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Today’s Proteomics. Session II. 台大生技教改暑期課程. What’s “proteomics” ?. "The analysis of the entire prote in complement expressed by a gen ome , or by a cell or tissue type.“

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Today s proteomics

Today’s Proteomics

Session II

台大生技教改暑期課程


What s proteomics
What’s “proteomics” ?

"The analysis of the entire protein complement expressed by a genome, or by a cell or tissue type.“

Wasinger VC et al Progress with gene-product mapping of the mollicutes: Mycoplasma genitalium. Electrophoresis 16 (1995) 1090-1094

  • Two MOST related technologies:

  • 2-D electrophoresis: separation of complex protein mixtures

  • Mass spectrometry: Identification and structure analysis


T oday s topics
Today’s topics

  • Introduction to Proteomics

  • Technology of Proteomics

  • Applications of Proteomics



Move over genome on to proteomics
Move over Genome…on to Proteomics

  • If the genome is the blueprint of an organism---who reads it?

  • At this point no computer algorithm can solve this

    • A computer can decode all 6 reading frames of an organism

    • A computer can compare these.. But then what?


Genomic dna
Genomic DNA

  • Structure

  • Regulation

  • Information

  • Computers cannot determine which of these 3 roles DNA play solely based on sequence… (although we would all like to believe they can)

Those are what we need

to know about protein


Definitions of proteomics
Definitions of Proteomics

Classical - restricted to large scale analysis of gene products involving only proteins

Inclusive - combination of protein studies with analyses that have genetic components such as mRNA, genomics, and yeast two-hybrid

  • Don’t forget that the proteome is dynamic, changing to reflect the environment that the cell is in


1 gene 1protein
1 gene = 1protein?

  • 1 gene is no longer equal to one protein

  • In fact, the definition of a gene is debatable..(ORF, promoter, pseudogene, gene product, etc)

  • 1 gene=how many proteins?



Differential protein expression
Differential protein expression

Stimulus

Stimulus

RNA

Protein

DNA

Transcription

Translation

x4

x1

Scenario 1: can be analyzed by microarray technology

RNA

Protein

DNA

Transcription

Translation

x3

Scenario 2: can be solved by proteomics technology

RNA

Protein

DNA

Transcription

Translation

x3


Co and post translational modification
Co- and Post-translational modification

Co-translational modified

Post-translational modified


Why proteomics summary
Why Proteomics? (summary)

  • Annotation of genomes, i.e. functional annotation

    • Genome + proteome = annotation

  • Protein Function

  • Protein Post-Translational Modification

  • Protein Localization and Compartmentalization

  • Protein-Protein Interactions

  • Protein Expression Studies

    • Differential gene expression is not the answer


Types of proteomics
Types of Proteomics

  • Protein Expression

    • Quantitative study of protein expression between samples that differ by some variable

  • Structural Proteomics

    • Goal is to map out the 3-D structure of proteins and protein complexes

  • Functional Proteomics

    • To study protein-protein interaction, 3-D structures, cellular localization and PTMS in order to understand the physiological function of the whole set of proteome.



Technolog y of proteomics
Technology of Proteomics

  • Separation of proteins

    • 1DE (convention)

    • 2DE (modern)

    • Multi-dimensional HPLC (modern)

  • Analysis of proteins

    • Edman Sequencing (convention)

    • Mass Spectrometry (modern)

  • Database utilization



Traditional rna technique northern blotting

Isolated RNA

Electrophoresis

Blotting

Labelling on probes !!

Developing

Probing

Traditional RNA technique : Northern blotting

1. Estimated time to get results: 2-3days

2. Expressed Gene (mRNA) checked: 1-8 species

3. Accuracy: Low to moderate


High throughput method microarray

Clustered

experiments

Clustered genes

High-throughput method: Microarray

Labelling on sample mRNA as probe

cDNA or oligonucleotide spotted on chips

data analysis

1. Estimated time to get results: 5-7 days

2. Expressed Gene (mRNA) checked: thousands

3. Accuracy: moderate to high


Traditional protein technique peptide sequencing
Traditional Protein technique: peptide sequencing

Cut desired band

Database searching for homolog

Peptide N terminal sequencing

  • Protein purification: necessary

  • Protein idetified: 1 per purified sample


High throughput technique 2d electrophoresis mass spectrometry
High throughput technique: 2D electrophoresis + Mass spectrometry

  • Protein purification: not necessary

  • Protein idetified: up to thousands per unpurified sample


In our course we will focus much on a 2 de b mass spectrometry
In our course, we will focus much on A. 2-DE B. Mass spectrometry





Major technique in proteomic research 2 d electrophoresis separation
Major technique in proteomic research:2-D electrophoresis (separation)

  • First dimension:

  • denaturing isoelectric focusing

  • separation according to the pI

  • 2. Second dimension:

  • SDS electrophoresis (SDS-PAGE)

  • Separation according to the MW

Interested spot

MS analysis

Digest to peptide fragment




The principle of ief
The principle of IEF

The IEF is a very high resolution separation method, and the pI of a protein can be measured.


Immobilized ph gradient strips ipg strips
Immobilized pH gradient strips (IPG strips)

  • Introduced by Gorg. A.

  • Ref: Gorg. A (1994), Westermeier (2001)

  • Dried gel strips can be stored at -20 to -80 from months to years.



2 de instruments 1st dimension
2-DE instruments, 1st dimension

Amersham Biosciences

Bio-Rad



2 de instruments 2nd dimension
2-DE instruments, 2nd dimension

Amersham Biosciences

23 x 20 cm

8 x 10 cm

16 x 16 cm




Examples of 2 de results
Examples of 2-DE results

D

Healthy control

Patient

MS analysis

Digest to peptide fragment



Major technique in proteomic research mass spectrometry analysis
Major technique in proteomic research:Mass Spectrometry (analysis)

Ion source

Ion separator

detector

Ion source: substance to ion gas

Mass analysis: according to mass/charge (m/z)

Detection: femtomole –attomole (10-15 – 10-18 mole)


Commonly used mass spectrometer in proteomics
Commonly used Mass Spectrometer in Proteomics

MALDI-TOF

Matrix Assisted Laser Desorption Ionization Time Of Flight

ESItandem MS (with HPLC, LC tandem MS or LC MS/MS)

  • Electro Spray Ionization MS Quadrupole


Commercial available maldi tof
Commercial available MALDI-Tof

Microflex ™, Bruker

Voyager DE-PRO™, ABI

MALDI micro™, Micromass









Peptide fingerprinting with maldi tof
Peptide fingerprinting with MALDI-TOF

Gel

Database

Protein

?

1

2

3

tryptic

stored data or theoretical? peptides

digestion

mass

spectrometry

compare:

?? is identical to ??


Esi quadrupole ms
ESI Quadrupole MS

  • Nano electrospray: >30 min spray time for 1 mL sample

  • Highly charged molecules are selected by ac modulation of transverse fields



Typical result from esi quadrupole ms
Typical result from ESI Quadrupole MS

From Eckerskorn in “Bioanalytik”, Lottspeich and Zorbas (Eds)


Triple quadrupole mass spectrometer
Triple Quadrupole Mass Spectrometer

CID: Collision Induced Dissociation

for acquiringMolecular weight and Structural information





Commercial lc ms ms
Commercial LC/MS/MS

API 4000, API

Q-Tof ultima API, Micromass

HCT plus, Bruker



Structure information resolved by lc ms ms
Structure information resolved by LC/MS/MS

  • Peptide sequenc

  • Post translational modifications

    • Proteolytic processing, truncation

      • Trypsin, Endoproteinase mapping

    • Acylation

      • Missing of N-terminal peptide

    • Phosphorylation

      • Differential mapping /phosphatase treatment

      • Fe 3+ -loaded IMAC column

    • Glycosylation

      • Neuraminidase treatment


Mass spectrometry analyzer i
Mass Spectrometry: Analyzer (I)

MALDI-TOF:

TYPE: TOF analyzer

Sample status: solid phase.

PROs: (1) easy

(2) fast

(3) high-through

(4) sensitive.

CONs: (1) only fingerprint of protein, no sequence information

(2) results is highly dependent on sample quality.


Mass spectrometry analyzer ii
Mass Spectrometry: Analyzer (II)

(LC) -tandem MS:

TYPE: (1) Triple quadrupole

(2) Ion trap

(3) Q-TOF

Sample status: Liquid phase.

PROs: (1) de novo sequencing data available.

(2) high sensitivity

CONs: (1) Lower through put

(2) pricey



Applications of proteomics
Applications of Proteomics

1. Protein Complexes Mining

2. Yeast Two-hybrid system ( in vivo PIP)

3. Phage display system (in vitro PIP)

4. Protein Arrays

5. SELDI protein chips (Ciphergen)


1 proteome complex mining
1. Proteome Complex Mining

  • A “functional” proteomics approach

  • A Proteome Mine Example

    • ATP is immobilized to beads in “protein kinase” conformation

    • Total protein is mixed the beads and the mixture “washed”

    • Remaining proteins isolated and identified…protein kinases, and purine dependent metabolic enzymes

  • Immobilize a putative drug to bead and test for a cellular ligand



2 yeast two hybrid system in vivo
2. Yeast Two-Hybrid System (in vivo)

  • Interaction of bait and prey proteins localizes the activation domain to the reporter gene, thus activating transcription.

  • Since the reporter gene typically codes for a survival factor, yeast colonies will grow only when an interaction occurs.

Activation

Domain

Prey

Protein

Reporter mRNA

Bait

Protein

Reporter mRNA

Reporter mRNA

Reporter mRNA

Binding

Domain

Reporter mRNA

Reporter Gene




3 phage display system in vitro
3. Phage display system (in vitro)

Biopanning

Phage minor coat protein

SCIENCE VOL 298 18 OCTOBER 2002



4 protein micro a rrays
4. Protein (micro) arrays

  • Another Functional Proteomics Approach

  • Same concept as a DNA Array

  • Has been published in a peer-reviewed journal

  • Too much expectation lies in with.


Protein microarray g macbeath and s l schreiber 2000 science 289 1760
Protein MicroarrayG. MacBeath and S.L. Schreiber, 2000, Science 289:1760

arrayIT TM

Spotting platform and protein microarray


What protein microarray can do
What protein microarray can do?

  • Protein / protein interaction

  • Enzyme / substrate interaction (transient)

  • Protein / small molecule interaction

  • Protein / lipid interaction

  • Protein / glycan interaction

  • Protein / Ab interaction

Reference: 1. G. MacBeath and S.L. Schreiber, 2000, Science 289:1760

2. H.Zhu et al, 2001 Science 293:2101

3. Ziauddin J and Sabatini DM, 2001 Nature 411:107



Face the real world
Face the real world

The true spot quality from real experiment


5 seldi protein chip ciphergen
5. SELDI protein chip (Ciphergen)

SELDI – surface enhanced laser desorption/ ionization

Protein chips




Seldi protein chip ciphergen application
SELDI protein chip (Ciphergen), application

Representative “raw” spectra and “gel-view” (grey-scale) of serum from a normal donor, and from patients with either BPH (benign prostate hyperplasia) or prostate cancer (PCA) using the IMAC3-Cu chip chemistry (Virginia Prostate Center).


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