching sensitivities of ～1 ng/mL. © 2010 Wiley Periodicals, Inc., Mass Spec Rev 30:685–732, 2011     

Proteins in longissimus muscle of Korean native cattle and their relationship to meat quality

Proteomic profiling by two-dimensional gel electrophoresis and mass spectrometry of longissimus dorsi muscle tissue from Korean native cattle identified seven proteins that are differentially expressed in animals producing low and high quality grade beef. The expression level of alpha actin is increased in high quality grade beef and the expression levels of T-complex protein 1 (TCP-1), heat shock protein beta-1 (HSP27), and inositol 1,4,5-triphosphate receptor type1 (IP3R1), a new protein to be associated with meat quality, are increased in low quality grade beef. In particular, the quantitation of HSP27 and IP3R1 by both silver staining and immunoblotting correlated well with intramuscular fat content, meat tenderness, and free calcium levels. The data suggest that HSP27 and IP3R1 are potential meat quality biomarkers and their identification provides new insight into the molecular mechanisms and pathways associated with overall beef quality.     

乙醇胁迫对啤酒酵母生长及蛋白表达的影响

研究了乙醇胁迫对啤酒酵母生长的影响,应用光镊拉曼光谱(LTRS)技术获得并分析酵母单细胞拉曼光谱,从分子水平分析酿酒酵母细胞内的蛋白质组变化。结果表明乙醇可抑制酵母生长,随着乙醇浓度的提高,酵母细胞直径变小、稳定期推迟、生物量和蛋白质含量也呈减少趋势;通过光镊拉曼光谱分析可了解酵母细胞内的乙醇浓度和生化组成的相对含量等信息;在不同乙醇浓度下,采用SDS变性凝胶电泳(SDS-PAGE)共检测到22个明显的差异条带,并对其中7个差异条带进行质谱鉴定,发现这7个差异蛋白的功能主要与端粒稳定性、细胞自溶及代谢相关;不同乙醇浓度可诱导酵母特定蛋白质表达发生变化,如HSP104等蛋白质,说明这些蛋白质所参与的代谢途径在啤酒酵母乙醇耐性中具有普遍作用。     

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.     

The impact of genomics on crops for industry

The complete sequence of the genomes of two model plants is now available together with technology for the parallel analysis of thousands of genes, the proteins that they encode and the metabolites whose production they control. This review focuses on application of these technologies to engineer output characteristics of crops specifically relevant to non‐food use. Following a brief introduction to the resources available for genomic analysis, some examples are provided of how genomics is enabling both transgenic and non‐GM manipulation of agricultural materials such as carbohydrates, oils, fibres and speciality chemicals for use as industrial feedstocks. Copyright © 2006 Society of Chemical Industry