Km Usuhs Class Improved1.2 Copy

INTERACTIVE PROTEOMICS – TECHNIQUES FOR
EXPLORING THE SOCIAL NETWORK OF CELLS

Karobi Moitra (Ph.D)
NCI Frederick , NIH
Cancer Inflammation Program
Human Genetics Section
Frederick MD.

Proteome: the entire protein complement of a cell , tissue, or organism

The proteome is DYNAMIC !

Why is the proteome dynamic ?

Proteins can be:

Synthesized

Modified by post-translational modifications

Undergo translocations within the cell

Degraded

Examination of the proteome of a cell is like
taking a “snapshot” of the protein environment
at any given time

Proteome: the entire protein complement of a cell , tissue, or organism

Proteomics: is the large scale characterization of this
proteome

Why do we need to characterize the proteome?

• To obtain a more global and integrated view of biology by
studying all the proteins of a cell rather than each one
individually

• To create a complete three-dimensional (3-D) map of the cell
indicating where proteins are located

The Google Earth Analogy

Global Protein
Landscapes

Different areas of study are now grouped under
the rubric of proteomics include:

Protein modifications

Protein function

Protein localization

Protein-protein interactions
(Interactive proteomics)

WHAT IS INTERACTIVE PROTEOMICS OR PROTEIN –
PROTEIN INTERACTIONS?

THE ABILITY OF A PROTEIN TO BIND OR INTERACT WITH ANOTHER PROTEIN
OR PROTEINS
Types of protein interactions :

Permanent interactions

Transient interactions

From the need for more -omics came the term
interactome

THE INTERACTOME
COMPLETE PROTEIN INTERACTION NETWORK OF A CELL OR
AN ORGANISM

Just as humans don’t thrive when isolated from other humans - the same can be
said for proteins !

Proteins interact with other proteins to provide :

Structural integrity to the cell (e.g., actin filaments)
Transport molecules (e.g.,Transporters)
Propagate signals (e.g., kinases)
Transcribe DNA, translate other proteins etc.

….there is no protein discovered yet that acts on its own
without interacting with any other entity !

The entire protein complement of a cell, tissue, or organism is called
the PROTEOME

The proteome is DYNAMIC

Proteins can interact or bind with other protein(s)

This ‘social’ network is called the INTERACTOME

….in the real world you have to interact with people to learn the
‘social dynamic’

…. in the protein world you would have to know how proteins
(and other components of a cell) interact with each other in order to explore
the ‘cellular’ dynamic

You have been asked to find the interacting partners of
a protein named ‘C3PO’. Your first task is to find out
everything you need to know about this protein in order to
undertake this study.

Your tool is the internet, which sites you would go to and
what information might you obtain from these sites to get
the relevant background knowledge you would need to
carry out the study?

Partial List of potential websites:

www.google.com

www.ncbi.nlm.nih.gov/

http://www.ensembl.org/index.html

www.expasy.ch

http://www.expasy.org/links.html

And a lot more links from this page………

1.Clone and express the protein (C3PO) in an expression
system of your choice

2. Optimize protein expression

3. Decide which techniques you would use to study
protein-protein interactions

TECHNIQUES USED TO STUDY
PROTEIN-PROTEIN INTERACTIONS

A. Standard techniques to probe protein-protein interactions
Affinity purification
Mass Spectrometry
Two-Hybrid Assay
Phage Display

B. In Vivo Imaging
Fluorescence Microscopy

C. Biophysical Approaches
Protein Co-crystallization

D. Microarrays
High Density Protein Microarray

E. Computational/Bioinformatics Methods
Computer programs that simulate protein-protein interactions
Prediction of co-evolved protein pairs based on similar
phylogenetic trees


Affinity purification
Basic Principle:
Historically affinity purification
was based on a specific biological
interaction such as
enzyme-substrate.
In a broader sense it may mean
Chemical/biological affinity.

Stationary/solid phase
Dynamic/liquid phase

Immunoprecipitation

Immunoprecipitation (IP) is the technique of precipitating a protein antigen
out of solution using an antibody that speciﬁcally binds to that particular
protein.

Immunoprecipitation/coimmunoprecipitation

Basic Principle:
B lysate

X A
Y B
A
X
Y

B
Cell lysis
X A Freeze thaw
Y Lysis buffer
Hypotonic
Mild detergent ProteinA/G beads
(Ripa, NP40) X (binds to Fc of Ab)
A Y

Post- ip
Run gel
Visualize protein
Excise band elute wash
Digest
Lysis buffer A
X Low pH(change pH)
MS SDS loading buffer
Y

Disadvantage: An antibody to the specific protein of interest is required

Solution: We can tag our protein of interest with an epitope tag

Epitope Tagging :
Antibody recognizes a specific portion of the protein - epitope.

Target protein Flag tag
Anti-Flag Ab
coupled to
beads
Associated proteins

(i) Single Tag

FLAG tag , c-Myc tag, GST tag, His tag etc.

(ii) Tandem affinity purification
TAP tag

Attaching the Tag :

Note:
Tags can be N
terminal or C terminal
depending on where the
functional region of the
protein is located. Tagging
close to the functional region
may interfere with binding
sites.

Clone into vector

Transfect into cells to express the protein

Histidine Tag
Imidazole groups
Imidazole can form a coordinate
covalent bond with metals
groups

Nickel column

Or
Imidazole

Transfect cells
Controls :
48-72hrs
Transfection:
(for peptide purification-
Untransfected cells
(Complex pulldown) antibody production Vector transfected
Also complex pulldown) Known positive control

Pulldown:
Vector transfected cells
Known positive control
Or
Mechanical lysis
Freeze-thaw method

His-tagged protein
binds to Ni column

(Low conc. to wash
out non-specific binding)

(Compete off His tagged
Protein)

(ii) Tandem Affinity Purification :

2 step purification :

1. Purify through Protein A tag on a IgG-
sephrose column

2. Purify through Calmodulin binding domain
on a Calmodulin-sepharose column

2 step purification removes a lot of the background / non-specific protein binding

Tandem affinity purification (TAP)

TAP TAG

(tobacco etch virus)

IgG-sepharose
bead

Step 1 TEV cleavage
Purify protein by passing through
IgG column and elute with TEV

IgG-sepharose
bead

Step 2
Purify protein by passing
Through Ca + calmodulin column
And elute with EDTA

Elute
(EGTA)
IgG-sepharose
Target protein
bead

Calmodulin-sepharose beads

Evaluation of a Co-IP Captured interaction

1.Confirm that the co-precipitated protein is obtained only by the antibody against
the target , try and use monoclonal antibodies , if using polyclonals purify
the antibody using an affinity column containing pure target

2. Use an antibody against the co-precipitated protein to co-IP the same complex

3. Determine that the interaction takes place in the cell and not as a consequence
of cell lysis, use co-localizatiion or mutation studies to confirm interaction.

4. Run a negative control IP with unrelated antibodies.

(i) Single Tag 
(ii) Tap tag 
(iii)Photochemical/chemical crosslinking

Photochemical / Chemical Crosslinking of Proteins

The interactions or proximity of proteins can be studied by the clever use
of crosslinking agents. Protein A and B may be quite close to each other
in a cell and a chemical crosslinker can be used to probe the protein-protein
interaction by linking them together, disrupting the cell and detecting the
crosslinked proteins.

B
A

Diazirine based photo crosslinking

Cells grown with photoreactive diazirine compounds

Diazirine incorporated into protein

UV light A B

Diazirines activated and bind to interacting proteins (within a few angstroms)

Chemical Crosslinking
• Covalently links distinct chemical functional groups & can detect both stable and
transient interactions

• If 2 proteins physically interact with each other they can be covalently crosslinked

Crude cellular extract + crosslinking agent (maleimides -SH reactive groups
would form disulphide bonds between proteins )

IP
A B

Recover complexes

Cleave with DTT, BME which would break disulphide bonds

Example : SMCC, succinimidal trans -4 (maleimidemethyl) cyclohexane-1- carboxylate

(a) Epitope tagging
(b) Chemical cross-linking
(c) TAP tag approach.

You have your putative protein -complex of interest

how would you identify the individual proteins that make up

this complex ?

SCHEMATIC DIAGRAM OF PROTEIN IDENTIFICATION

(can ID only 50-60 aa)

MASS SPECTROMETERY

Protein structural information : peptide mass
amino acid sequences

Type and location of post-translational modifications

Experimental Design

Immuno-precipitation

Examine complexes by SDS-PAGE

Mass Spectrometry

LC- TANDEM MS

In-gel digestion
with trypsin (K/R)

Extract peptides

MALDI-TOF/TOF

PROTEIN IDENTIFICATION BY PEPTIDE MAPPING
(MALDI-TOF)

MALDI-TOF
Matrix assisted laser desorption ionisation- time of flight

Soft ionisation technique suitable for fragile biomolecules like peptides

Basic Principle of Mass Spectrometry

How it works :

The amount of deflection for a
sideways force depends on the
Mass of the ball (acceleration constant)

Acceleration - known
Force - known
Mass - can be calculated
Force= mass x acceleration

Peptides + matrix (matrix protects peptides from the direct laser beam
and help absorption of laser energy)
Spotted onto a target plate

Ionised by laser beam (charge needed for deflection by electric field)
Ionised particles enter flight tube

Charged peptides move to other side of tube according to mass

Peptides hit the detector and time of flight (TOF) is recorded (to calculate speed)

Opposite charge

(speed)

(known force)

The computer generates a mass spectrum, with each peak representing the
mass to charge ratio (m/z) as a function of the % relative intensity (abundance)
of the detected peptide
The list of experimental peptide masses is compared against the theoretical
tryptic digest of every protein in a protein database.
When the experimental data matches the theoretical, the protein is identified.
A probability based scoring system is used for the search, indicating the
‘hit’ is not a random event.

PROTEIN IDENTIFICATION BY TANDEM MASS SPECTOMETRY (MS/MS)

If the protein cannot be identifed via the peptide mass profile (eg the
protein may not be listed in any database) then Tandem (MS/MS) may
be used to obtain an amino acid sequence.

Q1- 1st mass analyser (quadrupole) isolates peptide ion of interest

Q2- Collision chamber peptide ion collides with neutral gas molecules (helium,nitrogen
or argon) and fragments into smaller pieces

Q3- 2nd analyser (TOF) leads to detector which gives a product profile (aa sequence)
Fragments the peptides into the smallest length to ID short sequences

Affinity purification 
Mass Spectrometry 
Two-Hybrid Assay
Phage Display

B. In Vivo Imaging


D. Microarrays

phylogenetic trees

Yeast 2-hybrid assay

• Test the association of two specific proteins that
are believed to interact on the basis of other
criteria.
• Define domains or amino acids that are critical for the
interactions of two proteins that are known to interact
• Screen libraries for proteins that interact with a
specific protein.

Activating
Basic Principle of 2- Hybrid Assays domain
Binding
domain
The basic premise of a 2- hybrid assay
is that a prey protein is detected with
the help of a bait protein.

A transcription factor is split into 2
parts a DNA binding domain - BD and
an activation domain AD.

The BD is engineered to bind to the
bait and the AD is engineered to
bind to the prey.

Only if the bait and the prey protein
interact will the transcription factor
come together and transcribe a reporter
gene.

Probing Protein-Protein Interactions
with the Yeast 2-Hybrid Assay

DNA Binding Domain Protein X + Activation Domain Protein Y

YES NO
HIS3 HIS+ his-

lacZ Blue white

ADE2 Whit red

Split-Ubiquitin Membrane Yeast Two-Hybrid System

Drawbacks of typical Y2H necessitated the split-ubiquitin Y2H

1.Hybrid proteins are directed towards the nucleus so proteins that fold
incorrectly in nucleus are excluded from the method (integral membrane
proteins).

2. Interactions dependent on post-translational modifications ( in ER) won’t
take place.

3. Interactions mediated by the amino-terminus may not work because the
transcription factor domain blocks accessibility.

Split-Ubiquitin Membrane Yeast Two-Hybrid System
1. Contains 2 fragments of ubiquitin brought
together upon interaction of the
2 proteins.
Prey Bait
2. The bait protein X is fused to the C-term of
ubiquitin (Cub) followed by a TF

3. The prey protein Y is fused to N-term of Y X
ubiquitin (NubG)

4. The 2 plasmids are introduced into yeast
L40 strain. Transcription factor

5. Interaction of X and Y leads to the
assembly of ubiquitin and the proteolytic
release of transcription factor (by ubiquitin
proteases).

6. The transcription factor activates the 2
reporter genes lacZ and His3 so
the interactions can be monitored by
growing yeast in histidine deficient
media or by performing an X-gal test for
the expression of beta galactosidase.

Two-Hybrid Assay 
Phage Display

B. In Vivo Imaging


D. Microarrays

phylogenetic trees

PHAGE DISPLAY

Basic Principle:
In phage display new genetic material is inserted into a phage gene and
the bacteria process the new gene so that a protein/peptide is made and
exposed on the phage surface (due to a tag which only expresses on the cell surface).

A population of bacteriophages display hundreds/millions of protein -
one protein per phage.This is called a phage display library.

This library can be exposed to an immobilized target protein and some members will
bind to the target. The immobilized target is then washed to remove non/loose binding
phages. The DNA of phages that bind can be sequenced to identify the gene/protein.

B. IN VIVO IMAGING

Basic Principle

Fluorescent molecules are irradiated
with high intensity light.

When these molecules absorb a photon of
light an electron is boosted up to a higher
energy orbit creating an excited state

When this electron returns to the ground state
a photon of light may be emitted- this is
called fluorescence.

Fluorophores have distinct excitation and
emission spectra.

How can we use fluorescence microscopy to study
protein-protein interactions?

1. FRET (Fluorescent Resonance Energy Transfer)

2. BRET (Bioluminescence Resonance Energy Transfer)

1. FRET (Fluorescent Resonance Energy Transfer)

Normally an excited photon returns to the ground state when a photon is emitted.
FRET results in the excitation of a nearby acceptor fluorophore which will emit a
photon when it goes back to the ground state.

The occurrence of FRET thus results in decreased donor emission and increased
acceptor emission.

Distance is everything !
FRET is extremely sensitive to the distance among fluorophores
For CFP and YFP the half maximum distance or Forster radius is 49-52 angstroms

Basic Principle of FRET

475

CFP YFP

One probable interaction partner is tagged with CFP the other with YFP.
If the 2 proteins interact emission will be observed at 530nm instead of 475nm

Problems of FRET

1. Tissues and cells may be damaged by excitation light

2. Some tissues like the retina and most plant tissues are photoresponsive

3. Photobleaching, autofluorescence or diect excitation of the acceptor
fluorophore may occur.

2. BRET (Bioluminescence Resonance Energy Transfer)

In BRET the excitation light is replaced by bioluminescent light from
Renilla luciferase (RLUC)
The luciferase is activated by its substrate coelenterazine.

Bioluminescent light

C. Biophysical Approach

1. Protein Co-crystallization


-grow crystal

-collect diffraction data

-calculate electron density

-trace chain & generate structure

SNL1 & YPD1 co-crystals

SNL1 and YPD1 are part of a phosphorelay
signal transduction pathway in yeast.
these protein can be co-crystalized by using
a phosphate analog (BeF3) which bind
covalently and activates respose regulator
proteins.

(Chooback . L 2003)

Co-Crystal structure of YPD1 and SLN1

YPD - Yellow
SLN1- Cyan

(Xu et. al 2003)

D. Microarrays


Microspots of the captured molecules
are immobilized in rows and columns
on a solid support

They are exposed to samples containing
the corresponding binding molecules.

Proteins interact

Readout systems based on fluorescence, chemiluminescence,mass
spectometry, radioactivity etc. can be used to detect
complex formation


1.Computer programs that simulate protein-protein interactions
ie Docking programs like Autodock.

2. Prediction of co-evolved protein pairs based on similar phylogenetic trees

This method involves using a sequence search tool such as BLAST for finding
homologues of a pair of proteins, then building multiple sequence alignments with
alignment tools such as Clustal. From these multiple sequence alignments,
phylogenetic distance matrices are calculated for each protein in the hypothesized
interacting pair. If the matrices are sufficiently similar they are deemed likely to
interact.

Two-Hybrid Assay 
Phage Display 

B. In Vivo Imaging
Fluorescence Microscopy 

Protein Co-crystallization 

D. Microarrays
High Density Protein Microarray 

Computer programs that simulate protein-protein interactions 
Prediction of co-evolved protein pairs based on similar 
phylogenetic trees

Humans do not thrive when isolated from others - the same can be
said for proteins !

TO MARGUERITE

by: Matthew Arnold (1822-1888)

‘Yes in the sea of life enisled,
With echoing straits between us thrown.
Dotting the shoreless watery wild,
We mortal millions live alone.
The islands feel the enclasping flow,
And then their endless bounds they know…’

Think-Pair- Share Activity

In light of what you have learnt about proteins today do you think
that a protein can function on its own isolated from other proteins?

For or Against

‘O then a longing like despair
Is to their farthest caverns sent!
For surely once, they feel, we were
Parts of a single continent.
Now round us spreads the watery plain--
O might our marges meet again!’

Km Usuhs Class Improved1.2 Copy

Empfohlen

Empfohlen

Weitere ähnliche Inhalte

Was ist angesagt?

Was ist angesagt? (20)

Ähnlich wie Km Usuhs Class Improved1.2 Copy

Ähnlich wie Km Usuhs Class Improved1.2 Copy (20)

Mehr von Karobi Moitra CFD, MS, PhD

Mehr von Karobi Moitra CFD, MS, PhD (18)

Km Usuhs Class Improved1.2 Copy