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Department of Biotechnology and Bioinformatics
Laboratory of Nano-Biotechnology and Artificial Bioengineering
1
Proteomics
Chapter 1.
from genomics to proteomics Ⅱ
Department of Biotechnology and Bioinformatics
Laboratory of Nano-Biotechnology and Artificial Bioengineering
2
Functional genomics
Functional genomics: study of relations of genomics to biological functions at systems level
However, it cannot explain any more than individual parts of the biological systems
Two powerful technological tools
-Large-scale mutagenesis
-RNA interference (RNAi)
Department of Biotechnology and Bioinformatics
Laboratory of Nano-Biotechnology and Artificial Bioengineering
3
Large-scale mutagenesis
To establish the function of a gene, one can mutate it and observe the effect on phenotype.
Two approaches:
– genome-wide mutagenesis by homologous recombination (gene knockout) -> for small genomes
– genome-wide random mutagenesis by irradiation -> can produce more subtle point mutations
Department of Biotechnology and Bioinformatics
Laboratory of Nano-Biotechnology and Artificial Bioengineering
4
RNA interference (RNAi)
For functional genomics, RNAi is useful because the introduction of a double-stranded RNA homologous to an endogenous gene
results in the rapid destruction of any corresponding mRNA => silencing of that gene.
Department of Biotechnology and Bioinformatics
Laboratory of Nano-Biotechnology and Artificial Bioengineering
5
Transcriptomics
Transcriptomics: study of mRNA expression profiles based on DNA sequencing (genes are turned on/off depending on the condition and environment)
-sequence sampling : (from slide no. 23)
-cDNA microarray is a typical tool for transcriptomics
(1) mechanical spotting of cDNA molecules
(2) in situ oligonucleotide chip
Department of Biotechnology and Bioinformatics
Laboratory of Nano-Biotechnology and Artificial Bioengineering
6
Department of Biotechnology and Bioinformatics
Laboratory of Nano-Biotechnology and Artificial Bioengineering
7
Sequence sampling techniques for
Global analysis of gene expression
(1) Random sampling of cDNA libraries
(2) Analysis of EST (expressed sequence tag) databases
(3) Differential display PCR
(4) Serial analysis of gene expression (SAGE)
(5) Massively parallel signature sequencing (MPSS)
Department of Biotechnology and Bioinformatics
Laboratory of Nano-Biotechnology and Artificial Bioengineering
8
Serial Analysis of Gene Expression
(SAGE)
-B : Biotin
-Pink circle
:Streptavidin-coated beads
-Linkers A & B
: differ in sequence except that have 3’CATG hanging
-Downward triangle
: recognition site for anchoring enzyme (NlaIII)
- Extended arrow
: recognition site for type IIs restriction enzyme (tagging enzyme)
Department of Biotechnology and Bioinformatics
Laboratory of Nano-Biotechnology and Artificial Bioengineering
9
Need for proteomics
Transcriptomics, mutagenesis, RNAi dominated functional genomics because of the technology used; high-throughput clone generation and sequencing.
Nucleic acids are only information carriers. We can only indirectly infer protein functions from
these studies. Proteins are the building block of body and biological
functions. Thus, direct study of proteins is needed.
Department of Biotechnology and Bioinformatics
Laboratory of Nano-Biotechnology and Artificial Bioengineering
10
Importance of proteomics (1)
(1) The function of a protein depends on its structure and interactions.
(2) Mutations and RNA interference are coarse tools
for large-scale functional analysis. (3) The abundance of a given transcript may not reflect
the abundance of the corresponding protein (4) Protein diversity is generated post-transcriptionally
(alternative splicing).
Department of Biotechnology and Bioinformatics
Laboratory of Nano-Biotechnology and Artificial Bioengineering
11
Importance of proteomics (2)
(5) Protein activity often depends on post-translational modifications.
(6) The function of a protein often depends on its localization.
(7) Some biological samples do not contain NA’s.
(8) Proteins are the most therapeutically relevant molecules in the body.
Department of Biotechnology and Bioinformatics
Laboratory of Nano-Biotechnology and Artificial Bioengineering
12
Scope of proteomics (1)
(1) Sequence and structural proteomics: databases
-protein sequencing (chap3), bioinformatics (chap5),
storage, presentation, comparison and prediction of
structure (chap6)
(2) Expression proteomics: microarrays
-Fig 1.9; 2D-GE (chap2), Protein quantitation (chap4), analysis of post-translational modification (chap8), use of protein chip for analysis and quantitation (chap9)
Department of Biotechnology and Bioinformatics
Laboratory of Nano-Biotechnology and Artificial Bioengineering
13
Department of Biotechnology and Bioinformatics
Laboratory of Nano-Biotechnology and Artificial Bioengineering
14
Scope of proteomics (2)
(3) Interaction proteomics
-Fig 1.10; yeast two hybrid system (chap7)
(4) Functional proteomics: direct test of proteins
High-throughput functional analysis (chap9)
Department of Biotechnology and Bioinformatics
Laboratory of Nano-Biotechnology and Artificial Bioengineering
15
Challenges of proteomics
No single technique is suitable for every application
Lack of an amplification method such as PCR for NA’s
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