abclt Proteomics Introduction Klaus Herick Klaus Herickperkinelmer com
abclt Proteomics Introduction Klaus Herick Klaus. Herick@perkinelmer. com
Genome to Proteome abclt clinical markers!
abclt Genome Sequencing Haemophilus influenzae Mycoplasma genitalium Methanococcus jannaschii Synechocystis sp. PCC 6803 Saccharomyces cerevisiae 1. 7 Mbp 0. 6 Mbp Escherichia coli Bacillus subtilis Caenorhabditits elegans 4. 7 Mbp 4. 2 Mbp 100 Mbp p Arabidopsis thaliana Drosophila melanogaster 100 -150 Mbp p Homo sapiens 3000 Mbp p p 1995 p p 1998 p p p 2000 1. 8 Mbp 3. 6 Mbp 12. 1 Mbp
Why Proteome / Proteomics? p abclt Genomics: sequencing of genomes, i. e. the total DNA of human, plant, animals, insects, bacteria, etc. • Aim: find new drugs, understand cell actions p The hypothesis “One-Gene-one-Protein” (Beadle & Tatum) does not work. • One level down to the proteins, to the PROTEOME
Limitations of Genomics Challenge of Proteomics p co-translational modifications – differential m. RNA splicing p post-translational modifications (PTMs) – C-terminal GPI anchor – phosphorylation – sulfation – glycosylation – N-myristoylation – hydroxylation – N-methylation – carboxymethylation – signal peptidase site. . . . abclt
The Proteome evolves p abclt “The total PROTEin complement of a gen. OME. ” • M. Wilkins et al. Electrophoresis 1995, 16, 1090 -1094 p “The analysis of the entire protein complement expressed by a genome, or by a cell or tissue type. ”. ” • S. Fey, P. Mose-Larsen, Centre for Proteome Analysis, Odense, Denmark p Pharmaceutical proteomics: “Proteome approach to the interaction of drugs with biological systems. ” • L. Anderson, Large Scale Biology, MA, USA
Definition of Proteomics abclt p The study of protein propertieson a large scale to obtain a global, integrated viewof disease processes, cellular processes and networks at the protein level. p Properties: • • p expression level, post- & co-translational modifications interactions, structure, location time in cell cycle, development of cell extracellular & intracellular conditions Proteoms are dynamic.
Limitations of Genomics Challenge of Proteomics p p p abclt 30, 000 known human diseases some 100 s commercially rewarded only 2 % of human diseases result from a single gene defect • i. e. the absence of a protein product or presence of an altered protein p 98 % complex diseases like cancer are reflected in a modified protein network • PROTEOMICS = short-cut to understand this network
Why is Proteomics important? p Parallel analysis of multiple proteins • including their post-translational modifications p Discovery of disease-specific proteins • candidate targets • clinical markers p p p Analysis of signaling pathways, multi-protein complexes, dynamic of protein expression Molecular toxicology Mode-of-action studies abclt
What is Proteomics? A Glossary p abclt Proteome: • the total protein complement of a genome p Expression Proteomics: • quantitative expression of 1000 s of proteins: 2 D-gel/ image analysis central – surrogate markers, drug action, target validation p Cell Map Proteomics: • protein-protein interactions, potentially scaleable, MS central – functional analysis/target validation
Identification of Protein Spots p p p Cutting of the protein spots out of the gel Proteolytic digestion of the proteins into peptides Sequencing of peptides by Mass Spectroscopy • Matrix Assisted Laser Desorption Ionisation MALDI • Electrospray ES – Peptide Mass – Fingerprinting Bioinformatics Cell map construction abclt
What is meant by a Cell Map? abclt p Physical map - sum of protein/protein interactions, structure and location in cell p Perturbations to cell state cause translocation, dissociation, phosphorylation. . . “difference maps” p Sparse sampling right now! But will one day be at Proteome level. Need: Fluorescence staining!!!!
Proteomics, Databases and Things-in-between ORGANISM external influence sample preparation 2 D PAGE separation quantitative & qualitative gel image analysis single tissue whole cell protein identification 2 D gel databasis gene deletions & mutagenisis Genome Proteome genome sequencing post-translational modification studies protein database abclt genome sequence database
abclt Applications in Proteomics I. General items Markets, Applications, Customers
Expression Proteomics p Disease markers - correlation of protein level with disease • e. g. bladder cancer, cardiac rejection p Drug action - toxicology, mechanism • e. g. PPAR agonists, cyclosporin p Target validation- cellular pathways • e. g. diabetes abclt
Proteomics contribute to Target. Ident. and Validation abclt Knowledge of direct interactions partners, location, expression & modifications helps describe a protein’s function within the cell HUMAN GENOME DISEASE ASSOCIATIONS PUTATIVE TARGETS TOXICOLOGY LEAD OPTIMISATION EFFICACY/ P. O. C. TARGET FUNCTION IN CELL DEVELOPMENT HITS TO LEADS BIOLOGICALLY VALIDATED TARGETS HIT IDENTIFICATION Network of interactions and pathway expression predicts toxicology, required drug characteristics and side effects.
Clinical & Biomedical Applications p abclt Analysis of body fluids and tissue biopsies • identifying the origin of body fluid samples (spinal, cystic, serum, pleural, ascitic etc. ) or the origin of a tissue biopsy p Analysis of protein phenotypes and post translational modifications in fluid, cell or tissues • e. g. apolipoprotein E and J, haptoglobin p Examining the clonality of immunoglobulins which are not seen with conventional techniques • e. g. multiple scleorsis, haemolytic anaemia p Monitoring disease processes and protein expression • e. g. in inflammation, nutrition disorders, toxicology p Discovering new disease markers and/or pattern
Disease Diagnostic from Body Fluids and Biopsies abclt p Discovery of new disease markers and/or pattern p Creutzfeld-Jakob disease (CJD) • unusual proteins (no. 130 and 131) in spinal fluid = Tau chain p Non-invasive detection of acute rejection after solid organ transplantation • M. Dunn, National Heart and Lung Institute, London
Toxicology and Pharmaceuticals p abclt Multiple overlapping pathwaysare influenced by toxins or drug treatment • simultaneous identification, characterisation and quantification of numerous of gene products and their PTMs • massively parallel approach offered by Proteomics p Retinoic acid • • • p used in dermatology and onco-haematology retinoic acid acylation of proteins (PTM) detection of this protein retinoylation with proteomics Phosphorylation • “on” or “off” signals of biochemical pathways by kinases and phosphatases, complex networks p etc. .
Cancer p abclt Carcinogenic productsact similarly to pharmaceutical agents, affecting the PTMs and the level of expression of numerous proteins • oncogene product alterations & cell cycle specific protein modifications play important role in tumorgenesis and cancer progression p studies are going on in • brain, thyroid, breast, lung, colon, kidney, bladder, ovary, bone marrow
Biological Applications p abclt Proteome maps • • • starting point for major study in genomics questions of interest: How much of the genome is transcribed and translated in the living organism? • What effect different growth conditions have on the proteome? p studies are going on in • Eukaryotes like Humans – extensively modify their proteins by N- or C-terminal cleavages – decorate them with sugars and/or phosphates, sulfates. . . PTMs – Yeast Saccharomyces cerevisiae, . . – Fruit fly Drosophila melanogaster, . . – Plant Arabidopsis thaliana, . .
Biological Applications, cont. p abclt Tracking complexity • host-pathogen or host-parasite interactions – nitrogen fixation in legumes by association with bacteria (Rhizobium) to form nodules – infection of flax by flax rust p Immunogetic proteins • identifying proteins from infectious disease agents recognised by the immune system • vaccine candidate for microbial pathogens (e. g. Chlamydia trachomatisinfection) • allergy research: which grass pollens are most immunogenic? • identification of allergens (proteins) in Latex (gloves) by a 2 D PAGE run using Latex as a sample!
Biological Applications, cont. p abclt Improved agricultural products • engineering resistance to pathogens/parasites into various plants • most of these resistance mechanisms involve expression of toxic or protective proteins • discovery of new toxic or protective proteins • wool proteome project to investigate economically important characters like colour and fibre strength p Value added agricultural products • remanufacture low value products – proteinaceous whey as a by-product of cheese manufacture – investigation whether this whey can be used to grow recombinant bacteria for biotechnological production
Biological Applications, cont. p abclt Quality control • Is the hamburger mince sold as beef really beef or a mixture of beef and kangaroo or even buffalo? • proteome technology brings precision and definition to a new level in protein-based products p For further applications please refer to the Pro. XPRESS PIP on the intranet • forensic sciences • microbiology • epidemiology • taxonomy
abclt Applications in Proteomics II. Pro. XPRESS specific items sensitivity, dynamic range multiple (pre-) labelling high resolution
Challenges for 2 D Technology p p p abclt Whole cell extracts are too complex. How can complexity be reduced? Abundant & soluble proteins are easily characterized. How can rare and membrane proteins be found? Large format gels are difficult to handle and slow. Is there an alternative?
Challenges for 2 D Technology p abclt Abundant & soluble proteins are easily characterized. How can rare and membrane proteins be found? p p Increase sensitivity by fluorescent dye labelling! Use Pro. XPRESS for gel imaging!
Challenges remain with Gels p abclt Gels & staining limit S/N, dynamic range • today’s standard staining methods – silver staining: 10 to 70 -200 ng/spot (enough for MS) – Coomassie Blue: less sensitive – fluorescent dyes expectations: 0. 1 ng to 10 mg/spot (necessary for faint protein amounts) p Gels can be selective p Membrane proteins need special conditions p Low copy number proteins are missed – fluorescent dyes expectations: 0. 1 ng to 10 mg/spot (necessary for faint protein amounts)
Instrument design: 50 Micron spatial resolution 200 micron scan Progressive zoom 50 micron scan Progressive zoom 8 minute scan 16 minute scan abclt
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