Post-translational modifications

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Post-translational modifications

a
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.

• Harini Chandra

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Master Layout (Part 1)
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Commonly observed PTMs
This animation consists of 4 parts Part 1
Overview of PTMs Part 2 Gel-based detection
techniques for PTMs Part 3 MS-based detection
techniques for PTMs Part 4 Microarray-based
detection techniques for PTMs
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Cytosol
Endoplasmic reticulum (ER)
Signal sequence
PTMs
Protein translation
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Cleaved protein
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Source Modified from Biochemistry by
A.L.Lehninger, 4th edition (ebook)
3
Definitions of the componentsPart 1 Overview
of PTMs
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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
post-translational modifications. These are
essential for normal functioning of the protein.
Some of the most commonly observed PTMs
include 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. 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. c) Acylation The process by which an
acyl group is linked to the side chain of amino
acids like aspargine, glutamine or lysine. 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. 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.
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Definitions of the componentsPart 1 Overview
of PTMs
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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 the newly
synthesized polypeptide chains. 6. Cleaved
protein The protein product obtained after
removal of certain amino acid sequences such as
N- or C-terminal sequences, signal sequence etc.
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Part 1, Step 1
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Process of post-translational modification
Translated Protein
mRNA
Ribosome
Cytosol
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Endoplasmic reticulum (ER)
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Protein folding PTMs
Removal of certain N- and C-terminal residues
Cleaved protein
Protease
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Action
Description of the action
Audio Narration
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.
First the mRNA ribosome must be shown in
the cytosol. The ribosome must move across
the mRNA as shown and as it moves, the protein
must appear slowly as though it is growing out of
the ribosome (not depicted here). Next, this
protein must enter the ER through the green
channels shown. Next, the pie-shaped protease
must appear which must cut the pink strand
followed by the red strand followed by appearance
of text on the left. Next, the arrows must appear
one at a time with their respective figures on
the right.
As shown in animation.
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Source Modified from Biochemistry by
A.L.Lehninger, 4th edition (ebook)
6
Part 1, Step 2
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Different types of PTMs their modification sites
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Action
Description of the action
Audio Narration
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.
First show the pie chart as depicted. Next, each
segment must be highlighted sequentially along
with appearance of the corresponding text in the
boxes as depicted.
As shown in animation.
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Part 1, Step 3
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Increased complexity of proteome due to PTMs
Gene sequence
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Expected protein structure
Actual protein structure
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Action
Description of the action
Audio Narration
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.
First show the gene sequence on top followed by
the arrow to the left and the blue structure.
This must be zoomed into to show the inset below.
The red cross must then appear on this arrow.
Next, the arrow to the right must appear followed
by the pink structure which must again be zoomed
into to show the inset below.
As shown in animation.
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Part 1, Step 4
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Phosphorylation reactions
Kinase
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Amino acid residue
Phosphorylated residue
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Ser
Thr
Tyr
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Action
Description of the action
Audio Narration
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.
First show the figure on the top left entering
followed by the box below having the various
three arrows from R. Next the arrow must ease
in along with the curved arrow below. Finally,
the figure on the right must appear.
As shown in animation.
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Source Modified from Biochemistry by
A.L.Lehninger, 4th edition (ebook)
9
Part 1, Step 5
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Glycosylation reactions
N-linked Glycosylation
Sugar residues
Glycosyl transferase
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Asn
N-linked amino acid
3
O-linked Glycosylation
Glycosyl transferase
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Ser/Thr
O-linked amino acid
Action
Description of the action
Audio Narration
First show appearance of the figure on top let
with heading followed by arrow and finally
product on right. Similar animation must be
carried out for the second reactions.
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 O-linked
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, N-acetylglucosamine, N-acetylgalactosamie
as well as fucose.
As shown in animation.
5
Source Modified from Biochemistry by
A.L.Lehninger, 4th edition (ebook)
10
Master Layout (Part 2)
1
This animation consists of 4 parts Part 1
Overview of PTMs Part 2 Gel-based detection
techniques for PTMs Part 3 MS-based detection
techniques for PTMs Part 4 Microarray-based
detection techniques for PTMs
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1. Pro-Q-diamond
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2. Immunoblotting
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Definitions of the componentsPart 2 Gel-based
detection techniques for PTMs
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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. a)
Gel staining The process by which the protein
bands on an electrophoresis gel are stained by
suitable dyes for visualization. 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. 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.
a) Electrophoresis Electrophoresis is a
gel-based analytical technique that is used for
separation and visualization of biomolecules like
DNA, RNA and proteins based on their fragment
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.
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Definitions of the componentsPart 2 Gel-based
detection techniques for PTMs
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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.
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Part 2, Step 1 (a)
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Pro-Q-diamond staining
Completed 2-DE gel
2
Tubing connected outlet opened
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Tray with fixing solution (methanol acetic acid)
Pro-Q-diamond stain
Washing solution (methanol acetic acid)
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Action
Description of the action
Audio Narration
First show appearance of the 2-DE gel on top
along with tray having light grey solution. Gel
must be placed horizontally in the tray after
which the dialogue box must appear. Next the
solution must be drained out as shown and then
re-filled with the red solution. Again the
dialogue box must appear after which the solution
is drained out and the tray re-filled with light
blue solution. The last dialogue box then appears
and this solution is then drained out.
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.
As shown in animation.
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Part 2, Step 1 (b)
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Gel scanning
Gel scanner
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Emission maxima 580 nm
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Decreasing molecular weight
Phosphoprotein image
Decreasing pH
4
Description of the action
Action
Audio Narration
PLEASE REDRAW ALL IMAGES. First show the
scanner and the stained gel image. The lid of
the scanner must be opened and the gel must be
placed horizontally in it lid must be closed.
Next the yellow beam must appear followed by the
image below and all the labels. Next the scanner
lid must be opened and the gel must be removed
from it.
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.
As shown in animation.
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Part 2, Step 1 (c)
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Dual staining with SYPRO-Ruby Red
Tubing connected outlet opened
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Washing solution (methanol acetic acid)
SYPRO-Ruby red staining solution
4
Description of the action
Action
Audio Narration
The gel from the previous slide, after being
removed from the scanner is placed in a tray
containing the red SYPRO-Ruby Red stain as
shown. The dialogue box must appear after which
the tubing must appear and the solution must be
drained. The light blue solution must then be
added to the tray and the dialogue box on top
must appear after which the solution must again
be drained as depicted.
The scanned gel is then removed from the scanner
and placed in the SYPRO-Ruby 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.
As shown in animation.
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Part 2, Step 1 (d)
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Gel scanning
Gel scanner
2
Phosphoprotein image
Emission maxima 610 nm
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Total protein image
Decreasing molecular weight
A comparative profile between total protein image
and phosphoprotein image enables detection of
phosphorylated proteins.
Decreasing pH
4
Description of the action
Action
Audio Narration

• 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.

PLEASE REDRAW ALL IMAGES. First show the
scanner and the stained gel image. The lid of
the scanner must be opened and the gel must be
placed horizontally in it lid must be closed.
Next the red beam must appear followed by the
image below and all the labels. The graphs along
with the phosphoprotein image must then appear
followed by the green text box.
As shown in animation.
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Part 2, Step 2 (a)
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Immunoblotting
Proteins focused on IPG strip
2-D Electrophoresis
SDS-PAGE
Sample loading
-
Cathode
2
Direction of migration
Protein mixture
Direction of migration
Anode

Acrylamide gel
Buffer
3
Completed stained gels
4
Description of the action
Action
Audio Narration
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.
PLEASE REDRAW ALL IMAGES. First show the setup on
the left and right with their labels. Next the
hand must appear on the left and the strip on
the right. The hand must move into one of the
wells and the thick blue band must appear. The
strip on the right must be placed on the light
blue surface as shown. Next, the downward arrows
must appear along with text followed by the blue
bands on the left and the spots on the right.
Finally the images below must appear.
As shown in animation.
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Part 2, Step 2 (b)
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Immunoblotting
Proteins phosphorylated at Tyr residues
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Specific phospho-tyrosine antibodies added
Completed gels
Nitrocellulose sheet
Detection using labeled secondary antibodies
Blotting
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Proteins phosphorylated at Tyr residues
4
Description of the action
Action
Audio Narration
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.
PLEASE REDRAW ALL IMAGES. First show the two blue
gel images on the left. Next show the
nitrocellulose sheet which must be superimposed
on the gel. When this happens, the blue bands and
blue spots must appear on these sheets. These
must then be removed from the gel. The pink
inverted Y objects must then be added to the
sheets which must bind to the blue bands and
spots as depicted. Next, the brown inverted Y
with the green label must be added which must
glow upon contact with the pink inverted Y
objects.
As shown in animation.
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Master Layout (Part 3)
1
This animation consists of 4 parts Part 1
Overview of PTMs Part 2 Gel-based detection
techniques for PTMs Part 3 MS-based detection
techniques for PTMs Part 4 Microarray-based
detection techniques for PTMs
2
3
1. MALDI-TOF analysis
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2. LC-MS/MS approach
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Definitions of the componentsPart 3 MS-based
detection techniques for PTMs
1
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.
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Definitions of the componentsPart 3 MS-based
detection techniques for PTMs
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• 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.
• 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.

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Part 2, Step 3 (a)
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MALDI-TOF analysis Digestion sample spotting
Trypsin digestion
2
Digested protein
PTM modified protein of interest
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4
196 well MALDI Plate
Description of the action
Action
Audio Narration
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 a-cyano-4-hydroxycinnamic acid,
sinapinic acid etc. and then spotted on to a
MALDI plate.
PLEASE REDRAW ALL IMAGES. First show the tube on
the left followed by appearance of the arrow and
then the tube on the right. The hand must be
shown to enter this tube and must place a drop of
liquid from this tube onto the grey plate shown
below. Once this happens, that spot must be
zoomed into to show the image above.
As shown in animation.
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Part 2, Step 3 (b)
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MALDI-TOF analysis Ionization detection
Laser
Matrix analyte
2
Detector
Flight tube
3
MALDI
Target plate
4
Description of the action
Action
Audio Narration
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.
First show the entire grey apparatus with all the
labels and the violet rectangle laser source.
Next show a beam emerging from the rectangular
box and falling on the white semicircular region.
Once this happens, the colored ions must emerge
from the white surface as shown in animation.
Once the colored circles have appeared, they must
move towards the detector. The smallest blue
circles must move the fastest followed by the
orange circles and then the green circles.
As shown in animation.
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Part 2, Step 3 (c)
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MALDI-TOF analysis Data interpretation
Expected peptide masses
Observed peptide masses
80 Da implies presence of 1 phosphate group!
Superimposed image
80 Da
2
160 Da
Presence of 2 phosphate group!
3
4
Description of the action
Action
Audio Narration
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.
First show the graphical representation on the
left followed by the graph on the right. These
two must then merge together and be superimposed
to give the third figure as shown in animation.
The pink text and double headed arrows must then
appear followed by the blue arrows and pink text
boxes shown on the right. Finally the text below
must appear.
As shown in animation.
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Part 2, Step 4 (a)
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LC-MS/MS based approach Liquid chromatography
Phosphorylated residue of protein
Metal ions
Buffer solution 2
Buffer solution
Miniaturized immobilized metal affinity columns
2
Phosphorylated protein remains bound
Direction of migration
3
IMAC Ga3, Zn2, Fe3 Other affinity columns
TiO2
Sample protein mixture
TiO2 columns were found to have better
selectivity and sensitivity of detection for
phosphorylated peptide binding when compared to
IMAC.
Purified phosphorylated protein
4
Description of the action
Action
Audio Narration
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.
First show the protein mixture along with the
column. This mixture should be poured into the
column after which the buffer solution must be
placed on the column. Liquid must flow into the
column in the direction indicated. The column
must be zoomed into to show the inset image on
the right where the green and violet circles must
move towards each other, along with the text
below. Next, the pink and brown shapes must move
from the column into the tube below after which
the buffer solution must be replaced (change
color). Finally the green figure must also move
down from the column into the tube below.
As shown in animation.
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Part 2, Step 4 (b)
1
LC-MS/MS based approach Tandem mass spectroscopy
MS/MS analysis
Digested protein
Purified protein
2
LASER
Detector
3
TOF 2 (RF mode)
TOF 1 (scanning mode)
Reflector
4
Collision cell
Action
Audio Narration
Description of the action
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 MALDI-TOF-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.
First show the reaction on top appearing after
which an arrow from the tube on the right must
indicate that this sample enters the instrument
below. Show the instrument below as depicted
followed by appearance of a red beam from the red
box which must strike the dotted line. Next, the
colored circles must appear which must move into
the pink box with the smallest moving the fastest
vice versa. The yellow circle must then move to
the other end of the collision cell followed by
appearance of three smaller circles and
disappearance of the large yellow circle. These
smaller circles must move towards the detector,
with the smallest moving the fastest.
As shown in animation.
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Master Layout (Part 4)
This animation consists of 4 parts Part 1
Overview of PTMs Part 2 Gel-based detection
techniques for PTMs Part 3 MS-based detection
techniques for PTMs Part 4 Microarray-based
detection techniques for PTMs
1
2
3
1. Protein microarrays
4
2. Antibody microarrays
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Definitions of the componentsPart 4
Microarray-based detection techniques for PTMs
1

• 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.

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Definitions of the componentsPart 4
Microarray-based detection techniques for PTMs
1
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
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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Part 4, Step 1 (a)
1
Protein microarrays
2
3
Kinase enzyme
Protein substrate
Phosphorylated protein
Proteome array containing potential substrates
for phosphorylation
Kinase enzyme
g-33P ATP solution
4
Action
Audio Narration
Description of the action
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.
First show the proteome array surface along
with the flast with pink solution on the right.
The hand must appear and pipette out the solution
from the flask on to the array surface such that
the entire surface gets covered with a layer of
pink solution. Next, the blue solution must
appear and similar procedure must be carried out
with that. Once this is done, one of the spots on
the array surface must be zoomed into to depict
the inset image on the right hand side in which
the reaction components must appear sequentially
as shown in animation.
As shown in animation.
5
31
Part 4, Step 1 (b)
1
Phosphorylated proteins
Protein microarrays
Washing
2
Proteome array
33P
33P
33P
33P
33P
3
Radioactive emissions
Detection- Autoradiography film
4
Developed image
Action
Audio Narration
Description of the action
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.
First show the label washing and disappearance
of the blue and pink solutions coating the array
surface and only certain sites as shown must
contain a star label. Next a gray surface must
appear and the black surface must be placed in
contact with this gray surface as shown. This
must be zoomed into to show the inset on the left
along with the animation as depicted.
As shown in animation.
5
32
Part 4, Step 2 (a)
1
Labeled protein mixture added
Antibody microarrays
2
3
Polymer coated glass slide
Specific binding of phosphorylated proteins
Anti-phospho serine, threonine and tyrosine
antibodies immobilized on array
4
Action
Audio Narration
Description of the action
First show the gray surface (which must be
horizontal) on the left followed by binding of
the Y shaped brown objects as shown. Next, the
orange cloud along with the flagged shapes must
appear on top of this surface. The green circles
must be shown to bind to the brown shapes as
depicted while the rest must remain unbound.
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.
As shown in animation.
5
33
Part 4, Step 2 (b)
1
Antibody microarrays
Microarray scanner
Array washing
Unbound proteins removed
2
3
Bound phosphorylated proteins
Array image
4
Action
Audio Narration
Description of the action
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.
Please re-draw all images. The blue solution must
appear on top of the existing components and must
then be removed from the array surface such that
only the green circles remain bound to the
surface as shown. The microarray scanner image
must then appear and the array must shrink and be
placed on the extended grey region as depicted in
animation. Next the orange beam must appear which
must fall on the array surface followed by
appearance of the computer with the image pattern
as shown.
As shown in animation.
5
34
Interactivity option 1Step No 1
1
Based on the two MALDI-TOF-MS spectrums shown
below, determine the total number of
phosphorylated sites present in the protein.
Expected peptide masses
Observed peptide masses
2
3
A) 4
C) 5
B) 6
Answers
D) 7
4
Results
Interacativity Type Options
The correct answer is B. Once the user chooses an
answer, a sign must first be displayed saying
correct or incorrect depending on the answer
given. Next, the colored boxes along with the
double headed arrows must appear sequentially as
depicted in animation.
User must be shown the two graphs on top followed
by the question below and the answers. User must
be allowed to choose one of the four options.
5
Choose the correct answer.
35
Questionnaire
1

• 1. Proline most commonly undergoes which of the
  following PTMs?
• Answers a) Glycosylation b) Phosphorylation c)
  Hydroxylation d) Acylation
• 2. N-linked glycosylation most commonly occurs on
  which of the following amino acids?
• Answers a) Serine b) Threonine c) Aspartic
  acid d) Aspargine
• 3. Which of the following fluorescent dyes is
  used for specific detection of phosphoproteins?
• Answers a) Pro-Q-diamond
• b) SYPRO-Ruby Red
• c) SYPRO-Ruby Orange
• d) Coomassie brilliant blue
• Difference in molecular weight of 240 Da between
  two peptide fragments obtained by MALDI-TOF-MS is
  indicative of presence of how many
  phosphorylation sites on the protein?
• Answers a) 2 b) 3 c) 0 d) 5
• 5. Which of the following metal ions is not used
  for separation of phosphorylated proteins during
  liquid chromatography?
• Answers a) Zn2 b) Fe3 c) Ga3 d) K

2
3
4
5
36
Links for further reading

• Research papers
• Berggren, K. N. et al. An improved formulation of
  SYPRO Ruby protein gel stain Comparison with the
  original formulation and with a ruthenium II tris
  (bathophenanthroline disulfonate) formulation.
  Proteomics 2002, 2, 486-498.
• Reinders, J. Sickmann, A. Modificomics
  Posttranslational modifications beyond protein
  phosphorylation and glycosylation. Biomolecular
  Engineering 2007, 24169-177.
• Zhang, H. et al., Phosphoprotein Analysis Using
  Antibodies Broadly Reactive against
  Phosphorylated Motifs. J Biol. Chem. 2002,
  277(42)39379-39387.
• Delom, F. Chevet, E. Phosphoprotein analysis
  from proteins to proteomes. Proteome Science
  2006, 4!5.
• Andersson L, Porath J Isolation of
  phosphoproteins by immobilized metal (Fe3)
  affinity chromatography. Anal Biochem1986,
  154250-254.
• Posewitz MC, Tempst P Immobilized gallium(III)
  affinity chromatography of phosphopeptides. Anal
  Chem 1999, 712883-2892.
• Neville DC, Rozanas CR, Price EM, Gruis DB,
  Verkman AS, Townsend RR Evidence for
  phosphorylation of serine 753 in CFTR using a
  novel metal-ion affinity resin and matrix
  assisted laser desorption mass spectrometry.
  Protein Sci 1997, 62436-2445.
• Larsen MR, Thingholm TE, Jensen ON, Roepstorff P,
  Jorgensen TJ Highly selective enrichment of
  phosphorylated peptides from peptide mixtures
  using titanium dioxide microcolumns. Mol Cell
  Proteomics 2005, 4873-886.
• Oda Y, Nagasu T, Chait BT Enrichment analysis of
  phosphorylated proteins as a tool for probing the
  phosphoproteome. Nat Biotechnol 2001, 19379-382.
• Janek K, Wenschuh H, Bienert M, Krause E
  Phosphopeptide analysis by positive and negative
  ion matrix-assisted laser desorption/ionization
  mass spectrometry. Rapid Commun Mass Spectrom
  2001, 151593-1599.

37
Links for further reading

• Research papers
• Wilm M, Mann M Analytical properties of the
  nanoelectrospray ion source. Anal Chem 1996,
  681-8.
• Erdjan Salih, Phosphoproteomics by mass
  spectrometry and classical protein chemistry
  approaches. Mass Spectrometry Reviews 2005,
  24828-846.
• Morelle, W. et al., The use of mass spectrometry
  for the proteomic analysis of glycosylation.
  Proteomics 2006, 63993-4015.
• Ptacek, J. et al., Global analysis of protein
  phosphorylation in yeast. Nature Letters 2005,
  438679-684.
• Zhu, H. et al., Global Analysis of Protein
  Activities Using Proteome Chips. Science 2001,
  2932101-2105.
• Books
• Biochemistry by A.L.Lehninger et al., 4th edition
• Post-Translational Modification Phosphorylation
  and Phosphatases. Current Protocols in Protein
  Science Supplement 31, Chapter 13. (Cold Spring
  Harbour Proteomics Manual)
• Post-Translational Modification Glycosylation.
  Current Protocols in Protein Science Supplement
  26, Chapter 12. (Cold Spring Harbour Proteomics
  Manual)
• Websites
• Steinberg et al. Pro-Q Diamond phosphoprotein
  stain a new reagent for detection of
  phosphoproteins and phosphopeptides in
  polyacrylamide gels and in microarrays.
• http//www.biomax.us/antibody-arrays.php