Post on 17-Dec-2015
description
Proteomics Based Approaches for
Target Identification
Moazur Rahman
PhD in Molecular / Membrane
& Structural Biology
Proteomics Study of complete profile of proteins of the genome under a
defined state which involves analysis of changes in the
quantities and post-translational modification of proteins
Applications
Target Identification
Drug discovery Cancer research Biomarkers discovery Metabolic pathways Signal transduction etc.
The term proteome - 1994
Genomes, complete chromosomes and plasmids
sequenced to-date
Data were taken from NCBI website
Genomic research led to Proteomics discipline
Proteomics Clinical Applications (2007) 1: 417
Categories of proteomics
What is an amino acid ?
Structure of common L- amino acids
Peptide and peptide bond ?
Target Identification
Finding a protein of desired properties/interest
Approaches
2D-PAGE 2D-difference gel electrophoresis (2D-DIGE) Mass spectrometry Isotope coded affinity tagging Multi-dimensional protein identification technology Protein microarrays Chemical proteomics
Literature recommended
Karla K. Kopec, Donna Bozyczko-Coyne, Michael Williams (2005) Target
identification and validation in drug discovery: the role of proteomics,
Biochemical Pharmacology 69:11331139
Elzbieta Pastwa, Stella B. Somiari, Malgorzata Czyz and Richard I. Somiari (2007)
Proteomics in human cancer research,
Proteomics Clinical Applications 1: 417
Cho,William C.S (2007) Proteomic approaches to cancer target identification. Drug
Discovery Today: Therapeutic Strategies. 4:245-250
Standard proteomics workflow for target identification
http://en.wikibooks.org/wiki/Proteomics/Protein_Separations-_Electrophoresis/Two_Dimensional_Polyacrylamide_Gel_Electrophoresis(2D-PAGE)
Two dimensional gel electrophoresis
http://www.bioc.cam.ac.uk/uto/lilley.html
Detection using fluorescent label probes
SDS-PAGE
Component Function Structure
Acrylamide polymer
N,N'-methylene bisacrylamide Cross linker
Ammonium persulphate Free radical
generation
NNN'N'-
tetramethylethylenediamine
(TEMED)
Accelerator
/ stabilizer
SDS Solublizing
agent
Reservoir buffer
25 mM Tris, 190 mM Glycine,
0.1% SDS, pH 8.3
Sample buffer
Staining /Destaining buffer
Two Dimensional Gel Electrophoresis
2D-PAGE separation based on charge followed by size 2D-DiGE Cyanine or Alexa dye labeling
Minimal or saturation labeling
Mass spectrometry (MS) techniques
Electrospray Ionization- MS
Liquid chromatography-MS (LC-MS)
Matrix-assisted laser desorption/ionization time-of-flight MS
Surface enhanced laser desorption/ionization time of flight MS
Inductively coupled plasma MS
Capillary electrophoresis MS
Quadrupole ion trap - MS
Drug Discovery Today: Therapeutic Strategies Vol. 4, No. 4 2007
Isotope affinity tagging
Tags
-Thiol specific group-linker (Eight hydrogen atoms)-biotin (light version)
-Thiol specific group-linker (Eight deuterium atoms)-biotin (heavy version)
Protein sample
+ Light version tag Heavy version tag
Avidin based
purification
Avidin based
purification
Analyzed by MS
- 13C linker give high resolution
Limitation: Only cysteine containing peptides can be identified
Multi-dimensional protein identification technology
based on multidimensional LC/LC coupled with an MS/MS
-Chromatographic column:
-upstream packed with strong cation exchange resin
-down stream packed with reverse phase matrix
-Peptides eluting from LC column directly subjected to MS/MS analysis
-Can be used to identify around 1500 proteins
Protein microarrays
based on affinity protein and antibody
First approach
+
Protein immobilized
on a solid support
Target proteins are
visualized by
labeled antibodies
Second Third
Antibodies are
immobilized on a
solid support
Apply complex of
proteins
Target protein is
visualized by a
second antibody
Antibodies immobilized
on a solid support
Target proteins are
detected using
chemically labeled
complex protein
mixtures
Chemical Surfaces
(Hydrophobic) (Anionic) (Metal Ion) (Normal Phase) (Cationic)
ProteinChip Arrays
(Antibody - Antigen) (Receptor - Ligand) (DNA - Protein) (Immobilized enzyme)
Biological Surfaces
Chemical proteomics
based on use of small, drug like molecules
Small molecule biotinylation First strategy
Biotinylated compounds tethered
to resin (avidin agarose beads)
and packed into a column
Elute bound proteins and analyse
for potential drug target
Apply complex of
proteins
Second strategy
Immobilized complex mixture of
proteins on a chip
Resolve by 2DGE followed and
identify target by western blotting
using avidin-Horse reddish
peroxidase conjugate
Expose Biotinylated compounds
to protein chips
Limitations and challenges
Proteome heterogeneity different protein expression profile of the same genome
Low abundance such as membrane proteins
Reproducibility
Techniques compatibility