TECHNIQUES INVOVED IN PROTEOMICS,GENOMICS,TRANSCRIPTOMICS…….

2. TECHNIQUES INVOVED IN PROTEOMICS,GENOMICS,TRANSCRIPTOMICS……. AIN US SABA 15

3. OUTLINE • BASIC PRINCIPLE OF ALL OMICs • GENOMICS • TRANSCRIPTOMICS • PROTEOMICS • METABLOMICS • APPLICATIONS • CONCLUSION

4. BASIC PRINCIPLE OF OMICS

5. GENOMICS • The actual term 'genomics' is thought to have been coined by Dr. Tom Roderick • Genomics is • study of the genomes of organisms. • determine • the entire DNA sequence of organisms • fine-scale genetic mapping • studies of intragenomic phenomena • aim of this genetic • pathway • functional information analysis response to the entire genome's networks

6. HISTORY • The first genomes to be sequenced, a virus and a mitochondrion, and were done by Fred Sanger. • In 1972, Walter Fiers and his team were the first to determine the gene for Bacteriophage MS2 coat protein. • The first free-living organism to be sequenced was that of Haemophilus influenzae (1.8 Mb) in 1995 • As of September 2007, the complete sequence was known of about • 1879 viruses • 577 bacterial • 23 eukaryote • A rough draft of the human genome was completed by the Human Genome Project in early 2001

9. Whole genome sequencing • cutting the chromosomes into large pieces,physical mapping • Several copies of the genome are randomly cut into pieces • Each fragments is inserted into a BAC-a bacterial artificial chromosome. • These pieces are fingerprinted to give each piece a unique identification tag that determines the order of the fragments. • Making M13 library &sequencing • These sequences are fed into PHRAP that looks for common sequences that join two fragments together. • Multiple copies of the genome are randomly shredded into pieces • Each 2,000 and 10,000 bp fragment is inserted into a plasmid • Both the 2,000 and the 10,000 bp plasmid libraries are sequenced. • Computer algorithms assemble the millions of sequenced fragments into a continuous stretch resembling each chromosome. BAC to BAC Sequencing Shotgun Sequencing

14. Trancriptomics • The transcriptome is the set of all RNA molecules, including mRNA, rRNA, tRNA, and other non-coding RNA produced in one or a population of cells. • Study of transcriptome is called transcriptomics. • Techniques • Expression profiling • DNA microarrays • SAGE(serial analysis of gene expression)

16. PROTEOMICS • The term "proteomics“ • was first coined in 1997 • to make an analogy with genomics • "proteome" is a blend of "protein" and "genome“ • coined by Marc Wilkins in 1994 • Proteomics is the large-scale study of proteins •  structures  • functions.

17. PROTEOME • Theproteome  • is the entire complement of proteins •  including the modifications made to a particular set of proteins • This will vary with • time • distinct requirements • or stresses, that a cell or organism undergoes.

18. COMPLEXITY OF PROBLEM • proteomics is considered the next step in the study of biological systems. • It is much more complicated than genomics • organism's genome is more or less constant •  proteome differs from cell to cell and from time to time. • done by mRNA analysis, • correlate with protein content • now known that mRNA is not always translated into protein • SO,Proteomicsconfirms the presence of the protein and provides a direct measure of the quantity present. • PTM

19. Protein Chemistry Assay Techniques • Affinity capture • Phosphorylation • Protein Binding • Receptors • Complexes • Mass Spectrometry • Accurate molecular weight • Gel Electrophoresis • Isoelectric point • Molecular weight • Liquid Chromatography • Fluorescence • Staining

20. TECHNIQUES • develop an antibody which is specific to that modification. • For glycosylation lectins • two-dimensional gel electrophoresis“ •  PROTOMAP which combines •  SDS-PAGE with shotgun proteomics

21. AFFINITY CAPTURE • Antibodies are adding to the protein mixture • Antibodies bind to proteins that have modified • Proteins of interest can be separated based on the modification.

23. 2D Gel-Electrophoresis • Protein separation • Molecular weight (Mw) • Isoelectric point (pI) • Staining • Birds-eye view of protein abundance

25. PROTOMAP • PRoteinTOpography and Migration Analysis Platform • Is combination of • SDS-PAGE with shotgun proteomics • is performed by • resolving control and experimental samples in separate lanes of a 1D SDS-PAGE gel • proteins in these bands are sequenced using shotgun proteomics. • Sequence information from all of these bands are bioinformatically integrated into a visual format called a peptograph • which plots gel-migration in the vertical dimension (high- to low-molecular weight, top to bottom) • sequence coverage in the horizontal dimension (N- to C-terminus, left to right).

27. Sample + _ Detector Ionizer Mass Analyzer Mass Spectrometer ElectronMultiplier(EM) Time-Of-Flight (TOF) Quadrapole Ion-Trap MALDI Electro-SprayIonization (ESI)

28. Enzymatic Digest and Fractionation LC/MS for Peptide Abundance

29. Liquid Chromatography LC/MS for Peptide Abundance Mass Spectrometry LC/MS: 1 MS spectrum every 1-2 seconds

30. The NMR Spectrometer =>

31. METABLOMICS • Newborn cousin to genomics and proteomics • study of chemical processes involving metabolites. • "systematic study of the unique chemical fingerprints that specific cellular processes leave behind • The metabolome • represents the collection of all metabolites in a biological cell, tissue, organ or organism, which are the end products of cellular processes • metabolic profiling can give an instantaneous snapshot of the physiology of that cell.

32. HISTORY • In 2005, first metabolite database(called METLIN) • In January 2007, scientists at the University of Alberta and the University of Calgary completed the first draft of the human metabolome. • They catalogued approximately • 2500 metabolites, • 1200 drugs • 3500 food components • This information, available at the Human Metabolome Database • www.hmdb.ca • over 50,000 metabolites have been characterized from the plant kingdom

35. APPLICATIONS • Toxicology • Nutrition • Medical profiling • Pharmacology • Diagnostics……….

36. CONCLUSION • High-dimensional biology (HDB) refers to the simultaneous study in health and disease • The fundamental premise is that the evolutionary complexity of biological systems renders them difficult to comprehensively understand using only a reductionist approach. • Such complexity can become tractable with the use of "omics" research. • This term refers to the study of entities in aggregate. • The two major advances that have made HDB possible are technological breakthroughs that allow simultaneous examination of thousands of genes, transcripts, and proteins, etc., with high-throughput techniques and analytical tools to extract information.