Proteomik, Dr. Oeke Yunita, gasal 2014.pdf

8/16/2019 Proteomik, Dr. Oeke Yunita, gasal 2014.pdf

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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 othersubstrate Hemoglobin

Hormonal proteins Coordination of and

organism’s activities

Insulin

Receptors proteins Response of cell to

chemical stimuli

Receptor in nerve

transmit route

Contractile proteins Movement Actin, MyosinDefensive proteins Protecton against

disease

Antibodys

Enzymatic proteins Selective acceleraton

of chemical reactions

Trypsin, ATPase,

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 ofproteins, including sequence, quantity, state of

modification, interactions with other proteins,

activity, subcellular distribution and structure.



DNA

RNA

Protein

Transcription

Translation

Comparative genomics

Functional genomics

Comparative andfunctional proteomics

Genome sequencing projects

New avenue to study biology

Metabolites Metabonomics





In vitro/ In vivocell-based assays

Bioinformatics

Proteomics

TranscriptomicsCGTCCAACTGACGTCTACAAGTTCCTAAGCT

Genomics

Integrated view of the

complex biological systems

Integration of Omics

validation



PROTEOMICS

is inherently more challenging than

genomics/transcriptomics

Nucleic acids / genomics

– NA’s can be amplified

– NA’s show uniform behaviorin purifying and handling

– NA’s are self -complimentary

– NA’s have limited (butincreasingly appreciated)modifications

– NA’s 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 samplesthat 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 functionof 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

Proteins are

transferred

(blotted) onto the

surface of a

membrane

Strips are

incubated with

patient serumand antihuman

IgG conjugated

with an enzyme

(and

chromagen)

The membrane is

cut into strips



Western Blot Banding

*

*

*



2D Gels

• Proteins are first separated according to isoelectricpoint

– pH gradient is applied (usually horizontally)

– Each protein is charged except at it’s isoelectric point

• Proteins are then denatured in sodium dodecylsulfate (SDS)

– Unfolds them into straight molecules

– Binds SDS molecules roughly proportional to the length ofthe denatured protein

– Electric current then separates the proteins according tomass, similar to a regular agarose gel







2D-GE







Protein Data Bank (PDB)

Global data collection (>30000 records)

– www.pdb.org

– 3D structures

– experimental data

– biological and chemical information

0

5 000

10 000

15 000

20 000

25 000

30 000

35 000

40 000

1 9 7

6

1 9 7 7

1 9 7

8

1 9 7

9

1 9 8 0

1 9 8 1

1 9 8 2

1 9 8 3

1 9 8 4

1 9 8 5

1 9 8 6

1 9 8 7

1 9 8 8

1 9 8 9

1 9 9 0

1 9 9 1

1 9 9 2

1 9 9 3

1 9 9 4

1 9 9 5

1 9 9 6

1 9 9 7

1 9 9 8

1 9 9 9

2 0 0 0

2 0 0 1

2 0 0 2

2 0 0 3

2 0 0 4

2 0 0 5

2 0 0 6

P D B e

n r t i e s

total

per year

Proteins NA Complexes Other Total

X-ray 27335 807 1270 85 29497

NMR 4421 674 118 17 5230

El. Microsc. 77 9 27 0

113Other 70 4 3 0 77

Total 31903 1494 1418 102 34917

Molecule TypeMethod



• Analyze the

proteome of both

diseased and healthy

cells

• Find changes in:

– Cell or tissues

–

Subcellularstructures

– 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 ofpossible drugs

Clinical Proteomics







Differential Protein Expression Profiling

Identification of proteins in a sample as a function of a particularstate: differentiation, stage of development, disease state, response

to drug or stimulus

Normal DiseasedWhich proteins are upor down regulated ?

Biomarkers or drug targets





LUNG Ca

BIOMARKER

DISCOVERY

ClinicalProteomics



Overview of Proteomic Approach











Documents

Proteomik, Dr. Oeke Yunita, gasal 2014.pdf