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

Structural proteins

Support

Storage proteins
Transport proteins
Hormonal proteins
Receptors proteins
Contractile proteins
Defensive proteins
Enzymatic proteins

EXAMPLE

Collagen, Elastin,
Keratin
Storage of amino acid Ovalbumin,
Casein
Transport of other
Hemoglobin
substrate
Coordination of and Insulin
organisms activities
Response of cell to
Receptor in nerve
chemical stimuli
transmit route
Movement
Actin, Myosin
Protecton against
Antibodys
disease
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

Protein

Comparative and
functional proteomics

Translation

Metabolites

Metabonomics

Integration of Omics
Genomics

Proteomics
Transcriptomics

CGTCCAACTG
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
NAs can be amplified
NAs show uniform behavior
in purifying and handling
NAs are self-complimentary
NAs have limited (but
increasingly appreciated)
modifications
NAs are stable to drying,
spotting, etc.

Proteins/proteomics
No
No
No
No
conditional

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
separated by
size using gel
electrophoresis

The membrane is
cut into strips

Proteins are
transferred
(blotted) onto the
surface of a
membrane

Strips are
incubated with
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

30 000

PDB enrties

25 000

www.pdb.org
3D structures
experimental data
biological and chemical information

total
per year

Molecule Type
20 000

15 000

10 000

Method

Proteins

NA

Complexes

Other

Total

X-ray

27335

807

1270

85

29497

NMR

4421

674

118

17

5230

El. Microsc.

77

27

113

Other

70

77

Total

31903

1494

1418

102

34917

5 000

0
2006

2005

2004

2003

2002

2001

2000

1999

1998

1997

1996

1995

1994

1993

1992

1991

1990

1989

1988

1987

1986

1985

1984

1983

1982

1981

1980

1979

1978

1977

1976

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

Which proteins are up

or down regulated ?

Biomarkers or drug targets

Diseased

Clinical
Proteomics

LUNG Ca
BIOMARKER
DISCOVERY

Overview of Proteomic Approach