Proteomics Profiling Techniques

Proteomics Profiling Techniques

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Description: Paper is about proteomics Introduction. Common techniques in proteomics, The pioneer of electrophoresis, Advantage of using zinc reverse staining, Principle of mass spectrometry in proteomics, Typical result from MALDI-Tof (spectrum), Typical result from ESI Quadrupole MS, Other proteomic techniques, Protein Complexes Mining, Yeast Two-Hybrid System, Phage display system, Protein Arrays, Core Technologies in Protein Chip, SELDI protein chip, Multi-dimensional HPLC.

 
Author: 陳翰民 (Fellow) | Visits: 2428 | Page Views: 2430
Domain:  Medicine Category: Equipment Subcategory: Proteomics 
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Common techniques in proteomics

Separation of proteins
1DE (convention) 2DE (modern) Multi-dimensional HPLC (modern)

Analysis of proteins
Edman Sequencing (convention) Mass Spectrometry (modern)

Database utilization

About the old techniques

The pioneer of electrophoresis

The Nobel Prize in Chemistry 1948

Arne Wilhelm Kaurin Tiselius was born August 10,1902, in Stockholm. After the early loss of his father,the family moved to Gothenburg where he went to school, and after graduation at the local "Realgymnasium" in 1921, he studied at the University of Uppsala, specializing in chemistry. He became research assistant in The Svedberg's laboratory in 1925 and obtained his doctor's degree in 1930 on a thesis "The moving-boundary method of studying the electrophoresis of proteins" (published in Nova Acta Regiae Societatis Scientiarum Upsaliensis, Ser. IV, Vol. 7, No. 4) and was appointed Docent (Assistant Professor) in Chemistry from 1930 on.

Operation of SDS-PAGE

cathode

Anode

SDS-PAGE

Poly-acrylamide gel electrophoresis Has not changed in the 32 years since its inception..the question answered is still the same Resolution depends on gel length Most convenient technique to separate protein

Edman degradation

Introduced in 1949
1. Run PAGE 2. Digest whole protein mixture and start sequencing

Only works with high abundance proteins

Edman degradation
1953, Frederick Sanger worked out the sequence of insulin 1958 Nobel Prize in Chemistry 1980 Nobel Prize in Chemistry: DNA sequencing

Edman degradation

Only identify the first a.a.

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Technology, Now and then

Traditional RNA technique : Northern blotting

Isolated RNA

Electrophoresis Blotting

Labelling on probes !! Developing Probing

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

Labeling on sample mRNA as probe cDNA or oligonucleotide spotted on chips

Clustered genes

data analysis

Clustered experiments

1. Estimated time to get results: 5-7 days 2. Expressed Gene (mRNA) checked: thousands 3. Accuracy: moderate to high

Traditional Protein technique: protein purification and edman degradation

Cut desired band

Database searching for homolog

Peptide N terminal sequencing

1. Protein purification: necessary 2. Protein idetified: 1 per purified sample


High throughput technique: 2D electrophoresis + Mass spectrometry

1. Protein purification: not necessary 2. Protein idetified: up to thousands per unpurified sample


Major techniques in modern proteomics

A. Two dimensional electrophoresis, 2-DE B. Mass spectrometry

Separation identification



The pioneer of two dimensional electrophoresis

Patrick H. O'Farrell, PhD Professor, Biochemistry and Biophysics, UCSF O'Farrell PH (1975)

High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250: 4007. A technique has been developed for the separation of proteins by two-dimensional polyacrylamide gel electrophoresis. Due to its resolution and sensitivity, this technique is a powerful tool for the analysis and detection of proteins from complex biological sources.

The pioneer of two dimensional electrophoresis
The Nobel Prize in Chemistry 2002

John B. Fenn is the chemist who invented the electrospray method. Today it is used in laboratories all over the world. Fenn has worked mainly as Professor of Chemistry at Yale University, USA, and at Virginia Commonwealth University, USA.

Koichi Tanaka worked as a research engineer at Shimadzu Corp. in Kyoto, Japan . Tanaka's idea was to use the energy from laser light, ingeniously transferred to the proteins, to get them to let go of one another and hover freely. It worked!

Automation process for 2-DE + mass spectrometry
Image Analysis Gel hotel Spot picker

2-D Gel
Digester Spotter

MS

The old way to find protein markers
Takes months to years

Sample homogenization

Prepare for chromatography

Run Chromatography

Prepare for Chromatography again

Run Chromatography again

XT

C1

C2 C3

cut

?
Sequence N termini (Edman degradation) Search database for homologous match

Run SDS-PAGE and Transfer to membrane

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 applied technologies: 1. 2-D electrophoresis: separation of complex protein mixtures 2. Mass spectrometry: Identification and structure analysis

The new way to find protein markers



How proteomic techniques help?
Identification of protein markers from patient samples

patient Mass

Healthy people

Global profiling proteomics

The identities of proteins

The ID for proteins
Name: hemoglobin
MW: 20kDa pI: 5.67 Hydrophobicity: -20?

Size: molecular weight (utilized in 2-DE) Charge: pI (utilized in 2-DE) Hydrophobicity

Two dimensional electrophoresis (2-DE)

1. First dimension: denaturing isoelectric focusing separation according to the pI 2. Second dimension: SDS electrophoresis (SDS-PAGE) Separation according to the MW

Interested spot

Digest to peptide fragment

MS analysis

Run 2-DE, step 1

Run 2-DE, step 2

Run 2-DE, step 3

Run 2-DE, step 4

The theory of IEF

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

How pH gradient forms, Immobilized pH gradient, IPG

First developed by Righetti ,(1990). Immobilized pH gradient generated by buffering acrylamide derivatives (Immobilines) Immobilines are weak acid or weak base. General structure
CH2 CN C O N H R CH2 CN C O N H H

R = amino or carboxylic groups

Acrylamide

Schematic drawing of IPG matrix

First dimension electrophoresis instrument

Amersham Biosciences

Bio-Rad

Run IEF, STEP 1

1. Remove protective film from ImmobilineTM DryStrip gel.

Run IEF, STEP 2

2 . Apply rehydration solution to the Strip Holder.

Run IEF, STEP 3

3. Wet entire length of IPG strip in rehydration solution by placing IPG strip in strip holder (gel facing down).

Run IEF, STEP 4

4. Gently lay entire IPG strip in the strip holder, placing the end of IPG strip over cathodic electrode.

Run IEF, STEP 5

5. Protein sample can be applied at sample application well following the rehydration step if the protein sample was not included in the rehydration solution.

Run IEF, STEP 6

6. Carefully apply DryStrip Cover Fluid along entire length of IPG strip.

Run IEF, STEP 7

7. Place cover on strip holder.

Run IEF, STEP 8

8. Place assembled strip holder on EttanTM IPGphorTM platform

From IEF to SDS-PAGE

Choice of electrophoresis systems

Amersham Biosciences

23 x 20 cm 8 x 10 cm

16 x 16 cm

Choice of electrophoresis systems

Bio-Rad

Protein gel staining methods

Advantage of using zinc reverse staining

1. 2. 3. 4. 5. 6. 7. 8.

Fast (5mins) Convenient (only 2 reagents) Sensitivity (1ng) Compatible to mass spectrometry Re-stainable by all other methods Recover of protein is possible Economic Less toxic (require no heavy metal or organic solvent)

Comparison of staining sensitivity of four methods

2008 Proteomics Lin et. al,

Find the different protein spots on 2-DE gels
Normal C Patient D

Prepare the protein spots for analysis by mass

1. Washing staining material from the gel 2. Equilibrium of gel to protease buffer condition 3. Reduction and alkylation of proteins 4. Digestion of proteins to peptides 5. Extraction of peptides 6. Purification of peptides (Optional)

Principle of mass spectrometry in proteomics

Ion source

Mass analyzer

detector

Ion source: ionize peptide/protein to gas ions Mass analysis: analyze ion according to mass/charge (m/z) Detection: detect the prescence of ions femtomole �attomole (10-15 � 10-18 mole)

Commonly used Mass Spectrometer in Proteomics

MALDI-TOF
Matrix Assisted Laser Desorption Ionization Time Of Flight

ESI tandem MS (with HPLC, LC tandem MS or LC MS/MS)
Electro Spray Ionization Mass Spectrometry

Commercial available MALDI-TOF

Microflex TM, Bruker

Voyager DE-PROTM, ABI

MALDI microTM, Micromass

Principle of MALDI-TOF mass spectrometry
peptide mixture embedded in light absorbing chemicals (matrix)

pulsed UV or IR laser (3-4 ns)

detector

+ + + + + + +

vacuum
+
+
strong electric field

+

Vacc

cloud of protonated peptide molecules

Time Of Flight tube

Typical result from MALDI-Tof (spectrum)

Peptide mass fingerprinting (PMF)

Gel
?
tryptic digestion mass spectrometry

Database
1 2 3

Protein

stored data or theoretical? peptides

compare: ?? is identical to ??

ESI Quadrupole MS

N Gold-coated glass capillary pray
+ + + +

2

vacuum
+ + + + ++ + ++ +

HPLC
Vacc

opening: 1 - 2 m

Orifice
+ ++ ++ + +++ + + + + + + +

Quadrupole detector (multiplier)

solvents

+ + + + + ++ + + +++

Charged droplets

Taylor cones

Ions

Nano electrospray: >30 min spray time for 1 L sample Highly charged molecules are selected by ac modulation of transverse fields

Principle of Quadrupole mass filter

-U -V cos 2 f t
ions with corresponding m/z

U + V cos 2 f t

ions collide with the rods

Scanning of measuring range: U and V are varied ac and dc voltages U (DC voltage) V (peak amplitude of a radio frequency)

Detector

Typical result from ESI Quadrupole MS

From Eckerskorn in "Bioanalytik", Lottspeich and Zorbas (Eds)

Triple Quadrupole Mass Spectrometer

Interface
NH 2

1. ion scanner ac only for proper ion entrance

collision chamber
Argon atoms

2. ion scanner

multiplier

NH 2

Quadrupole 0

Quadrupole 1

Quadrupole 2

Quadrupole 3 Detector

CID: Collision Induced Dissociation for acquiring Molecular weight and Structural information

Nomenclature for CID fragments

CID mass spectrum

Note: Not all b or y ions will present in the spectrum

Commercial LC/MS/MS

API 4000, API

Q-Tof ultima API, Micromass

HCT plus, Bruker

Identification of protein (MASCOT) http://www.matrixscience.com/

Other proteomic techniques

Applications of Proteomics

1. Protein Complexes Mining 2. Yeast Two-hybrid system (in vivo PIP) 3. Phage display and cell surface display system (in vitro PIP) 4. Protein Arrays 5. SELDI protein chips (Ciphergen) 6. Multi-dimensional HPLC (MDLC)

1. Protein Complexes Mining

1. Proteome Complex Mining

A "functional" proteomics approach A proteome complex mining example (purification of kinases)
1. ATP is immobilized to beads in "protein kinase" conformation 2. Total protein is mixed the beads and the mixture "washed" 3. Remaining proteins isolated and identified...protein kinases, and purine dependent metabolic enzymes

Proteome complex mining by affinity capture

Affinity capture methods

Coimmunoprecipitation (Adams et al. 2002, eg. Anti
p53 antibody)

Coprecipitation (Seraphin et al. 2003, eg. V5 epitope) Protein affinity-interaction chromatography
(Einarson and Orlinick 2002, eg. GST fusion protein)

Isolation of intact multi-protein complexes (eg.
Nuclear pore complexes, ribosome complexes, and spliceosomies)

Affinity capture methods

Coimmunoprecipitation

Coprecipitation

Protein affinity-interaction

Isolation of intact multiprotein complexes

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

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 Bait Protein Binding Domain

Reporter Gene

Yeast 2 hybrid system, contd.

X

Y1, Y2, Y3, Y5, Y6............(all genome)

X Y4

More complex Yeast 2/3/4 hybrid system

3. Phage display and cell surface display system (in vitro PIP)

3. Phage display system (in vitro)

3. Phage display system (in vitro)

Biopanning

Phage minor coat protein GIII

SCIENCE VOL 298 18 OCTOBER 2002

Applications for Phage display system

Human antibody techniques

The growth and potential of human antiviral monoclonal antibody therapeutics Wayne A Marasco & Jianhua Sui Nature Biotechnology 25, 1421 - 1434 (2007) Published online: 7 December 2007

4. Protein Arrays

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.

Technological Components for Protein Chips

Protein Microarrays

Science, 289, 1760, 2000

Microspotting of proteins on aldehyde glass slide 150~200 �m in diameter (100 �g/mL) 10,799 spots of Protein G (1,600 spots/cm2) A single spot of FRB (FKBP12-rapamycin binding)

Protein Microarray
G. MacBeath and S.L. Schreiber, 2000, Science 289:1760

Spotting platform and protein microarray

What protein microarray can do?

1. 2. 3. 4. 5. 6.

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

Application of protein microarray

Protein microarrays (Ab arrays)

Face the real world

The true spot quality from real experiment

Class of capture molecule for protein microarray

Core Technologies in Protein Chip
Protein chips for practical use

Detecting the biomolecular interaction with Detecting the biomolecular interaction with high sensitivity and reliability high sensitivity and reliability How to construct the monolayers of biomolecules on a solid surface Maximizing the binding efficiency Most difficult parts Maximizing the fraction of active biomolecules Minimizing the nonspecific protein binding

5. SELDI protein chips

5. SELDI protein chip

SELDI � surface enhanced laser desorption/ ionization

Protein chips

Types of protein chip

IMAC30immobilized metal affinity capture array with a nitriloacetic acid (NTA) surface with an updated hydrophobic barrier coating. IMAC3mmobilized metal affinity capture array with a nitriloacetic acid (NTA) surface. CM10weak cation exchange array with carboxylate functionality, with an updated hydrophobic barrier coating WCX2weak cation exchange array with carboxylate functionality. Q10strong anion exchange array with quaternary amine functionality, with an updated hydrophobic barrier coating. SAX2strong anion exchange array with quaternary amine functionality. H50bind proteins through reversed phase or hydrophobic interaction chromatography with an updated hydrophobic barrier coating H4 mimic reversed phase chromatography with C16 functionality. NP20mimic normal phase chromatography with silicate functionality Auold chips to be used directly for MALDI-based experiments

Experimental procedure of SELDI protein chip

SELDI protein chip, 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).

6. Multi-dimensional HPLC (MDLC)

Configuration of MDLC

2nd RP

1st SCX

From: N341 Institute of Biomedical Sciences , Academia Sinica

An analysis result by MDLC

From: N341 Institute of Biomedical Sciences , Academia Sinica

Agilent 1100 series

MDLC

MASS