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

The term proteome - 1994

Applications
Target Identification
Drug discovery
Cancer research
Biomarkers discovery
Metabolic pathways
Signal transduction etc.

Genomic research led to Proteomics discipline

Genomes, complete chromosomes and plasmids
sequenced to-date

Data were taken from NCBI website

Categories of proteomics

Proteomics Clinical Applications (2007) 1: 417

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

Two dimensional gel electrophoresis

http://en.wikibooks.org/wiki/Proteomics/Protein_Separations-_Electrophoresis/Two_Dimensional_Polyacrylamide_Gel_Electrophoresis(2D-PAGE)

Detection using fluorescent label probes

http://www.bioc.cam.ac.uk/uto/lilley.html

SDS-PAGE
Component

Function

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

Structure

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

Second
Antibodies are
immobilized on a
solid support

Third
Antibodies immobilized
on a solid support

+
Target proteins are
visualized by
labeled antibodies

Apply complex of
proteins

Target protein is
visualized by a
second antibody

Target proteins are

detected using
chemically labeled
complex protein
mixtures

ProteinChip Arrays

Chemical Surfaces

(Hydrophobic)

(Anionic)

(Cationic)

(Metal Ion)

(Normal Phase)

Biological Surfaces

(Immobilized enzyme)

(Antibody - Antigen)

(Receptor - Ligand)

(DNA - Protein)

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

Apply complex of
proteins

Elute bound proteins and analyse

for potential drug target

Second strategy
Immobilized complex mixture of
proteins on a chip

Expose Biotinylated compounds

to protein chips

Resolve by 2DGE followed and

identify target by western blotting
using avidin-Horse reddish
peroxidase conjugate

Limitations and challenges

Proteome heterogeneity different protein expression profile of the
same genome

Low abundance such as membrane proteins

Reproducibility

Techniques compatibility