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Proteomics Introduction

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Title: Introduction Proteomics Author: Klaus Herick Last modified by: kh Created Date: 1/13/1998 9:07:30 AM Document presentation format: Overhead-Folien – PowerPoint PPT presentation

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Title: Proteomics Introduction


1
Proteomics Introduction
  • Klaus Herick
  • Klaus.Herick_at_perkinelmer.com

2
Genome to Proteome
3
Genome Sequencing
  • Haemophilus influenzae 1.7 Mbp
  • Mycoplasma genitalium 0.6 Mbp
  • Methanococcus jannaschii 1.8 Mbp
  • Synechocystis sp. PCC6803 3.6 Mbp
  • Saccharomyces cerevisiae 12.1 Mbp
  • Escherichia coli 4.7 Mbp
  • Bacillus subtilis 4.2 Mbp
  • Caenorhabditits elegans 100 Mbp
  • Arabidopsis thaliana 100-150 Mbp
  • Drosophila melanogaster 100-150 Mbp
  • Homo sapiens 3000 Mbp

1995
1998
2000
4
Why Proteome / Proteomics?
  • Genomics sequencing of genomes, i.e. the total
    DNA of human, plant, animals, insects, bacteria,
    etc.
  • Aim find new drugs, understand cell actions
  • The hypothesis One-Gene-one-Protein (Beadle
    Tatum) does not work.
  • One level down to the proteins, to the PROTEOME

BUT
5
Limitations of Genomics Challenge of Proteomics
  • co-translational modifications
  • differential mRNA splicing
  • post-translational modifications (PTMs)
  • C-terminal GPI anchor
  • phosphorylation
  • sulfation
  • glycosylation
  • N-myristoylation
  • hydroxylation
  • N-methylation
  • carboxymethylation
  • signal peptidase site........

6
The Proteome evolves
  • The total PROTEin complement of a genOME.
  • M. Wilkins et al. Electrophoresis 1995, 16,
    1090-1094
  • 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
  • Pharmaceutical proteomics Proteome approach to
    the interaction of drugs with biological
    systems.
  • L. Anderson, Large Scale Biology, MA, USA

7
Definition of Proteomics
  • The study of protein properties on a large scale
    to obtain a global, integrated view of disease
    processes, cellular processes and networks at the
    protein level.
  • Properties
  • expression level, post- co-translational
    modifications
  • interactions, structure, location
  • time in cell cycle, development of cell
  • extracellular intracellular conditions
  • Proteoms are dynamic.

8
Limitations of Genomics Challenge of Proteomics
  • 30,000 known human diseases
  • some 100s 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
  • 98 complex diseases like cancer are reflected
    in a modified protein network
  • PROTEOMICS short-cut to understand this network

9
Why is Proteomics important?
  • Parallel analysis of multiple proteins
  • including their post-translational modifications
  • Discovery of disease-specific proteins
  • candidate targets
  • clinical markers
  • Analysis of signaling pathways, multi-protein
    complexes, dynamic of protein expression
  • Molecular toxicology
  • Mode-of-action studies

10
What is Proteomics? A Glossary
  • Proteome
  • the total protein complement of a genome
  • Expression Proteomics
  • quantitative expression of 1000s of proteins
    2D-gel/ image analysis central
  • surrogate markers, drug action, target validation
  • Cell Map Proteomics
  • protein-protein interactions, potentially
    scaleable, MS central
  • functional analysis/target validation

11
Identification of Protein Spots
  • 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

12
What is meant by a Cell Map?
  • Physical map - sum of protein/protein
    interactions, structure and location in cell
  • Perturbations to cell state cause translocation,
    dissociation, phosphorylation...... difference
    maps
  • Sparse sampling right now! But will one day be at
    Proteome level. Need Fluorescence staining!!!!

13
Proteomics, Databases and Things-in-between
14
Applications in Proteomics
  • I. General items
  • Markets, Applications, Customers

15
Expression Proteomics
  • Disease markers - correlation of protein
    level with disease
  • e.g. bladder cancer, cardiac rejection
  • Drug action - toxicology, mechanism
  • e.g. PPAR agonists, cyclosporin
  • Target validation - cellular pathways
  • e.g. diabetes

16
Proteomics contribute to Target Ident. and
Validation
BIOLOGICALLY VALIDATED TARGETS
TARGET FUNCTION IN CELL
PUTATIVE TARGETS
HUMAN GENOME
DISEASE ASSOCIATIONS
LEAD OPTIMISATION
HIT IDENTIFICATION
HITS TO LEADS
TOXICOLOGY
Network of interactions and pathway expression
predicts toxicology, required drug characteristics
and side effects.
EFFICACY/ P.O.C.
DEVELOPMENT
17
Clinical Biomedical Applications
  • 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
  • Analysis of protein phenotypes and post
    translational modifications in fluid, cell or
    tissues
  • e.g. apolipoprotein E and J, haptoglobin
  • Examining the clonality of immunoglobulins which
    are not seen with conventional techniques
  • e.g. multiple scleorsis, haemolytic anaemia
  • Monitoring disease processes and protein
    expression
  • e.g. in inflammation, nutrition disorders,
    toxicology
  • Discovering new disease markers and/or pattern

18
Disease Diagnostic from Body Fluids and Biopsies
  • Discovery of new disease markers and/or pattern
  • Creutzfeld-Jakob disease (CJD)
  • unusual proteins (no. 130 and 131) in spinal
    fluid Tau ? chain
  • Non-invasive detection of acute rejection after
    solid organ transplantation
  • M. Dunn, National Heart and Lung Institute, London

19
Toxicology and Pharmaceuticals
  • Multiple overlapping pathways are 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
  • Retinoic acid
  • 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
  • etc....

20
Cancer
  • Carcinogenic products act 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
  • studies are going on in
  • brain, thyroid, breast, lung, colon, kidney,
    bladder, ovary, bone marrow

21
Biological Applications
  • 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?
  • 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, ....

22
Biological Applications, cont.
  • 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
  • Immunogetic proteins
  • identifying proteins from infectious disease
    agents recognised by the immune system
  • vaccine candidate for microbial pathogens (e.g.
    Chlamydia trachomatis infection)
  • allergy research which grass pollens are most
    immunogenic?
  • identification of allergens (proteins) in Latex
    (gloves) by a 2D PAGE run using Latex as a sample!

23
Biological Applications, cont.
  • 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
  • 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

24
Biological Applications, cont.
  • 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
  • For further applications please refer to the
    ProXPRESS PIP on the intranet
  • forensic sciences
  • microbiology
  • epidemiology
  • taxonomy

25
Applications in Proteomics
  • II. ProXPRESS specific items
  • sensitivity, dynamic range
  • multiple (pre-) labelling
  • high resolution

26
Challenges for 2D Technology
  • 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?

27
Challenges for 2D Technology
  • Abundant soluble proteins are easily
    characterized. How can rare and membrane proteins
    be found?
  • Increase sensitivity by fluorescent dye
    labelling! Use ProXPRESS for gel imaging!

28
Challenges remain with Gels
  • Gels staining limit S/N, dynamic range
  • todays 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)
  • Gels can be selective
  • Membrane proteins need special conditions
  • Low copy number proteins are missed
  • fluorescent dyes expectations 0.1 ng to 10
    mg/spot (necessary for faint protein amounts)

29
Applying Technology to Applications
Over expressed proteins as indicators of disease
Typical experiments involve 200,000
such comparisons
Missing proteins show genetic variances
30
Instrument design 50 Micron spatial resolution
200 micron scan Progressive zoom
8 minute scan
50 micron scan Progressive zoom
16 minute scan
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