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蛋白質體在生物醫學上之應用 Genomics • study of molecular organization of genomes, their information content, and gene products they encode • divided into three areas – structural genomics • physical nature of genomes – functional genomics • how genome functions – comparative genomics • compares genomes of different organisms Functional Genomics • determination of how genome works • three common approaches – genome annotation – study of RNA-level gene expression – study of protein-level gene expression Genome Annotation Nature Genetics 21:10-14, Supplement (1999) Evaluation of Protein-Level Gene Expression • proteome – entire collection of proteins that an organism produces • proteomics – study of the proteome Types of proteomic analysis • functional proteomics – study of function of cellular proteins, how they interact, and ways they are regulated • structural proteomics – using known protein structures to predict three-dimensional structures of other proteins and protein complexes Evaluation of Protein-Level Gene Expression… • Two-dimensional gel electrophoresis coupled with mass spectrometry (Gel-based proteomics) • Two-dimensional liquid chromatography (2-D LC) coupled with mass spectrometry (Chromatography-based proteomics) • SELDI Protein chip coupled with mass spectrometry • Antibody array Two-dimensional gel electrophoresis • when coupled with mass spectrometry, used to: – determine mass of each protein – determine amino acid composition or sequence of each protein – identify protein Pandey et.al., Nature 405 (2000) Silver stain Silver stain • Sensitivity: 1-2 ng • Use formaldehyde or glutaraldehyde as reductant and fixative • Aldehydes form Shiff’s bases with 1 and 2 amines • Not suitable for Edman degradation and mass I.D. • Omit aldehydes: lost sensitivity • Narrow linear range over saturation SYPRO Ruby • • • • Sensitivity: 1-2 ng Large linear range Speckle: mistaken as spots Metal contamination LavaPurple • Epicocconone Isolated from Epicoccum nigrum Enamine Primary amine, Lysine, Arginine, Two-dimensional difference in gel electrophoresis