Download Post-translational Modification Learning Objective Post

Proteomics
Post-translational Modification
Post-translational Modification
Many proteins undergo chemical modifications at certain
amino acid residues following translation. These modifications
are essential for normal functioning of the protein and are
carried out by one or more enzyme catalyzed reactions.
Learning Objective
In this Learning Object, the learner will be able to,
1.Recall what Post translation modifications are.
2.Describe Gel-based detection techniques for Post translation
modifications.
3.Define MS-based detection techniques for Post translation
modifications,
4.Describe Microarray-based detection techniques for Post
translation modifications.
Proteomics
Post-translational Modification
Overview of PTMs
Once the protein has been synthesized
by
the
ribosome
from
its
corresponding mRNA in the cytosol,
many proteins get directed towards
the endoplasmic reticulum for further
modification. Certain N and C terminal
sequences are often cleaved in the ER
after which they are modified by
various enzymes at specific amino acid
residues. These modified proteins
then undergo proper folding to give
the functional protein.
Proteomics
Post-translational Modification
Overview of PTMs
There are several types of post
translational modifications that can
take place at different amino acid
residues.
The
most
commonly
observed
PTMs
include
phosphorylation,
glycosylation,
methylation as well as hydroxylation
and acylation. Many of these
modifications,
particularly
phosphorylation, serve as regulatory
mechanisms for protein action.
Proteomics
Post-translational Modification
Overview of PTMs
The final structure of functional
proteins most often does not correlate
directly with the corresponding gene
sequence. This is due to the PTMs that
occur at various amino acid residues in
the protein, which cause changes in
interactions between the amino acid
side chains thereby modifying the
protein
structure.
This
further
increases the complexity of the
proteome as compared to the genome.
Proteomics
Post-translational Modification
Overview of PTMs
Phosphorylation of amino acid residues
is carried out by a class of enzymes
known as kinases that most commonly
modify side chains of amino acids
containing a hydroxyl group.
Phosphorylation requires the presence
of a phosphate donor molecule such as
ATP, GTP or other phoshorylated
substrates. Serine is the most
commonly phosphorylated residue
followed by threonine and tyrosine.
Removal of phosphate groups is
carried out by the phosphatase
enzyme and thus this forms one of the
most important mechanisms for
regulation of proteins.
Proteomics
Post-translational Modification
Overview of PTMs
Glycosylation involves the enzymatic
addition of saccharide molecules to
amino acid side chains. This can be of
two types – N-linked glycosylation,
which links sugar residues to the
amide group of aspargine and Olinked glycosylation, which links the
sugar moieties to the hydroxyl groups
of serine or threonine. Suitable
glycosyl transferase enzymes catalyze
these reactions. Sugar residues that
are attached most commonly include
galactose, mannose,
glucose, Nacetylglucosamine,
Nacetylgalactosamie as well as fucose.
Proteomics
Post-translational Modification
Gel-based detection techniques for PTMs
Protein phosphorylation can be
detected using a novel gel-based
detection
technique.
Proteins
separated on a 2-DE gel are first
placed in a fixing solution containing
methanol and acetic acid which fixes
the protein bands on to the gel and
minimizes any diffusion. They are
then stained using the Pro-Q-diamond
staining solution which selectively
stains only phosphoproteins on the
gel. The excess stain is then washed
off with a solution of methanol and
acetic acid.
Proteomics
Post-translational Modification
Gel-based detection techniques for PTMs
The stained gel is then scanned at its
excitation wavelength using a gel
scanner. The gel image obtained shows
the protein bands corresponding to
only the phosphoproteins present. This
image is saved and the gel is then
removed from the scanner for
treatment with the second stain, a
procedure known as dual staining.
Proteomics
Post-translational Modification
Gel-based detection techniques for PTMs
The scanned gel is then removed from
the scanner and placed in the SYPRORuby Red fluorescent dye solution.
This dye stains all the protein spots
present on the gel thereby providing a
total protein image with sensitivity
down to nanogram level. Excess dye is
then washed off using a solution of
methanol and acetic acid.
Proteomics
Post-translational Modification
Gel-based detection techniques for PTMs
The gel stained with SYPRO-Ruby Red
is then scanned in the gel scanner at
its excitation maxima. The image
produced will have more number of
spots since all proteins present on the
gel are detected. This dual staining
procedure
provides
a
useful
comparative
profile
of
the
phosphoproteins and the total proteins
on the gel, thereby enabling detection
of the phosphorylated proteins.
Proteomics
Post-translational Modification
Gel-based detection techniques for PTMs
Protein
mixture
containing
phosphorylated as well as other
unmodified proteins can be separated
by
a
suitable
electrophoresis
technique.
SDS-PAGE
and
two
dimensional gel electrophoresis are
most commonly used for protein
separation. These separated proteins
on the gel are used for further
analysis.
Proteomics
Post-translational Modification
Gel-based detection techniques for PTMs
The separated protein bands are then
blotted
onto
a
nitrocellulose
membrane. These membranes are
then probed either by means of
specific
anti-phospho-amino
acid
antibodies or more recently, by motif
antibodies that specifically bind to
proteins having phosphorylation at a
particular amino acid residue. This
binding interaction can then be
detected by means of suitably labeled
secondary
antibodies
or
by
autoradiography using a radioactive
probe.
Thus,
the
use
of
immunoblotting technique has been
shown to be extremely effective for
detection of PTMs.
Proteomics
Post-translational Modification
MS-based detection techniques for PTMs
Post translational modifications can be
detected
by
means
of
mass
spectrometry due to the unique
fragmentation
patterns
of
phosphorylated seine and threonine
residues.. The modified protein of
interest is digested into smaller
peptide fragments using a suitable
enzyme like trypsin. This digest is
then mixed with a suitable organic
matrix
such
as
-cyano-4hydroxycinnamic acid, sinapinic acid
etc. and then spotted on to a MALDI
plate.
Proteomics
Post-translational Modification
MS-based detection techniques for PTMs
The target plate containing the
spotted matrix and analyte is placed
in a vacuum chamber with high
voltage and short laser pulses are
applied. The laser energy gets
absorbed by the matrix and is
transferred to the analyte molecules
which undergo rapid sublimation
resulting in gas phase ions. These ions
are accelerated and travel through
the flight tube at different rates. The
lighter ions move rapidly and reach
the detector first while the heavier
ions migrate slowly. The ions are
resolved and detected on the basis of
their m/z ratios and a mass spectrum
is generated.
Proteomics
Post-translational Modification
MS-based detection techniques for PTMs
Identification of PTMs by MS largely
lies in the interpretation of results.
Comparison of the list of observed
peptide masses from the spectrum
generated with the expected peptide
masses enables identification of those
peptide fragments that contain any
PTM due to the added mass of a
modifying group. In this hypothetical
example, two peptide fragments are
found to have different m/z values,
differing by 80 daltons and 160
daltons. It is known that the added
mass of a phosphate group causes an
increase in m/z of 80 daltons.
Therefore, this principle of mass
difference enables detection of
modified fragments.
Proteomics
Post-translational Modification
MS-based detection techniques for PTMs
Liquid chromatography coupled with mass
spectrometry serves as a useful technique
for enrichment and identification of
proteins having a particular type of PTM
from a complex mixture. The complex
protein sample is loaded onto a
miniaturized affinity column which will
interact specifically with proteins having
the PTM of interest. Here, we depict the
use
of
immobilized
metal
affinity
chromatography columns containing ions
such as Ga3+, Zn2+, Fe3+ or TiO2 which have
been found to specifically chelate the
phosphorylated proteins. Unwanted proteins
are removed by washing the column with a
suitable buffer solution after which the
phosphorylated protein of interest is eluted
out by modifying the buffer solution.
Proteomics
Post-translational Modification
MS-based detection techniques for
PTMs
The protein purified by liquid
chromatography is then subjected to
typtic digestion followed by analysis
using tandem mass spectrometry. Here
we demonstrate the use of MALDITOF-TOF-MS for resolution of the
generated ion fragments. Separation is
based on the flight time of the ions
and greater resolution is achieved due
to the presence of two mass
analyzers. The peptide ion spectrum
generated is analyzed by comparing it
with the expected spectrum, thereby
allowing determination of modified
peptides having different m/z values.
Proteomics
Post-translational Modification
Microarray-based detection techniques for PTMs
PTMs can also be detected by means
of protein microarrays using a kinase
assay. Potential substrates for protein
phosphorylation are immobilized on a
suitably coated array surface. To this,
kinase enzyme and gamma P-32
labeled ATP are then added and the
array is incubated at 30oC. The
phosphorylation reaction occurs at
those sites containing proteins that
can be modified.
Proteomics
Post-translational Modification
Microarray-based detection techniques for PTMs
After sufficient incubation, excess
unbound ATP and enzyme are washed
off the array surface. Detection is
carried
out
by
means
of
autoradiography
wherein
a
photographic film is placed in contact
with
the
array
surface.
The
radioactive
emissions
from
the
phosphate label present at the
phosphorylated protein sites strike the
film. Upon development, the positions
at which phosphorylation has occurred
can be clearly determined. Thus
proteome chip technology offers a
useful platform for detection of
phosphporylated proteins.
Proteomics
Post-translational Modification
Microarray-based detection techniques for PTMs
Antibodies specific to phosphorylated
serine, threonine or tyrosine residues
as well as motif antibodies can be
immobilized on to a suitably coated
microarray surface and used for
detection of PTM. The complex
protein mixture containing modified
and unmodified proteins is labeled
with a suitable fluorescent tag
molecule and added to the array
surface. Specific binding interactions
occur between the phosphorylated
proteins and their corresponding
antibodies.
Proteomics
Post-translational Modification
Microarray-based detection techniques for PTMs
The array is then washed to remove
any excess unbound proteins from the
surface. This is followed by scanning
of the array using a microarray
scanner at a suitable wavelength to
detect the fluorescent tag of the
bound proteins. This method offers
sensitive and simultaneous detection
of large number of post translationally
modified proteins.
Proteomics
Post-translational Modification
Overview of PTMs
1. Post-translational modification (PTM): The
chemical modifications that take place at
certain amino acid residues after the protein is
synthesized by translation are known as posttranslational modifications. These are essential
for normal functioning of the protein. Some of
the most commonly observed PTMs include:
c) Acylation: The process by which an acyl group
is linked to the side chain of amino acids like
aspargine, glutamine or lysine.
a) Phosphorylation: The process by which a
phosphate group is attached to certain amino
acid side chains in the protein, most commonly
serine, threonine and tyrosine.
e) Hydroxylation: This PTM is most often found
on proline and lysine residues which make up
the collagen tissue. It enables crosslinking and
therefore strengthening of the muscle fibres.
b) Glycosylation: The attachment of sugar
moieties to nitrogen or oxygen atoms present in
the side chains of amino acids like aspargine,
serine or threonine.
d) Alkylation: Addition of alkyl groups, most
commonly a methyl group to amino acids such as
lysine or arginine. Other longer chain alkyl
groups may also be attached in some cases.
Proteomics
Post-translational Modification
Overview of PTMs
2. Protein translation: The process by which
the mRNA template is read by ribosomes to
synthesize the corresponding protein molecule
on the basis of the three letter codons, which
code for specific amino acids.
3. Cytosol: A cellular compartment that serves
as the site for protein synthesis.
4. Signal sequence: A sequence that helps in
directing the newly synthesized polypeptide
chain
to
its
appropriate
intracellular
organelle. This sequence is most often cleaved
following protein folding and PTM.
5. Endoplasmic reticulum: A membrane-bound
cellular organelle that acts as a site for post-
translational modification of
synthesized polypeptide chains.
the
newly
6. Cleaved protein: The protein product
obtained after removal of certain amino acid
sequences such as N- or C-terminal sequences,
signal sequence etc.
Proteomics
Post-translational Modification
Gel-based detection techniques for PTMs
1. Pro-Q-diamond: This fluorescent dye is
capable of detecting modified proteins that
have been phosphorylated at their serine,
threonine or tyrosine residues. They are
suitable for use with electrophoretic
techniques or with protein microarrays and
offer sensitivity down to few ng levels,
depending upon the format in which they are
used. This dye can also be combined with
other staining procedures thereby allowing
more than one detection protocol on a single
gel.
b) Gel scanning: The visualization of the stained
protein bands on an electrophoresis gel by
exciting it at a suitable maximum wavelength
such that the dye absorbs the light and emits its
own characteristic light at another emission
wavelength.
a) Gel staining: The process by which the
protein bands on an electrophoresis gel are
stained by suitable dyes for visualization.
a) Electrophoresis: Electrophoresis is a gelbased analytical technique that is used for
separation and visualization of biomolecules like
DNA, RNA and proteins based on their fragment
2. Immunoblotting: This process, also known as
Western blotting, is a commonly used analytical
technique for detection of specific proteins in a
given mixture by means of specific antibodies to
the given target protein.
Proteomics
Post-translational Modification
Gel-based detection techniques for PTMs
lengths or charge-to-mass ratios using an
electric field. The protein mixture is first
separated
by
means
of
a
suitable
electrophoresis technique such as SDS-PAGE or
Two-dimensional Electrophoresis.
b) Blotting: The process by which the proteins
separated on the electrophoresis gel are
transferred on to another surface such as
nitrocellulose by placing them in contact with
each other.
c) Nitrocellulose sheet: A membrane or sheet
made of nitrocellulose onto which the protein
bands separated by electrophoresis are
transferred for further probing and analysis.
d) Specific probe antibodies: Antibodies that
are specific to a particular protein modification
can be used as probes to detect those proteins
containing that particular PTM. Protein
phosphorylation is commonly detected using
anti-phosphoserine,
phosphothreonine
or
phosphotyrosine antibodies. Recently, specific
motif antibodies have also been developed
which detect a particular sequence of motif of
the protein that contains a PTM.
e) Labeled secondary Abs: Antibodies labeled
with a suitable fluorescent dye molecule are
used to detect the interaction between the
modified protein and its antibody by binding to
another domain of the probe antibody.
Proteomics
Post-translational Modification
MS-based detection techniques for PTMs
1. MALDI-TOF-MS: A mass spectrometry
instrument that produces charged molecular
species in vacuum, separates them by means of
electric and magnetic fields and measures
the mass-to-charge
ratios and relative
abundances of the ions thus produced. It has
the following components:
a) Ion source: The ion or ionization source is
responsible for converting analyte molecules
into gas phase ions in vacuum. The technology
that enables this is termed soft ionization for
its ability to ionize non-volatile biomolecules
while ensuring minimal fragmentation and thus,
easier interpretation. In MALDI-TOF-MS, the ion
source used is MALDI, in which the target
analyte is embedded in dried matrix-
sample and exposed to short, intense pulses
from a UV laser.
b) Flight tube: Connecting tube between the ion
source and detector within which the ions of
different size and charge migrate to reach the
detector. The Time-of-Flight mass analyzer
correlates the flight time of the ion from the
source to the detector with the m/z of the ion.
c) Detector: The ion detector determines the
mass of ions that are resolved by the mass
analyzer and generates data which is then
analyzed. The electron multiplier is the most
commonly used detection technique.
Proteomics
Post-translational Modification
MS-based detection techniques for PTMs
2. LC-MS/MS approach: LC-MS/MS a common
analytical tool that combines physical
separation by liquid chomatography with mass
analysis and resolution by mass spectrometry. It
is capable of separating and identifying
complex mixtures for proteomics studies.
a) Liquid chromatography: This is a
chromatographic separation technique that
separates molecules based on their differential
adsorption and desorption between the
stationary matrix phase in the column and the
mobile phase.
b) Affinity columns: Columns that make use of
specific affinity interactions between the
analyte of interest and the bound stationary
phase matrix thereby successfully separating this
component
from
a
complex
mixture.
Immobilized Metal ion Affinity Chromatography
(IMAC) is one such affinity technique that relies
on the formation of specific coordinate-covalent
bonds between certain amino acid residues of
the protein (like histidine) and the immobilized
metal ions. Phosphorylated proteins have been
found to bind specifically to ions such as iron,
gallium and zinc, thus facilitating their
separation by IMAC. Recently, titanium dioxide
(TiO2) columns have proved to be extremely
useful for specific separation of phosphorylated
proteins.
Proteomics
Post-translational Modification
MS-based detection techniques for PTMs
c) Tandem MS: This is a mass spectrometry
technique that makes use of a combination of
ion source and two mass analyzers, separated
by a collision cell, in order to provide improved
resolution of the fragment ions. The mass
analyzers may either be the same or different.
The first mass analyzer usually operates in a
scanning mode in order to select only a
particular ion which is further fragmented and
resolved in the second analyzer. This can be
used for protein sequencing studies.
Proteomics
Post-translational Modification
Microarray-based detection techniques for PTMs
1. Protein microarrays: These are miniaturized
arrays normally made of glass, onto which small
quantities
of
many
proteins
can
be
simultaneously immobilized and analyzed. For
detection of phosphorylation sites, potential
protein substrates are immobilized on to the
array.
a) Kinase enzyme: An enzyme that is
responsible for phosphorylation of specific
amino acid residues in the protein with the help
of ATP as a phosphate donor.
b) Phosphorylated proteins: Proteins that have
been phosphorylated at specific amino acid
residues.
c) Autoradiography: Radioactivity is the process
by which certain elements spontaneously emit
energy in the form of particles or waves due to
disintegration of the unstable atomic nuclei into
a more stable form. These radiations that are
given out can be detected by means of
autoradiography, wherein the radiations are
allowed to strike a photographic film which on
exposure shows the presence radioactive
emissions.
2. Antibody microarrays: An array onto which
different antibodies are spotted, which have
specific binding domains for detection of the
protein of interest from a complex mixture. For
detection of PTMs, antibodies against specific
protein motifs containing the PTM or against a
Proteomics
Post-translational Modification
Microarray-based detection techniques for PTMs
specific residue containing a phosphorylated
site may be used.
a) Labeled protein mixture: The protein
mixture containing the protein of interest is
labeled uniformly with a suitable fluorescent
dye which can be detected by scanning at the
appropriate wavelength. Cyanine dyes are
commonly used for such labeling purposes.
b) Array scanning: Once the binding
interactions have taken place on the array
surface and excess unbound material has been
washed away, the array is scanned using a
microarray scanner. This scans the array at a
suitable wavelength depending upon the
fluorescent dye used for labeling purposes to
generate an image depicting the array positions
at which binding has occurred.
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