Transcript Proteome
蛋白質體學 阮雪芬 Jul 18 & 25, 2003 Outline The characters of proteins Differences between protein chemistry & proteomics Why to study proteome Proteomics Introduction to proteomics Definitions of proteomics The major techniques in current proteomics Protein-protein interactions The characters of proteins DNA 和蛋白質合成的地方 Three Developments Formed the Foundation of the New Biology The growth of gene, expressed sequence tag (EST), and proteinsequence databases during the 1990s. The introduction of user-friendly, browser-based bioinformatics tools. The development of oligonucleotide microarray. Why to study proteome ? Why the Transcriptomic Analyses May Not Have Revealed All Proteins ? Lack of correlation between transcript and disease-associated protein levels Translocation of a protein in the disease state rather than simply differential levels of the transcript Novel/uncharacterized genes that are not highly represented within the "closed system" of a cDNA array Protein chemistry Individual proteins Complete sequence analysis Emphasis on structure and function Structural biology Proteomics Complex mixtures Partial sequence analysis Emphasis on identification by database matching Systems biology Introduction To Proteomics Genomics vs. Proteomics DNA Genome “Genomics” mRNA Proteins Cell functions Proteome “Proteomics” Generalized Proteomics Scheme Yarmush & Jayaraman, 2002 Definitions of Proteomics Definitions of Proteomics First coined in 1995 Be defined as the large-scale characterization of the entire protein complement of a cell line, tissue, or organism. Goal: -To obtain a more global and integrated view of biology by studying all the proteins of a cell rather than each one individually. Definitions of Proteomics The classical definition • Two-dimensional gels of cell lysate and annotation • Two-dimensional gels to visualize differential protein expression In the post-genomics era • • • • • • Protein Identification Post-translational modifications Determining Function Molecular Medicine Differential display by two-dimensional gels Protein-Protein Interactions Proteomics Origins In 1975, the introduction of the 2D gel by O’Farrell who began mapping proteins from E. coli. The first major technology to emerge for the identification of proteins was the sequencing of proteins by Edman degradation picomole MS technology has replaced Edman degradation to identify proteins femtomole How Proteomics Can Help Drug Development http://www.sciam.com.tw/read/readshow.asp?FDocNo=63&CL=18 Why is Proteomics Necessary? Having complete sequences of genome is not sufficient to elucidate biological function. A cell is normally dependent upon multitude of metabolic and regulatory pathways for its survival Modifications of proteins can be determined only by proteomic methodologies It is necessary to determine the protein expression level The localization of gene products can be determined experimentally Protein-protein interactions Proteins are direct drug targets. Jürgen Drews, 2000 Amgen（Applied Molecular Genetics） 成立日期：1980 年4 月8 日 CEO：Kevin W. Sharer 員工人數：6342 市場總值：698.4 億美元 產品項目：重組蛋白藥物 EPOGENR (Epoetin alfa) NEUPOGENR (Filgrastim) INFERGENR (Interferon alfacon-1) 資料來源：彭博資訊社、Zacks.com，6/14/2001 各項產品營業收入 資料來源：Amgen, Inc. The Major Techniques in Current Proteomics The Major Techniques in Current Proteomics Two-dimensional electrophoresis Mass Spectrometry IEF strip separation SDS-PAGE gel separation Protein sequencing Peptide mapping Others ICAT Yeast two hybrid assay Protein chips Two-dimensional Gel Approach Nature 2000, 405, 837-846 kDa 150 Image Matching Increase of 50% 70 60 Decrease of 50% 42 Unmatched spots Matched spots 10 pH 3.5 10 www.expasy.ch/ch2d http://www.expasy.ch/melanie/ Standard Proteome Analysis by 2DE-MS Mass Fingerprint Searching in http://www.expas ych/tools/peptide nt.html Current Opinion in Chemical Biology 2000, 4:489–494 Typical mass spectrometry scheme peptide mass mapping and tandem mass spectrometry Yarmush & Jayaraman, 2002 Ionization State as a Function of pH Two-dimensional Gel Electrophoresis First dimension: IEF (based on isoelectric point) Sample + acidic - basic SDS-PAGE (based on molecular weight) High MW Low MW Staining of Polyacrylamide Gels Silver staining Coomassie blue staining Sypro Ruby staining Image Analysis In-gel Digestion Enzyme: trypsin chymotrypsin Mass Spectrometric Identification of Proteins Mapping Peptide mass fingerprinting (PMF) or peptide mapping Trypsin * * * * * * * Protein Identification by MALDI-TOF 1. Cut protein spot 2. Protein digestion Protease 4. Spot onto MALDI chip 5. MALDI-TOF analysis 3. Peptide purification 6. Peptide fragment fingerprint How Does a Mass Spectrometer Work? Sample input Ionization Analyzer Detector How Does a Mass Spectrometer Work? • Sample Input: Gas Chromatography (GC), Liquid Chromatography (LC), Capillary Electrophoresis (CE), Solid crystal etc. • Ionization: Electrospray, Matrix-assisted Laser Desorption/Ionization (MALDI) etc • Analysis: quadrupole, time of flight(TOF), ion trap etc. • Detection: Ionization Electrospray Ionization Matrix-Assisted Laser Desorption/Ionization (MALDI) Matrix: - organic acids - benzoic acids Isotope-coded Affinity Tags (ICAT) Linker ICAT consists of a biotin affinity group, a linker region that can incorporate heavy or light atoms , and a thiolreactive end group for linkage to cysteines Avidin chromatography Biotin Thiolreactive end group A Strategy for Mass Spectrometric Identification of Proteins and Posttranslational Modifications NATURE, VOL 405, 15 JUNE 2000 Proteome chip ‘proteome chip’ composed of 6,566 protein samples representing 5,800 unique proteins, which are spotted in duplicate on a single nickelcoated glass microscope slide39. The immobilized GST fusion proteins were detected using a labeled antibody against GST. (MacBeath G. Nat Genet 2002 Dec;32 Suppl 2:526-32 ) Microarrays for Genomics and Proteomics DNA microarray are used for genetic analysis as well as expression analysis at the mRNA level. Protein microarrays are used for expression analysis at the protein level and in the expansive field of interaction analysis. Protein Microarrays In Medical Research Accelerate immune diagnostics. The reduction of sample volume----the analysis of multiple tumor markers from a minimun amount of biopsy material. New possibilities for patient monitoring during disease treatment and therapy will be develpoed based on this emerging technology. Clinical and Biomedical Applications of Proteomics An approach complementary to genomics is required in clinical situations to better understand epigenetic regulation and get closer to a "holisitic" medical approach. The potential clinical applications of 2-D PAGE, especially to the analysis of body fluids and tissue biopsies. Identifying the origin of body fluid samples or the origin of a tissue biopsy. Analyzing protein phenotypes and protein posttranslational modifications in fluid, cells, or tissues. Examining the clonality of immunoglobulins and detecting clones which are not seen with conventional techniques. Monitoring disease processes and protein expression. Discovering new disease markers and/or patterns in body fluids, cells, or tissues. Clinical applications of 2-D electrophoresis Body fluids Blood cell Plasma and serum Urine Cerebrospinal fluid Amniotic fluid Synovial fluid Saliva Sweat Tears Semen Solid tissue Heart Brain Thyroid Muscle Malignant diseases Tissue culture Malignant cells Bacterial proteins Young & Tracy Journal of Chromatography A, 698 (1995) 163-179 Protein-protein Interactions Protein-protein Interactions Introduction Mass Spectrometry Yeast Two-hybrid Assay Introduction Protein-protein interactions are intrinsic to every cellular process. Form the basis of phenomena DNA replication and transcription Metabolism Signal transduction Cell cycle control Secretion The Study of Protein-protein Interactions by Mass Spectrometry bait ? ? S14 ? ? SDS- PAGE * MASS * * * Yeast Two-hybrid System Useful in the study of various interactions The technology was originally developed during the late 1980's in the laboratory Dr. Stanley Fields (see Fields and Song, 1989, Nature). Yeast Two-hybrid System GAL4 DNAbinding domain GAL4 DNAactivation domain Nature, 2000 Yeast Two-hybrid System Library-based yeast twohybrid screening method Nature, 2000 Protein-protein Interactions on the Web Yeast http://depts.washington.edu/sfields/yplm/data/index.html http://portal.curagen.com http://mips.gsf.de/proj/yeast/CYGD/interaction/ http://www.pnas.org/cgi/content/full/97/3/1143/DC1 http://dip.doe-mbi.ucla.edu/ http://genome.c.kanazawa-u.ac.jp/Y2H C. Elegans http://cancerbiology.dfci.harvard.edu/cancerbiology/ResLabs/Vidal/ H. Pylori http://pim/hybrigenics.com Drosophila http://gifts.univ-mrs.fr/FlyNets/Flynets_home_page.html Pathway Software BIOCARTA http://biocarta.com/ Browse all pathway Pathway Software BIOCARTA Pathway Result 1: Enolase Pyruvate Cancer cells Acetyl-CoA ethanol lactate Glycolysis Pathway Result 2: Retinoic Acid Receptor RXR-alpha Useful BioWeb Site name URL Information available MOWSE http://srs.hgmp.mrc.ac.uk/cgi-bin/mowse Peptide mass mapping and sequencing ProFound http://prowl.rockefeller.edu/cgibin/ProFound Peptide mass mapping and sequencing PeptIdent http://www.expasy.ch/tools/peptident. Peptide mass mapping and sequencing PepSea http://195.41.108.38/PepSeaIntro.html Peptide mass mapping and sequencing MASCOT http://www.matrixscience.com/ Peptide mass mapping and sequencing PepFrag http://www.proteometrics.com/ Peptide mass mapping and sequencing Protein Prospector http://prospector.ucsf.edu/ Peptide mass mapping and sequencing FindMod http://www.expasy.ch/tools/findmod/ Posttranslational modification SEAQUEST http://fields.scripps.edu/sequest/ Uninterpreted MS/MS searching FASTA Search Programs http://fasta.bioch.virginia.edu/ Protein and nucleotide database searching Cleaved Radioactivity of http://fasta.bioch.virginia.edu/crp Protein phosphorylation site mapping Major Directions in Coming Proteomics Chemical proteomics (screens for activity and binding) Structural proteomics (target validation and development) Interaction proteomics (identification of new protein targets) Bioinformatics (annotation of the proteome) Major Directions in Coming Proteomics Protein structure prediction and modeling Assignment of protein structure to genomes Classifications of protein structures Drug discovery and development Types of Proteomics and Their Applications to Biology Proteomics Network Identify Proteins Protein-Protein Interactions Protein Functions Pathways Drug Discovery Structures Thank You!