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proteomics era
Chatchai Tayapiwatana
Div of Clinical Immunology
Fac of Associated Medical Sciences
Chiang Mai University
Copyright 2008
After 3x,xxx human genes
Genome sequences (DNA only) by themselves are not
as useful as genomes that are fully annotated.
Need to know where the protein coding sequences
are, and what they do: this is a very big challenge in
bioinformatics.
Functions of many processes reside 3D proteins, and
the structure of proteins is known for only few
sequences.
Definitions:
Computational biology: an interdisciplinary
field that applies the techniques of computer
science and applied mathematics to biology
Bioinformatics: applies algorithms and
statistical techniques to biological datasets,
typically large numbers of DNA, RNA, or
protein sequences
Immunoinformatics: bioinformatics applied to
the study of immune system and its function
Immunology is essentially a
combinatorial science
multi-stepprocessing pathways
network-type interactions
complex signalling
mechanisms for modulation of immune
responses
Immunoinformatics
Database technology for storage,
analysis, and modelling of
immunological data
Sequence analysis and various
statistical tools
Computational models to facilitate
research in immunology
molecular level models
system level models
Basic Clinical
immunology immunology
Networks,
pathways, -omics
and systems
IMMUNOINFORMATICS
Artificial
intelligence Cell biology
Physics/
Databases Chemistry
Algorithms Maths/Stats
Why Immunoinformatics?
UsingBioinformatics to address
problems in Immunology
Application of bioinformatics to accelerate
immune system research has the potential
to deliver vaccines and address
immunotherapeutics.
Computational systems biology of immune
response
Disease alleviation
1. Genome screening - marker detection
2. Transcriptomics/Proteomics of diseased
state
3. Sequence analysis of antigens/markers
4. Structure analysis of antigens
5. T cell epitope analysis
6. Antibody epitope analysis
7. Vaccine design
OMICS
Genomics
Transcriptomics
Proteomics
Immunomics
Others ie. Metabolomics,
Interactomics, …..
What is Proteomics?
Proteomics – An emerging field of life science research that
uses High Throughput (HT) technologies to display, identify
and/or characterize all the proteins in a given cell, tissue or
organism (i.e. the proteome).
molecular biology
chromatography
2D electrophoresis
mass spectrometry
X-ray crystallography
NMR spectroscopy
robotics
computational biology
Genomics vs. Immunomics
Genomics: solving the genome puzzle
104 genes coding for 106 products
Immunomics: understanding immune
response
102-103 genes leading to >1012 products
Enormous diversity in immunomics has
implications for immune function and
modulation
An enormous diversity in human immune
system
>1013 MHC class I haplotypes (IMGT-HLA)
MHC MHC
Class Class
II I
Modelling MHC-binding peptides
Antigen processing pathway:
peptides, MHC, T-cells
1. Degradation of antigen
2. Peptide binding to MHC
3. Recognition of peptide-MHC complex by T-cells
Yewdell et al. Ann. Rev Immunol (1999)
Protein 3D Structure
Active Site
Docking Search
ca.300,000
compounds
Compound Database
Computational models can help
identify T cell epitopes
COMPUTER
COMPUTER
SCIENCE IMMUNOLOGY
IMMUNOLOGY
SCIENCE
Learning
Algorithms, COMPUTATIONAL IMMUNOLOGY Design of
Pattern Experiments,
Recognition,
COMPUTATIONAL Data
Adaptive
DATABASES
DATABASES COMPUTATIONAL
Memories, MODELS Interpretation
MODELS
Intelligent
Agents
COMPUTATIONAL
COMPUTATIONAL
EXPERIMENTS
EXPERIMENTS
Specialist databases
target
Cy5 Cy3
Cy5 Cy3
Cy5 Cy3
from RNA
Hybridise
Probe printed
onto slide
Gene 1 Gene 2 Gene 3 Gene 4 Gene 5
Slide Scanned
Spot intensities
analysed
ENZ ENZ
CLONDIAG
Principles of the Luminex Technology
The flow cell – where it all happens..
The
After
In
Free
assays
The phycoerythrin
excess
another
withstreptavidin-
wash
highisstep
immune-complex/
The
A
PE primary
biotinylated,
Microsphere
primary
Streptavidin
analyte
excited
bindsby antibody
numbers
to antibody
the
the
PE analyte
is
reporter
non-
is a
excess
added
microsphere
5.6mM
binds
specifictopolystyrene
for
reporter
the the isanalyte
then
specificantibody
bead is
toexcited
biotinylated
specifically
laser
theand
assay.
emits
by bound
reporter
The
the a******
with
conjugated
analyte
is added
two–to
antibodies
biotinylated
fluorescence fluorescent
no the
to
bind the
assay
reporter
whichto bead
the
isdyes
laser. Thean
incorporated
surface
crossreactivity
after another
by bead
into
wash
amine
with specific
it step.
in
other
Strep-PE
antibody
quantified
emmission in
binds
leading
bya non-specific
theto to
onea
is quantified of
different
coupling
analytes
the
manner.
signal
Luminex reaction
occurs.
ratios.
byfour
theamplification.
available
reader. and
luminex sites.
the
bead identified.
Phage display technique
1976 Tonegawa ----> Ig genes rearrangement
1991 McCafferty (Winter group) and
Lerner + Burton (Scripps) ---> Phage Display
Phage display Ab libraries
Bacterial expression, E. coli
mRNA purification, cDNA amplification kits
PCR for Ig H and L chain genes
Phagemid vector; pComb3H
M13 filamentus helper phage
XL-1 BLUE E. coli host
Phagemid vector, pComb3HSS
Concenus Design of an Ankyrin
Revue:
Current Opinion in
Biotechnology 2005,
16:459–469
Towards protein chips?
Domaine Fn 3
Intrabody Therapy