KULIAH BIOTEKNOLOGI

PROTEOMIK

Dr. Oeke Yunita, S.Si., M.Si., Apt.

The birth of proteomics
 Importance of Proteins:

CATALYSTS
STRUCTURAL ELEMENTS
SIGNALS
RECEPTORS
KEY COMPONENTS OF THE MACHINERY
INVOLVED IN MANIPULATION OF DNA AND RNA
Proteins are the molecule tools for most cellular functions

TYPE FUNCTION EXAMPLE

Structural proteins Support Collagen, Elastin,
Keratin
Storage proteins Storage of amino acid Ovalbumin,
Casein
Transport proteins Transport of other Hemoglobin
substrate
Hormonal proteins Coordination of and Insulin
organisms activities
Receptors proteins Response of cell to Receptor in nerve
chemical stimuli transmit route
Contractile proteins Movement Actin, Myosin
Defensive proteins Protecton against Antibodys
disease
Enzymatic proteins Selective acceleraton Trypsin, ATPase,
of chemical reactions GAPDH
Diverse properties of proteins in a cell
 What is PROTEOMICS ?
The systematic analysis of the protein population in a
tissue, cell, or subcellular compartment.
"The analysis of the entire protein complement
expressed by a genome, or by a cell or tissue type.
Systematic determination of diverse properties of
proteins, including sequence, quantity, state of
modification, interactions with other proteins,
activity, subcellular distribution and structure.
 New avenue to study biology
Genome sequencing projects

DNA Comparative genomics

Transcription

RNA Functional genomics

Translation
Comparative and
Protein
functional proteomics

Metabolites Metabonomics
 Integration of Omics
Proteomics
Genomics
CGTCCAACTG Transcriptomics
ACGTCTACAA
GTTCCTAAGC
T

Bioinformatics

In vitro/ In vivo
cell-based assays
Integrated view of the
complex biological systems
validation
 PROTEOMICS
is inherently more challenging than
genomics/transcriptomics
Nucleic acids / genomics Proteins/proteomics
NAs can be amplified No
NAs show uniform behavior No
in purifying and handling
NAs are self-complimentary No
NAs have limited (but No
increasingly appreciated)
modifications
conditional
NAs are stable to drying,
spotting, etc.
 PROTEOMICS can answer
Protein identification
Protein Expression Studies
Protein Function
Protein Post-Translational Modification
Protein Localization and Compartmentalization
Protein-Protein Interactions
 General classification for
PROTEOMICS

Protein Expression comparison (beginning)

Quantitative study of protein expression between samples
that differ by some variable
Structural Proteomics (simulation)
Goal is to map out the 3-D structure of proteins and
protein complexes
Functional Proteomics (everything)
To study protein-protein interaction, 3-D structures,
cellular localization and posttranslational modifications
(PTMS) in order to understand the physiological function
of the whole set of proteome.
PROTEIN EXTRACTION
Current status of proteomic technologies

Two most applied technologies:

1. 2-D electrophoresis:
separation of complex protein mixtures
2. Mass spectrometry:
Identification and structure analysis
 GEL ELECTROPHORESIS

Denaturing SDS-PAGE
SDS gives uniform neg. charge
Separates proteins by size/mass
Non-denaturing
Separates based on charge and size/conformation
Often combined with Western blotting (using
antibodies specific for proteins of interest)
SDS-PAGE
 WESTERN BLOT

HIV lysate Proteins are

proteins are transferred
separated by (blotted) onto the
size using gel surface of a
electrophoresis membrane

Strips are
The membrane is incubated with
cut into strips patient serum
and antihuman
IgG conjugated
with an enzyme
(and
chromagen)
 Western Blot Banding

*
 2D Gels

Proteins are first separated according to isoelectric

point
pH gradient is applied (usually horizontally)
Each protein is charged except at its isoelectric point

Proteins are then denatured in sodium dodecyl

sulfate (SDS)
Unfolds them into straight molecules
Binds SDS molecules roughly proportional to the length of
the denatured protein
Electric current then separates the proteins according to
mass, similar to a regular agarose gel
2D-GE
 Protein Data Bank (PDB)
40 000
Global data collection (>30000 records)
35 000
www.pdb.org total
3D structures per year
30 000
experimental data
25 000 biological and chemical information
PDB enrties

Molecule Type
Method
20 000 Proteins NA Complexes Other Total

X-ray 27335 807 1270 85 29497

15 000
NMR 4421 674 118 17 5230
El. Microsc. 77 9 27 0 113
10 000
Other 70 4 3 0 77
Total 31903 1494 1418 102 34917
5 000

0
1976
1977
1978
1979
1980
1981
1982
1983
1984
1985
1986
1987
1988
1989
1990
1991
1992
1993
1994
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
 Clinical
Proteomics
Analyze the
proteome of both
diseased and healthy
cells

Find changes in:

Cell or tissues
Subcellular
structures
Protein complexes
Biological fluids
 Clinical Proteomics

Develop new biomarkers for disease diagnosis

and early detection

Identify new targets for drugs

Better evaluate the therapeutic effect of

possible drugs
 Differential Protein Expression Profiling

Identification of proteins in a sample as a function of a particular

state: differentiation, stage of development, disease state, response
to drug or stimulus

Normal Diseased
Which proteins are up
or down regulated ?

Biomarkers or drug targets

 Clinical
Proteomics

LUNG Ca
BIOMARKER
DISCOVERY
Overview of Proteomic Approach