Presentation is loading. Please wait.

Presentation is loading. Please wait.

Abclt Proteomics Introduction Klaus Herick

Similar presentations


Presentation on theme: "Abclt Proteomics Introduction Klaus Herick"— Presentation transcript:

1 abclt Proteomics Introduction Klaus Herick Klaus.Herick@perkinelmer.com

2 abclt Genome to Proteome

3 abclt Genome Sequencing p Haemophilus influenzae1.7 Mbp p Mycoplasma genitalium0.6 Mbp p Methanococcus jannaschii1.8 Mbp p Synechocystis sp. PCC68033.6 Mbp p Saccharomyces cerevisiae12.1 Mbp p Escherichia coli4.7 Mbp p Bacillus subtilis4.2 Mbp p Caenorhabditits elegans100 Mbp p Arabidopsis thaliana100-150 Mbp p Drosophila melanogaster100-150 Mbp p Homo sapiens3000 Mbp 1995 1998 2000

4 abclt Why Proteome / Proteomics? p Genomics: sequencing of genomes, i.e. the total DNA of human, plant, animals, insects, bacteria, etc. Aim: find new drugs, understand cell actionsAim: 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 PROTEOMEOne level down to the proteins, to the PROTEOME

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

6 abclt The Proteome evolves p “The total PROTEin complement of a genOME. ” M. Wilkins et al. Electrophoresis 1995, 16, 1090-1094M. 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, DenmarkS. 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, USAL. Anderson, Large Scale Biology, MA, USA

7 abclt Definition of Proteomics p 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. p Properties: expression level, post- & co-translational modificationsexpression level, post- & co-translational modifications interactions, structure, locationinteractions, structure, location time in cell cycle, development of celltime in cell cycle, development of cell extracellular & intracellular conditionsextracellular & intracellular conditions p Proteoms are dynamic.

8 abclt Limitations of Genomics Challenge of Proteomics p 30,000 known human diseases p some 100s commercially rewarded p only 2 % of human diseases result from a single gene defect i.e. the absence of a protein product or presence of an altered proteini.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 networkPROTEOMICS = short-cut to understand this network

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

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

11 abclt Identification of Protein Spots p Cutting of the protein spots out of the gel p Proteolytic digestion of the proteins into peptides p Sequencing of peptides by Mass Spectroscopy Matrix Assisted Laser Desorption Ionisation MALDIMatrix Assisted Laser Desorption Ionisation MALDI Electrospray ESElectrospray ES –Peptide Mass –Fingerprinting p Bioinformatics p Cell map construction

12 abclt What is meant by a Cell Map? 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!!!!

13 abclt Proteomics, Databases and Things-in-between ORGANISM Proteome Genome external influence gene deletions & mutagenisis whole cell single tissue sample preparation 2D PAGE separation quantitative & qualitative gel image analysis 2D gel databasis protein database genome sequence database protein identification post-translational modification studies genome sequencing ORGANISM

14 abclt Applications in Proteomics I. General items Markets, Applications, Customers

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

16 abclt Proteomics contribute to Target Ident. and Validation HUMAN GENOME DISEASE ASSOCIATIONS PUTATIVE TARGETS TARGET FUNCTION IN CELL BIOLOGICALLY VALIDATED TARGETS HIT IDENTIFICATION HITS TO LEADS LEAD OPTIMISATION TOXICOLOGY EFFICACY/ P.O.C.DEVELOPMENT Knowledge of direct interactions partners, location, expression & modifications helps describe a protein’s function within the cell Network of interactions and pathway expression predicts toxicology, required drug characteristics and side effects.

17 abclt Clinical & Biomedical Applications p 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 biopsyidentifying 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, haptoglobine.g. apolipoprotein E and J, haptoglobin p Examining the clonality of immunoglobulins which are not seen with conventional techniques e.g. multiple scleorsis, haemolytic anaemiae.g. multiple scleorsis, haemolytic anaemia p Monitoring disease processes and protein expression e.g. in inflammation, nutrition disorders, toxicologye.g. in inflammation, nutrition disorders, toxicology p Discovering new disease markers and/or pattern

18 abclt Disease Diagnostic from Body Fluids and Biopsies p Discovery of new disease markers and/or pattern p Creutzfeld-Jakob disease (CJD) unusual proteins (no. 130 and 131) in spinal fluid = Tau  chainunusual 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, LondonM. Dunn, National Heart and Lung Institute, London

19 abclt Toxicology and Pharmaceuticals p Multiple overlapping pathways are influenced by toxins or drug treatment simultaneous identification, characterisation and quantification of numerous of gene products and their PTMssimultaneous identification, characterisation and quantification of numerous of gene products and their PTMs massively parallel approach offered by Proteomicsmassively parallel approach offered by Proteomics p Retinoic acid used in dermatology and onco-haematologyused in dermatology and onco-haematology retinoic acid acylation of proteins (PTM)retinoic acid acylation of proteins (PTM) detection of this protein retinoylation with proteomicsdetection of this protein retinoylation with proteomics p Phosphorylation “on” or “off” signals of biochemical pathways by kinases and phosphatases, complex networks“on” or “off” signals of biochemical pathways by kinases and phosphatases, complex networks p etc....

20 abclt Cancer p 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 progressiononcogene 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 marrowbrain, thyroid, breast, lung, colon, kidney, bladder, ovary, bone marrow

21 abclt Biological Applications p Proteome maps starting point for major study in genomicsstarting point for major study in genomics questions of interest:questions of interest: How much of the genome is transcribed and translated in the living organism?How much of the genome is transcribed and translated in the living organism? What effect different growth conditions have on the proteome?What effect different growth conditions have on the proteome? p studies are going on in Eukaryotes like HumansEukaryotes 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 abclt Biological Applications, cont. p Tracking complexity host-pathogen or host-parasite interactionshost-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 systemidentifying proteins from infectious disease agents recognised by the immune system vaccine candidate for microbial pathogens (e.g. Chlamydia trachomatis infection)vaccine candidate for microbial pathogens (e.g. Chlamydia trachomatis infection) allergy research: which grass pollens are most immunogenic?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!identification of allergens (proteins) in Latex (gloves) by a 2D PAGE run using Latex as a sample!

23 abclt Biological Applications, cont. p Improved agricultural products engineering resistance to pathogens/parasites into various plantsengineering resistance to pathogens/parasites into various plants most of these resistance mechanisms involve expression of toxic or protective proteinsmost of these resistance mechanisms involve expression of toxic or protective proteins discovery of new toxic or protective proteinsdiscovery of new toxic or protective proteins wool proteome project to investigate economically important characters like colour and fibre strengthwool proteome project to investigate economically important characters like colour and fibre strength p Value added agricultural products remanufacture low value productsremanufacture 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 abclt Biological Applications, cont. p Quality control Is the hamburger mince sold as beef really beef or a mixture of beef and kangaroo or even buffalo?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 productsproteome technology brings precision and definition to a new level in protein-based products p For further applications please refer to the ProXPRESS PIP on the intranet forensic sciencesforensic sciences microbiologymicrobiology epidemiologyepidemiology taxonomytaxonomy

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

26 abclt Challenges for 2D Technology p Whole cell extracts are too complex. How can complexity be reduced? p Abundant & soluble proteins are easily characterized.How can rare and membrane proteins be found? p Large format gels are difficult to handle and slow. Is there an alternative?

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

28 abclt Challenges remain with Gels p Gels & staining limit S/N, dynamic range today’s standard staining methodstoday’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)

29 abclt Over expressed proteins as indicators of disease Missing proteins show genetic variances Typical experiments involve 200,000 such comparisons Applying Technology to Applications

30 abclt Instrument design: 50 Micron spatial resolution 200 micron scan Progressive zoom 8 minute scan 50 micron scan Progressive zoom 16 minute scan


Download ppt "Abclt Proteomics Introduction Klaus Herick"

Similar presentations


Ads by Google