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