1. APPLICATION OF PROTEOMICS AND GENOMICS
R.RAJAGOPAL M.Sc MARINE BIOTECHNOLOGY

2. Defination
PROTEOMICS:- comprehensive analysis of the identify ; interactions and location of proteins with in the cell.
GENOMICS:- comprehensive analysis of the complete genome sequences from different organism; used to assess evolutionary relations among species and to product the number and general types of proteins produced by an organism.

3. Types of proteomics and their application to biology

4. Proteomics - the challenge
The proteome :
- dynamic
- highly complex
- relative protein abundances in a cell can differ from 105 up to about 1010
Proteomics aims to analyze the levels and structure of all proteins present in a cell or a tissue including their post-translational modifications
Proteomics approaches include:
1) protein identification
2) protein quantitation or differential analysis
3) protein-protein interactions
4) post-translational modifications
5) structural proteomics

5. Proteomics - the classical definition
Two – dimensional gel electrophoresis 2D-PAGE of all lysate
Mass spectrometry +
generates global patterns of protein expression
 annotation
 large-scale visualization of differential
protein expression
Peptide mass fingerprinting
for protein identification
- High resolution 2D-PAGE first developed in 1975 (O’Farrell and Klose)
- Combination with biological mass spectrometry (1990s)
- Availability of genome sequences in databases
 central role in proteomic studies

6. First dimension: Isoelectrofocusing (IEF)
strip containing a pH gradient immobilized on a gel matrix (Garfin et al. 2000)

7. 9.5
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pI 4.5
pI 4.5
pI 4.5
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9.5
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Position of proteins before IEF
7.5
9.5
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9.5
pI 4.5
pI 3.5
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pI 3.5
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Position of proteins after IEF

8. Second dimension: SDS-PAGE
Proteins enter SDS-Polyacrylamide
gel and are dissolved according to
their molecular mass
Postelectrophoretic staining of the
proteins with:
Coomassie,
Silver,
Fluorescent stains (SYPRO Ruby) MW

9. 2D-PAGE based expression proteomics
Protein expression profiling: ~ 1000 proteins routinely detectable in a
2D-gel  global changes in the proteome readily detectable
posttranscriptional control mechanisms can influence protein expression
posttranslational modifications of a protein such as phosphorylation, glycosylation, processing of signal sequences or degradation can be visualized pI MW
SYPRO Ruby stained gel

10. Protein identification by peptide mass fingerprinting
from Graves and Haystead, 2002
The unknown protein is excised from a gel and converted to peptides by the action of a specific protease. The mass of the peptides produced is then measured in a mass spectrometer.
(B) The mass spectrum of the unknown protein is searched against theoretical mass spectra produced by computer-generated cleavage of proteins in the database.

11. Generation of protein expression reference maps
Link protein information with DNA sequence information from the genome projects,
comprehensive 2D-gel databases constructed for different cell types

12. 2D-PAGE based differential expression proteomics
2D-gel electrophoresis combined with mass spectrometry to get
qualitative and quantitative protein behavioural data
Most frequently used method in proteome analysis
from: Pandey and Mann, 2000

13. Workflow of differential expression proteomics
Sample preparation
Isoelectrofocusing (1.dimension)
Equilibration incl. reduction, alkylation
SDS-PAGE (2. dimension)
Staining
Imaging
Spot detection and matching
Normalization and quantification
Analysis
Cutting of selected spots
Trypsin digestion in-gel
Identification with mass spectroscopy
Database comparison
Steps to be practised during
the workshop

14. Difference in-gel 2D-PAGE system (DIGE)
Proteins are labeled prior to running the first dimension with up to three different fluorescent cyanide dyes (Unlu et al.1997)
Allows use of an internal standard in each gel which reduces gel-to-gel variation,
reduces the number of gels to be run
Adds 500 Da to the protein labelled
Additional postelectrophoretic staining needed
from Kolkman et al. 2005

15. Limitations and challenges of gel-based approaches
Dynamic range detectable on 2D-gels: 104, protein expression levels of a cell can vary between 105 (yeast) and even 1010(humans)
enrichment or prefractionation strategies needed to reach less abundant proteins
Resolution of 2D-gels has its limits
use narrow pH range gels and combine
Protein extraction and solubility during IEF can be a problem for poorly water-soluble proteins e.g. membrane proteins or nuclear proteins
Challenges for further development in gel-based proteomics:
improve sample preparation to be able to analyze extreme proteins (extremely basic or acidic, small or big, hydrophobic),
sensitivity, dynamic range, automation

16. 2D-gel based proteomics: the state-of-the-art versus the challenge
Proteome coverage
Copies/cell
- 106
- 20%
- 105
- 40%
- 104
- 60%
- 103
- 80%
- 102
- 101
- 100%

17. Other proteomic approaches
Liquid chromatography coupled to mass spectrometry
- Shotgun multidimensional protein identification technology MudPIT
- ICAT: isotope coded affinity tags (Gygi et al. 1999), cysteine biased
Peptide and protein arrays
Yeast two-hybrid system
Phage display

18. ICAT for measuring differential protein expression
ICAT consists of a biotin affinity group, a linker region
that can incorporate heavy (deuterium) or light (hydrogen)
atoms, and a thiol-reactive end group for linkage to
cysteines.
Proteins are labeled on cysteine residues with either the
light or heavy form of the ICAT reagent. Protein samples
are mixed and digested with a protease. Peptides labeled
with the ICAT reagent can be purified using avidin
chromatography.
ICAT-labeled peptides can be analyzed by MS to
quantitate the peak ratios and proteins can be identified
by sequencing the peptides with MS/MS.

19. 2D-PAGE in functional proteomics
Identify specific proteins in a cell that undergo changes in abundance, localization, or modification in response to a specific biological condition
Often combined with complementary techniques (protein biochemistry, molecular biology and cell physiology)
If:
- Monitoring quantitative changes in the biological process of
interest
- Quantitatively looking at protein modifications
Then:
2D-gel based proteomics is the method of choice

20. Zebrafish samples used for 2D-GE Experiment
Phenotypic differences
between untreated and
treated zebrafish embryos
Treatment startet at high oblong stage of development
Samples taken from 70-90% epiboly stage

21. Workflow of Differential Expression Proteomics
Sample preparation
Isoelectrofocusing (1.dimension)
Equilibration incl. reduction, alkylation
SDS-PAGE (2. dimension)
Staining
Imaging
Spot detection and matching
Normalization and quantification
Analysis
Cutting of selected spots
Trypsin digestion
Identification with mass spectroscopy
Database comparison

22. 2D-gel analysis software practical
-Introduction into the PDQuest software
package
-Demonstration of an comparative
analysis of gels from two different
sample types (wildtype, mutant)
-Practising PDQuest analysis of the gels
run during the workshop
-Compare:
a)control embryos vs.
ethanol treated embryos
b)control embryos vs.
selenium treated embryos
High performance analysis of 2D-gels

23. Genomics
Genomics Study of an organism's entire genome. The field includes intensive efforts to determine the entire DNA sequence of organisms and fine-scale genetic mapping efforts. Proteomics The study and Systematic analysis of protein expression of normal and diseased tissues that involves the separation, identification and characterization of all of the proteins in an organism

24. DNA contains the instructions needed for a living organisms grow and function.
DNA contains the instructions needed for a living organisms grow and function. It tells cells exactly what role they should play in the body. It holds instructions to make your: Heart cells beat. Limbs form in the correct place. Immune system fight infection. Digestive system digest your dinner.

25. Functional Genomics :
Functional Genomics Ambitious high-throughput approach to understanding the genome Highly automated Studies how genes and other sequences of DNA act in relation to the entire organism

26. Comparative Genomics :
Comparative Genomics Analyzing DNA sequence patterns of different organisms side by side to identify genes and determine functions Looking for similar genes in different species to determine possible relationships and genomic variations

27. STRUCTURAL GENOMICS
STRUCTURAL GENOMICS Sequence organization. Assigning loci to specific chromosomes. High-resolution chromosome maps. Physical mapping of genomes. Genome sequencing. Using genome maps in genetic analysis.

28. THANK YOU