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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
Master Layout (Part 1)
1
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
Phosphorylation
Glycosylation
Acylation
2
Alkylation
Hydroxylation
3
Cytosol
Endoplasmic reticulum (ER)
P
P
Signal
sequence
4
Protein
translation
PTMs
Cleaved protein
CH3
CH3
Glc
Glc
5
Source: Modified from Biochemistry by A.L.Lehninger, 4th edition (ebook)
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2
Definitions of the components:
Part 1 – 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:
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.
3
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.
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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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1
2
Definitions of the components:
Part 1 – 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.
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5. Endoplasmic reticulum: A membrane-bound cellular organelle that acts as a site for posttranslational 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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5
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Part 1, Step 1
Process of post-translational modification
Translated
Protein
mRNA
Ribosome
Cytosol
2
P
3
Endoplasmic
reticulum
(ER)
Removal of
certain N- and
Cleaved protein
C-terminal Protease
residues
P
Protein folding
& PTMs
CH3
CH3
Glc
Glc
4
Action Description of the action
5
As
shown
in
animatio
n.
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.
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.
Source: Modified from Biochemistry by A.L.Lehninger, 4th edition (ebook)
Part 1, Step 2
1
Different types of PTMs & their modification sites
Ser, Thr, Tyr
Pro, Lys
2
Phosphorylation
Asn, Glycosylation
Ser, Thr
Lys, Arg
3
Acylation
Alkylation
Hydroxylation
Asn, Gln, Lys
4
Action Description of the action
5
As
shown
in
animatio
n.
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.
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.
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Part 1, Step 3
Increased complexity of proteome due to PTMs
A A C G G U G C C G U G C A C G C A C U A C G C A C U
Expected protein
structure
2
Gene sequence
Actual protein
structure
3
Glc
P
CH3
4
Action Description of the action
5
As
shown
in
animatio
n.
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.
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.
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Part 1, Step 4
Phosphorylation reactions
COO-
COOKinase
2
C
H
NH3+
3
R
H
OH
ATP
Amino acid
residue
ADP
CH2
NH3+
C
R
O
PO43-
Phosphorylated
residue
Ser
CH
Thr
R
CH3
Tyr
CH2
4
Action Description of the action
5
As
shown
in
animatio
n.
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.
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.
th
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Part 1, Step 5
Glycosylation reactions
N-linked Glycosylation
COO-
2
H
Glycosyl transferase
C
CH2
H
C
CH2
CONH2
CON
NH3+
NH3+
Asn
3
Sugar residues
COO-
N-linked amino acid
O-linked Glycosylation
COO-
COOGlycosyl transferase
H
C
R
C
H
R
O
OH
NH3+
4
NH3+
Ser/Thr
O-linked amino acid
Action Description of the action
5
As
shown
in
animatio
n.
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.
Audio Narration
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, Nacetylglucosamine, N-acetylgalactosamie as well as fucose.
th
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
Gel scanning
Gel staining
2
3
Specific probe
antibodies
4
Electrophoresis
1. Pro-Q-diamond
Blotting
SDS-PAGE/
2-DE gel
5
Detection
Nitrocellulose sheet
2. Immunoblotting
Labeled
secondary
Abs
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2
Definitions of the components:
Part 2 – 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.
a) Gel staining: The process by which the protein bands on an electrophoresis gel are
stained by suitable dyes for visualization.
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4
5
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 Twodimensional Electrophoresis.
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2
Definitions of the components:
Part 2 – Gel-based detection techniques for PTMs
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.
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4
5
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)
Pro-Q-diamond staining
Dye
stains
the get
Protein
bands
Excess
dye
phosphorylated
fixed
on gel and
removed
protein
bands
only.
minimize
diffusion.
Completed 2-DE gel
2
Tubing connected &
outlet opened
3
Tray with fixing solution
Pro-Q-diamond
stain
Washing
solution
(methanol
+ acetic
acid)
(methanol + acetic acid)
4
Action Description of the action
5
As
shown
in
animatio
n.
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.
Audio Narration
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.
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Part 2, Step 1 (b)
Gel scanning
Gel scanner
2
Stained gel
Emission maxima
– 580 nm
Decreasing
molecular weight
3
Phosphoprotein
image
Decreasing pH
4
Action Description of the action
5
Gel removed
from scanner
As
shown
in
animatio
n.
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.
Audio Narration
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.
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Part 2, Step 1 (c)
Dual staining with SYPRO-Ruby Red
Dye
stains
all
Excess
dye
protein
bands.
removed
Tubing connected &
outlet opened
2
3
Washing solution
SYPRO-Ruby
red
(methanol
acetic acid)
staining+ solution
4
Action Description of the action
5
As
shown
in
animatio
n.
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.
Audio Narration
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.
Fluorescence
Gel scanning
Gel scanner
2
Phosphoprotein
image
Emission maxima
– 610 nm
Total protein
image
Decreasing
molecular weight
3
Decreasing pH
4
A comparative profile
between total protein image
and phosphoprotein image
enables detection of
phosphorylated proteins.
Action Description of the action
5
Phosphoprotein
image
Stained gel
As
shown
in
animatio
n.
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.
Fluorescence
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Part 2, Step 1 (d)
Total protein image by
SYPRO-Ruby Red
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.
1
Part 2, Step 2 (a)
Proteins focused on
IPG strip
Immunoblotting
SDS-PAGE
-
2-D Electrophoresis
Sample loading
Cathode
2
3
Direction of
migration
Protein
mixture
Acrylamide
gel
Direction of
migration
+ Anode
Buffer
Completed
stained gels
4
Action Description of the action
5
As
shown
in
animatio
n.
Audio Narration
PLEASE REDRAW ALL IMAGES. First show the setup on the Protein mixture containing phosphorylated as
left and right with their labels. Next the hand must appear on well as other unmodified proteins can be
the left and the ‘strip’ on the right. The hand must move into separated by a suitable electrophoresis
technique. SDS-PAGE and two dimensional
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 gel electrophoresis are most commonly used
shown. Next, the downward arrows must appear along with for protein separation. These separated
text followed by the blue bands on the left and the spots on proteins on the gel are used for further
analysis.
the right. Finally the images below must appear.
1
Part 2, Step 2 (b)
Immunoblotting
Proteins
phosphorylated at
Tyr residues
2
Completed
gels
Nitrocellulose
Blotting
sheet
3
4
5
Specific phosphotyrosine
antibodies added
Detection using
labeled secondary
antibodies
Proteins
phosphorylated at
Tyr residues
Action Description of the action
As
shown
in
animatio
n.
Audio Narration
PLEASE REDRAW ALL IMAGES. First show the two
The separated protein bands are then blotted onto a nitrocellulose
blue gel images on the left. Next show the ‘nitrocellulose membrane. These membranes are then probed either by means of
sheet’ which must be superimposed on the gel. When specific anti-phospho-amino acid antibodies or more recently, by
this happens, the blue bands and blue spots must
motif antibodies that specifically bind to proteins having
appear on these sheets. These must then be removed
phosphorylation at a particular amino acid residue. This binding
from the gel. The pink inverted Y objects must then be
interaction can then be detected by means of suitably labeled
added to the sheets which must bind to the blue bands
and spots as depicted. Next, the brown inverted Y with secondary antibodies or by autoradiography using a radioactive
the green label must be added which must glow upon probe. Thus, the use of immunoblotting technique has been shown
to be extremely effective for detection of PTMs.
contact with the pink inverted Y objects.
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
MALDI-TOF-MS
2
Ion source
Flight tube
Detector
3
1. MALDI-TOF analysis
Affinity columns
4
Liquid
chromatography
5
Tandem MS
2. LC-MS/MS approach
1
2
3
4
Definitions of the components:
Part 3 – 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.
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1
2
3
4
5
Definitions of the components:
Part 3 – 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.
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.
1
Part 2, Step 3 (a)
MALDI-TOF analysis – Digestion & sample spotting
Protein
Trypsin
digestion
Matrix
2
PTM modified
protein of
interest
Digested
protein
3
4
196 –well MALDI Plate
Action Description of the action
5
As
shown
in
animatio
n.
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.
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-4hydroxycinnamic acid, sinapinic acid etc. and then spotted on
to a MALDI plate.
1
Part 2, Step 3 (b)
MALDI-TOF analysis – Ionization & detection
Laser
Matrix & analyte
2
Detector
Flight tube
3
Target plate
MALDI
4
Action Description of the action
5
As
shown
in
animatio
n.
Audio Narration
The target plate containing the spotted matrix and analyte is placed
First show the entire grey apparatus with all the
in a vacuum chamber with high voltage and short laser pulses are
labels and the violet rectangle laser source. Next
applied. The laser energy gets absorbed by the matrix and is
show a beam emerging from the rectangular box
transferred to the analyte molecules which undergo rapid
and falling on the white semicircular region. Once
this happens, the colored ions must emerge from the sublimation resulting in gas phase ions. These ions are accelerated
and travel through the flight tube at different rates. The lighter ions
white surface as shown in animation. Once the
move rapidly and reach the detector first while the heavier ions
colored circles have appeared, they must move
migrate slowly. The ions are resolved and detected on the basis of
towards the ‘detector’. The smallest blue circles
must move the fastest followed by the orange circles their m/z ratios and a mass spectrum is generated.
and then the green circles.
72
32
81 107
25
72
80 Da
53
65
Observed peptide
masses
80 Da implies
152 of 1
presence
192
81phosphate
group!
107
152
164
25192
53
143
92 122
Presence
of 2
151 170
65
164
phosphate group!
143
92 122 151 170
m/z
160 Da
81 107
32
143
92 122 151 170
25
m/z
3
Superimposed image
164
53
65
Relative abundance
Expected peptide
masses
Relative abundance
2
MALDI-TOF analysis – Data interpretation
Relative abundance
1
Part 2, Step 3 (c)
m/z
Thus, examination and
comparison of list of observed
peptide masses with expected
peptide masses enables
simple detection of PTMs.
4
Action Description of the action
5
As
shown
in
animatio
n.
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.
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.
1
Part 2, Step 4 (a)
LC-MS/MS based approach – Liquid chromatography
Buffer
Buffer
solution
2
solution
2
3
PO43-
Phosphorylated
protein remains
bound
Metal ions
Miniaturized
immobilized metal
affinity columns
Direction of
migration
PO43-
M3+
M3+
PO43M3+
IMAC: Ga3+, Zn2+, Fe3+
Other affinity columns: TiO2
Sample protein
mixture
Purified phosphorylated protein
4
Action Description of the action
5
Phosphorylated
residue of
protein
As
shown
in
animatio
n.
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.
TiO2 columns were found to have
better selectivity and sensitivity of
detection for phosphorylated peptide
binding when compared to IMAC.
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.
1
2
Part 2, Step 4 (b)
LC-MS/MS based approach – Tandem mass spectroscopy
Purified protein
Digested protein
MS/MS
analysis
Analysis and
interpretation of data
is carried out as
described for MALDITOF-MS.
LASER
Detector
3
TOF 2
(RF
mode)
TOF 1
(scanning
mode)
4
Collision cell
Action Description of the action
5
Reflector
As
shown
in
animatio
n.
Audio Narration
First show the reaction on top appearing after which an arrow from The protein purified by liquid chromatography is then
the tube on the right must indicate that this sample enters the
subjected to typtic digestion followed by analysis using tandem
instrument below. Show the instrument below as depicted followed mass spectrometry. Here we demonstrate the use of MALDIby appearance of a red beam from the red box which must strike
TOF-TOF-MS for resolution of the generated ion fragments.
the dotted line. Next, the colored circles must appear which must
Separation is based on the flight time of the ions and greater
move into the pink box with the smallest moving the fastest & vice resolution is achieved due to the presence of two mass
versa. The yellow circle must then move to the other end of the
analyzers. The peptide ion spectrum generated is analyzed by
‘collision cell’ followed by appearance of three smaller circles and comparing it with the expected spectrum, thereby allowing
disappearance of the large yellow circle. These smaller circles must determination of modified peptides having different m/z values.
move towards the detector, with the smallest moving the fastest.
Master Layout (Part 4)
1
2
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
Autoradiography film
[g-33P]
ATP
ADP
Protein array
Kinase
enzyme
3
Phosphorylated
proteins
1. Protein microarrays
Array
scanning
`
4
Antibody microarrays
Phosphorylated
proteins
5
Labeled protein
mixture
2. Antibody microarrays
1
2
Definitions of the components:
Part 4 – 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.
3
4
5
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.
1
2
3
4
5
Definitions of the components:
Part 4 – Microarray-based detection techniques
for PTMs
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.
1
Part 4, Step 1 (a)
Protein microarrays
2
Ser
[g-33P]
ATP
ADP
Ser
Kinase enzyme
3
Protein
substrate
Proteome array containing
potential substrates for
phosphorylation
Phosphorylated
protein
33P] ATP
[g-Kinase
enzyme
solution
4
Action Description of the action
5
As
shown
in
animatio
n.
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.
Audio Narration
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.
1
Part 4, Step 1 (b)
Phosphorylated
proteins
Protein microarrays
Washing
2
Proteome array
P
33
P
33
P
33
P
33
3
P
33
Radioactive
emissions
4
Developed image
Action Description of the action
5
As
shown
in
animatio
n.
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.
DetectionAutoradiography
film
Audio Narration
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.
1
Part 4, Step 2 (a)
Antibody microarrays
Labeled protein
mixture added
2
3
Polymer coated
glass slide
Anti-phospho serine, threonine and
tyrosine antibodies immobilized on
array
Specific binding of
phosphorylated
proteins
4
Action Description of the action
5
As
shown
in
animatio
n.
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.
Audio Narration
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.
1
Part 4, Step 2 (b)
Antibody microarrays
Microarray scanner
Array washing
Unbound proteins
removed
2
3
Bound phosphorylated
proteins
Array image
4
Action Description of the action
5
As
shown
in
animatio
n.
Audio Narration
Please re-draw all images. The blue solution must appear on The array is then washed to remove any excess
top of the existing components and must then be removed
unbound proteins from the surface. This is followed by
from the array surface such that only the green circles remain scanning of the array using a microarray scanner at a
bound to the surface as shown. The ‘microarray scanner’
suitable wavelength to detect the fluorescent tag of the
image must then appear and the array must shrink and be
bound proteins. This method offers sensitive and
placed on the extended grey region as depicted in animation.
simultaneous detection of large number of post
Next the orange beam must appear which must fall on the
translationally modified proteins.
array surface followed by appearance of the computer with
the image pattern as shown.
1
Interactivity option 1:Step No: 1
Based on the two MALDI-TOF-MS spectrums shown below, determine the total
number of phosphorylated sites present in the protein.
3
77
40
25
81 107
164
53
96 122143
151 170
62
Relative abundance
2
Relative abundance
Expected peptide
masses
Observed peptide
masses
157
25
4
A) 4
Interacativity Type
5
Choose the correct
answer.
133
62
m/z
Answers:
200
81
164
96 122143 170
151
m/z
B) 6
Options
C) 5
D) 7
Results
The correct answer is B. Once the user
User must be shown the two
graphs on top followed by the chooses an answer, a sign must first be
question below and the answers.displayed saying ‘correct’ or ‘incorrect’
User must be allowed to choose depending on the answer given. Next, the
colored boxes along with the double
one of the four options.
headed arrows must appear sequentially as
depicted in animation.
267
1
Questionnaire
1. Proline most commonly undergoes which of the following PTMs?
Answers: a) Glycosylation b) Phosphorylation c) Hydroxylation d) Acylation
2
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?
3
Answers: a) Pro-Q-diamond
b) SYPRO-Ruby Red
c) SYPRO-Ruby Orange
d) Coomassie brilliant blue
4
4. 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
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+
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, 24:169-177.
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, 154:250-254.
Posewitz MC, Tempst P: Immobilized gallium(III) affinity chromatography of
phosphopeptides. Anal Chem 1999, 71:2883-2892.
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, 6:2436-2445.
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, 4:873-886.
Oda Y, Nagasu T, Chait BT: Enrichment analysis of phosphorylated proteins as a tool for
probing the phosphoproteome. Nat Biotechnol 2001, 19:379-382.
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, 15:1593-1599.
Links for further reading
Research papers:
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•
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•
Wilm M, Mann M: Analytical properties of the nanoelectrospray ion source. Anal Chem 1996,
68:1-8.
Erdjan Salih, Phosphoproteomics by mass spectrometry and classical protein chemistry
approaches. Mass Spectrometry Reviews 2005, 24:828-846.
Morelle, W. et al., The use of mass spectrometry for the proteomic analysis of glycosylation.
Proteomics 2006, 6:3993-4015.
Ptacek, J. et al., Global analysis of protein phosphorylation in yeast. Nature Letters 2005,
438:679-684.
Zhu, H. et al., Global Analysis of Protein Activities Using Proteome Chips. Science 2001,
293:2101-2105.
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