Today s proteomics
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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


1 introduction to proteomics

1. Introduction to 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?


Why proteomics

Why Proteomics?


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.


2 technology of proteomics

2. Technology of Proteomics


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


Technology now and then

Technology, Now and then


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


Common process for proteomics research

Common process for proteomics research


Today s proteomics

取材自台大微生物生化系莊榮輝教授網頁


A two dimensional electrophoresis 2 de

A. Two-dimensional Electrophoresis 2-DE


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


Run 2 de step by step

Run 2-DE, step by step


Run 2 de step by step1

Run 2-DE step by step


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.


Ief sample loading

IEF sample loading


2 de instruments 1st dimension

2-DE instruments, 1st dimension

Amersham Biosciences

Bio-Rad


Run 2 de step by step2

Run 2-DE step by step


2 de instruments 2nd dimension

2-DE instruments, 2nd dimension

Amersham Biosciences

23 x 20 cm

8 x 10 cm

16 x 16 cm


2 de instruments 2nd dimension1

2-DE instruments, 2nd dimension

Bio-Rad


Run 2 de step by step3

Run 2-DE step by step


Examples of 2 de results

Examples of 2-DE results

D

Healthy control

Patient

MS analysis

Digest to peptide fragment


B mass spectrometry

B. Mass spectrometry


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


Principal for maldi tof mass

Principal for MALDI-TOF MASS


Principal for maldi tof mass1

Principal for MALDI-TOF MASS


Two major types of maldi tof

Two major types of MALDI-TOF


Reflectron enhance the resolution

Reflectron enhance the resolution


Video for maldi tof

Video for MALDI-Tof


Video for maldi tof reflectron

Video for MALDI-Tof (reflectron)


Typical result from maldi tof

Typical result from MALDI-Tof


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


Quadrupole mass filter

Quadrupole Mass filter


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


Q tof mass spectrometer

Q-TOF Mass Spectrometer

Collision Cell


Nomenclature for cid fragments

Nomenclature for CID fragments


Cid mass spectrum

CID Mass Spectrum


Commercial lc ms ms

Commercial LC/MS/MS

API 4000, API

Q-Tof ultima API, Micromass

HCT plus, Bruker


Vedio for lc ms ms

Vedio for LC/MS/MS


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


3 application of proteomics

3. Application of Proteomics


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


Cell mapping

Cell mapping


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


Yeast 2 hybrid system contd

Yeast 2 hybrid system, contd.


Yeast 2 hybrid system contd1

Yeast 2 hybrid system, contd.


3 phage display system in vitro

3. Phage display system (in vitro)

Biopanning

Phage minor coat protein

SCIENCE VOL 298 18 OCTOBER 2002


Applications for phage display system

Applications for Phage display system


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


Protein microarrays ab arrays

Protein microarrays (Ab arrays)


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 contd

SELDI protein chip (Ciphergen), contd.


Video for seldi protein chip

Video for SELDI protein chip


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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