Vijay protiomics current

PROTEOMICS

Proteomics – in view of other fields

Proteome

Proteomics

Proteome

Proteomics

Genome

Genomics

Genome

Genomics

Structural proteome

Structural proteome

Database

Application

Database

Application

Molecular evolution

Molecular evolution

DataMining

DataMining

Chemistry

Cell Biology

Imaging

Biotechnology

Nanotechnology

Protein Science

Biochemistry

What is a Proteome?What is a Proteome?

All the proteins expressed by a genome.

“Functional Proteome” = all the proteins produced by a specific cell in a single time frame.

Why is the Proteome Why is the Proteome Important?Important? It is the proteins within the cell that:

Provide structure Produce energy Allow communication Allow movement Allow reproduction

Proteins provide the structural and functional framework of cellular life

Hemoglobin

Antibody

Epidermal Growth Factor Receptor

Proteins are molecular machines of many shapes and sizes

Rubisco

http://www.rcsb.org/pdb/explore.do?structureId=1AGW

What is Proteomics?What is Proteomics?

Proteomics refers to the systematic analysis of protein profiles of entire cells, tissues, organisms, or species.

It represents the protein counterpart to the analysis of gene function.

Proteomics vs GenomicsProteomics vs Genomics

DNA sequence does not predict if the protein is in an active form

RNA quantitation does not always reflect corresponding protein levels

Multiple proteins can be obtained from each gene (alternative splicing)

Genomics cannot predict post-translational modifications and the effects thereof

DNA/RNA analysis cannot predict the amount of a gene product made (if and when)

DNA/RNA analysis cannot predict events involving multiple genes

Why is Proteomics Why is Proteomics Important?Important?

Identification of proteins in normal and disease conditions

Investigating epidemiology and taxonomy of pathogens Analysis of drug resistance

Identification of pathogenic mechanisms Reveals gene regulation events involved in disease

progression

Promise in novel drug discovery via analysis of clinically relevant molecular events

Contributes to understanding of gene function

Proteomics: Proteomics: fundaments and fundaments and applicationsapplications

Susana CristobalBioinformatik, 4p. KTH

0utline0utline

1.1. The virtue of proteomicsThe virtue of proteomics

2.2. Two dimensional gel Two dimensional gel electrophoresiselectrophoresis

3.3. Detection technologyDetection technology

4.4. Identification methodsIdentification methods

5.5. Application of proteomicsApplication of proteomicsSusana CristobalBioinformatik, 4p. KTH

Proteome, the end product of the genome.

Proteome: dynamic entity.

Protein world: study of less abundant proteins

Transcriptomics: insufficient short-cut to study most functional aspects of genomics


Sampling biological materialSampling biological material

Factors:

In cell cultures: growth phase, culture conditions,strain employed

Cell from multicellular organisms: stage of differentiation

Tissues from biopsy: isolation of homogenous cell populations


Phases of a large scale analytical processPhases of a large scale analytical process

1. Separation of biomolecules of interest

• Extraction of protein sample

Cell culture

Organelle isolation

• Two dimensional electrophoresis

2. Molecular characterization

• Detection technology

• Identification of proteins

• Differential expression profiles


Different strategies for proteome purification and Different strategies for proteome purification and protein separation for identification by MSprotein separation for identification by MS

A. Separation of individual proteins by 2-DE.

B. Separation of protein complexes by non-denaturing 2-DE (BN-PAGE)

C. Purification of protein complexes by immuno-affinity chromatography and SDS-PAGE.

D. Multidimensional chromatography.

E. Organic solvent fractionation for separation of complex protein mixtures of hydrobhobic membrane proteins.

(van Wijk, 2001, Plant Physiology 126, 501-508)


http://www.plantphysiol.org/content/vol126/issue2/images/large/pp0616687001.jpeg

Organelle can be separated by differential velocity centrifugation

Rupture plasma membrane to prepare tissue/ cell homogenates:

• high speed blender

• Sonication

• tissue homogenize

• osmotic shock( Molecular cell biology.

Lodish. Fig 5-23)


Partially purified organelles can be better separated by equilibrium density gradient centrifugation

(Lodish fig 5-24)

How can you assess purity?

Organelle-specific markers

• Cytchrome c, mitochondria

• Catalase, peroxisome

• Ribosome, rough ER

• Esterase, microsomes


Organelle-specific antibodies are useful in preparing highly purified organelles

(Lodish fig 5-26)

Protein A o G is a bacterial molecule that selectively binds Igs.

Protein A:Ab:Ag complex collected and dissociated to release organelle.


Two-dimensional gel electrophoresisTwo-dimensional gel electrophoresis

Solubilization of proteins in 2D electrophoresis

Two dimensional electrophoresis with immobilized pH gradients

Detection of proteins on 2DE


Solubilization of proteins in two Solubilization of proteins in two dimensional electrophoresisdimensional electrophoresis

There is no universal solubilization protocol.

urea-reducer-detergent mixtures usually achieve disruption of disulfide bonds and non-covalent interactions.

Goals:

Breaking macromolecular interaction (disulfide bonds).

Preventing any artefactual modification of polypeptides in the solubilization medium.

Removal of substances that may interfere with 2DE.

Keeping proteins in solution during 2DE process. Susana Cristobal

Bioinformatik, 4p. KTH

Sample bufferSample buffer

Chaotropes: 8M Urea 2M Thiourea/ 7M Urea

Surfactants: 4% CHAPS 2 % CHAPS / 2 % SB-14

Reducing agents: 65 mM DTE (dithioerythritol) 100 mM DTT ( dithiothreitol) 2 mM tributyl phosphine

Ampholytes 2%Susana CristobalBioinformatik, 4p. KTH

How many quantities of samples can be How many quantities of samples can be loaded in one IPG strip?loaded in one IPG strip?

Identification of membrane proteins

(Govorun, 2002)Susana CristobalBioinformatik, 4p. KTH

Two-dimensional gel electrophoresisTwo-dimensional gel electrophoresis

Internet-sites: http://www.weihenstephan.de/blm/deg/manual/manualwork2html02testp6

htm and http://www.expasy.ch/ch2d/protocols. Susana CristobalBioinformatik, 4p. KTH

http://www.weihenstephan.de/blm/deg/manual/manualwork2html02testp6

http://www.weihenstephan.de/blm/deg/manual/manualwork2html02testp6

First dimension: IEFFirst dimension: IEFImmobilized pH gradients (IPGs)Immobilized pH gradients (IPGs)

IPG principle:

pH gradient is generated by a limited number (6-8) of well defined chemicals (immobilines) which are co-polymerized with the acrylamide matrix.

IPG allows the generation of pH gradients of any desired range ( broad, narrow, ultra-narrow) between pH 3 and 12.

sample loading capacity is much higher.

This is the method of choice for micropreparative separation or spot identification.


How many quantities of samples can be How many quantities of samples can be loaded in one IPG strip?loaded in one IPG strip?

(18 cm)(18 cm)Analytical run: 50-100 g Micropreparative runs: 0.5-10 mg


Two dimensional Two dimensional electrophoresiselectrophoresis

Running conditionsRunning conditionsSampleSample::Caenorhabditis elegansCaenorhabditis elegans

IEF: dry strips pH = 4-7IEF: dry strips pH = 4-7Hydratation conditionsHydratation conditions:urea, :urea, thiourea, CHAPS, DTT, thiourea, CHAPS, DTT, ampholytes, iodoacetamine. ampholytes, iodoacetamine. Passive 15h.Passive 15h. Isoelectrofocussing:Isoelectrofocussing:

200v 1h200v 1h500v 1h500v 1h1000v 1h1000v 1h5000v 3h5000v 3h

SDS-PAGE: 12%SDS-PAGE: 12%Silver stainingSilver staining


Detection technologies in proteome analysisDetection technologies in proteome analysis

General detection methods.

Differential display proteomics.

Specific detection methods for post-translational modifications.


General detection methods

Organic dye- and silver-based methods

Coomassie blue (R and G)

Silver

Radiactive labeling methods

Reverse stain methods

Flourescence methodsSusana CristobalBioinformatik, 4p. KTH

Differential display Differential display proteomicsproteomics

Detection techniques:

Difference gel electrophoresis Difference gel electrophoresis (DIGE).(DIGE).

Multiplexed proteomics (MP)Multiplexed proteomics (MP)

Isotope-coded affinity tagging Isotope-coded affinity tagging (ICAT)(ICAT)

Differential gel exposure.Differential gel exposure.


Summary of protein expression Summary of protein expression profile analysisprofile analysis


Difference gel electrophoresis (DIGE)Difference gel electrophoresis (DIGE)

(Unlu, 1997, electrophoresis 18, 2071)


Multiplexed proteomics (MP) technology Multiplexed proteomics (MP) technology platformplatform

(Steinberg, 2001, Proteomics 1,841, 2071)


Isotope-coded Isotope-coded affinity tagging affinity tagging (ICAT) technology (ICAT) technology platformplatform

(Smolka, 2002, Mol Cell Proteomics 1, 19-29)

Very successful technique for identification of integral membrane proteins

Differential gel exposure

Coelectrophoresis on 2DE of two protein samples.

In vivo labelling, using 14C and 3H -isotopes.

2DE separation.

Transfer on a PVDF membrane.

3H /14C ratio by exposure to two types of imaging plates.

Investigate changes in the rate of synthesis of individual proteins.

(Monribot-Espagne, 2002, Proteomics 2, 229-240)


Image analysisImage analysis

Software commomly used to manipulate the gel images:Software commomly used to manipulate the gel images:

•Imagemaster TM

•Melanie III TM

•Other functions:Other functions:

• QuantificationQuantification

• AlignmentAlignment

• ComparisonComparison

• MatchingMatching

• Synthetic image from the image of the sampleSynthetic image from the image of the sampleSusana CristobalBioinformatik, 4p. KTH

Example of data from differential Example of data from differential display proteomicsdisplay proteomics

(Chevatier, 2000, Eur.J. Biochem. 267, 4624-4634)Susana CristobalBioinformatik, 4p. KTH

http://www.ejbiochem.org/content/vol267/issue15/images/large/EJB1487.F3.jpeg

Protein profiling in response to various Protein profiling in response to various treatments at two different time-pointstreatments at two different time-points

(Chevatier, 2000, Eur.J. Biochem. 267, 4624-4634)Susana CristobalBioinformatik, 4p. KTH

http://www.ejbiochem.org/content/vol267/issue15/images/large/EJB1487.F4.jpeg

General scheme of proteomic analysis

Pick the protein gel spot from the gel

Pick up the protein gel spot from gel• Manual• Automatic

In-gel digestion:• Washing process• Dehydratation and drying• Trypsin digestion (50 ng trypsin,

37C 16h)• Extraction • Desalt and concentrate the

peptide

Identification methodsIdentification methods

Identification of proteins by mass spectrometry:

Identification of proteins by amino acid composition after acid hydrolysis

Identification of proteins by amino acid sequencing

FFlow chart for the analysis of proteomes by MSlow chart for the analysis of proteomes by MS

(van Wijk, 2001, Plant Physiology 126, 501-508)


http://www.plantphysiol.org/content/vol126/issue2/images/large/pp0616687002.jpeg

Identification of eluted protein spots by different MS approaches

Extraction of intact protein: (single Extraction of intact protein: (single peak)peak)

MALDI-TOF LINEAR mode Passive elution of proteins.

Analyze in a linear MALDI-TOF MS.

Peptide mass FINGERPRINT:Peptide mass FINGERPRINT: MALDI-TOF REFLECTRON mode

In situ tryptic digestion of spots.

Analyze in reflectron MALDI-TOF MS.

Fragments, SEQUENCE:Fragments, SEQUENCE: LC-ESI MS/MS

Separation in a C18 column.

MS/MS analysis in a Q-TOF.Susana CristobalBioinformatik, 4p. KTH

Comparison of MALDI-TOF and ESI-Comparison of MALDI-TOF and ESI-MS-MS approaches to protein MS-MS approaches to protein

identificationidentification

MALDI-TOF MSMALDI-TOF MS

Sample on a a slide (crystalline matrix).

Spectra indicate masses of the peptide ions.

Protein identification by peptide mass fingerprinting.

ESI-MS-MSESI-MS-MS

Sample in solution (high performance liquid chromatography).

MS-MS spectra reveal fragmentation patterns.

Protein identification by cross-correlation algorithms.


Schematic of the MALDI quadrupole Schematic of the MALDI quadrupole time of flight instrumenttime of flight instrument

Advantages:

Mixture are analysed easily.

It is highly tolerant to contaminants.

High sensitivity. (picomol range)

Good accuracy in mass determination.

Quick and not expensive analysis.

Disadvantages:

Low reproducibility and repeatability of single shot spectra. (Averaging )

Low resolution.

Matrix ions interfere in the low max range.


Comparison of MALDI-TOF and ESI-Comparison of MALDI-TOF and ESI-MS-MS approaches to protein MS-MS approaches to protein

identificationidentification

MALDI-TOF spectrum


Peptides that span exon splices will be missed when Peptides that span exon splices will be missed when matching uninterpreted MS-MS data to genomic DNAmatching uninterpreted MS-MS data to genomic DNA

(Jyoti, 2001, Trends 19, supp )


ESI-MS-MSESI-MS-MSPeptide sequencing by nano-electrospray Peptide sequencing by nano-electrospray MSMS


2DE gelIntact protein

Experimental proteolytic peptides

Experimental MS

Theorectical MSTheoretical

proteolytic peptides

DNA sequence database

Protein sequence database

COMPUTER SEARCH

Peptide identification using mapping Peptide identification using mapping fingerprint informationfingerprint information


Applications ?Applications ?

• Cancer proteomics

• Peptidomics: for profiling small proteins in the human fluids

• Neuroscience

•Toxicoproteomics: a new preclinical tool to revolutionary drug target discovery

• Environmental pollution assessment


Example Applications of Proteomics

• pecting the environment to identify proteins with desirable properties

• To better understand the role of proteins in human disease processes

• In anti-sports doping to catch drug cheats• Cancer proteomics• Peptidomics: for profiling small proteins in the human fluids• Neuroscience• Toxicoproteomics: a new preclinical tool to revolutionary drug target discovery• Environmental pollution assessment

Colorectal cancer chemotherapy

Can we a priori determine which patients will benefit from chemotherapy?

Acquire proteomic data Identify candidate biomarkers Validate biomarkers

• All Stage C patients undergo chemo, yet only 50% need it• Of those that need it, who will benefit?

Schedule of a proteomics experimentSchedule of a proteomics experiment

Day 1: Sample preparation and IEF

1. Load protein sample onto IPG strip (IEF)

2. Run the IEF (about 24 hours)

3. Polyacrylamide gel casting

Day 2: Equilibrium IPG strip and running SDS-PAGE

1. Remove IPG strip from IEF machine

2. Equilibrium IPG strip

3. Put IPG strip onto SDS-PAGE

4. Run the SDS-PAGE (overnight)Susana CristobalBioinformatik, 4p. KTH

Day 3: Staining, image scanning and image analysis

1. Remove the gel from the cassette

2. Stain the gel by SYPRO Ruby or silver

3. Scan the gel image

4. Image analysis

Day 4: In-gel digestion, MALDI-TOF and database search

1. Pick the protein gel spot from gel

2. In-gel digestion

3. Spot the sample onto MALDI chip

4. MALDI-TOF analysis

5. Database searchSusana CristobalBioinformatik, 4p. KTH

??? Questions ?????? Questions ???

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