Transcript Application 1

Applications of nanotechniques
in proteomics
Nanotechniques have found an increasing number of
applications for proteomic studies due to their
advantages over conventional approaches such as
assay miniaturization, high sensitivity, real-time
multiplexed analysis and low sample consumption.
Harini Chandra
Affiliations
Master Layout (Application 1)
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This animation consists of 3 parts:
Application 1 – Immunological studies
Application 2 – Protein interaction studies
Application 3 – Biomarker detection
COOH
Use of aptamer functionalized silicon
nanowire-field effect transistors for
detection of vascular endothelial
growth factor (VEGF).
COOH
Silicon nanowire
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VEGF sample
injection
CONH
CONH
Detection
4
Current
Anti-VEGF RNA aptamer
Gate voltage
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Zheng, G., Patolsky, F., Cui, Y., Wang, W. U., Lieber, C. M., 2005. Nat Biotechnol. 23, 1294-1301.
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Definitions of the components:
Application 1 – Immunological studies
1. Silicon nanowire: Si nanowires are semiconducting devices that can
be modified with suitable biomolecules and used for detection of analytes
by means of a measurable change in the localized current.
2. Anti-VEGF RNA aptamer: Aptamers are small oligonucleotide
molecules that can bind specifically to certain analytes. The RNA aptamer
is a short artificial oligonucleotide sequence that binds with high affinity
and specificity to Vascular Endothelial Growth Factor (VEGF), useful for
cancer diagnosis.
3. VEGF sample injection: The VEGF is a useful molecule for cancer
diagnosis and is applied to the functionalized silicon nanowire surface
where it is detected.
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4. Detection: Changes in current due to localized charge transfers
caused by binding events are monitored continuously.
1 Application 1, Step 1:
COOH
COOH
3’
Silicon nanowire
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N
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Anti-VEGF RNA aptamer
CONH
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CONH
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N
H
Si
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Action
The pink strip
must be
zoomed into
and figure on
the right
shown.
Description of the action
(Please redraw all figures.)
First show the figure on top with its
label followed by the appearance of a
pink strip on top of the blue strip. This
pink strip must be zoomed into and
figure on the right must be shown.
O
Audio Narration
Lee et al. (2009) innovatively made use of aptamers as
recognition elements for real-time, label-free detection
of cancer markers. These RNA aptamers showed
specificity for binding to VEGF, a useful cancer marker.
1 Application 1, Step 2:
CONH
CONH
VEGF sample
injection
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Current
3
Gate voltage
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Action Description of the action
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Liquid must be
shown to come
out of the
injection
followed by
appearance of
the graph.
(Please redraw all figures.)
First show the grey surface followed
by the injection with its label. This
injection must be pressed and the
pale orange liquid must appear on top
of the pink strip. Once this happens,
the curve on the graph must appear
simultaneously on the right.
Audio Narration
Binding of VEGF to the anti-VEGF RNA aptamer on
the silicon nanowire surface brought about localized
charge transfers which in turn caused a change in
current. This localized current change was detected
and was useful for monitoring such binding events.
Master Layout (Application 2)
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This animation consists of 3 parts:
Application 1 – Immunological studies
Application 2 – Protein interaction study
Application 3 – Biomarker detection
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Protein mixture
Use of carbohydrate encapsulated
gold nanoparticles as affinity probes
for protein separation and on-probe
detection by mass spectroscopy.
Proteolytic
digestion
Binding of
target protein
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Carbohydrate
encapsulated
gold nanoparticle
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Centrifugation
Unbound protein
MS analysis
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Bound protein
(pellet)
MALDI-TOF-TOF-MS
Chen, Y. J., Chen, S. H., Chien, Y. Y., Chang, Y. W., Liao, H. K., Chang, C. Y., Jan, M. D., Wang, K. T., Lin, C. C., 2005.
Chembiochem. 6, 1169-1173.
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Definitions of the components:
Application 2 – Protein interaction study
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1. Carbohydrate encapsulated gold nanoparticles: Functionalized gold
nanoparticles can serve as useful detection and targeting agents. These
AuNPs when encapsulated with carbohydrate ligands can serve as a
useful affinity probe for rapid, efficient separation of target proteins
followed by on-probe analysis using mass spectroscopy. This technique of
protein targeting allows mapping of carbohydrate-recognition peptide
sequences, which could provide very useful information on carbohydrate
interactions and their biological roles.
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2. Protein mixture: The mixture of proteins containing the target protein
of interest as well as other unwanted protein impurities.
3. Proteolytic digestion: The target proteins once bound to the
carbohydrate encapsulated gold nanoparticles are digested into small
peptide fragments using proteolytic enzymes.
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4. Centrifugation: The process by which the components of a mixture get
separated based on their relative densities. Here, the free peptides remain
in the supernatant while the bound peptides settle down in the form of a
pellet.
5. MALDI-TOF-TOF-MS: A mass spectrometry method that makes use of
a laser beam for sample ionization (MALDI) and time of flight (TOF) tube
for detection of peptide fragments based on the time taken to reach the
detector.
1 Application 2, Step 1:
Nanoparticle encapsulation &
addition of protein mixture
Protein mixture
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Carbohydrate
ligand
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Gold nanoparticle
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Action
5
The yellow circles
must appear
followed by their
red surface
features. The
protein mixture
must then move
down as shown.
Description of the action
(Please redraw all figures.)
First show the appearance of the
yellow circles followed by their red
surface structures and their
appropriate labels.
Then show the protein mixture on top
and show it moving down into the grey
container near the yellow circles.
Audio Narration
Gold nanoparticles functionalized with suitable
carbohydrate ligands were used by Chen et al. (2008)
for targeting specific proteins from a mixture. They
carried out separation of galactophilic lectins from
Pseudomonas aeruginosa by binding galactose and
another carbohydrate (Pk antigen) to the gold
nanoparticle surface.
1 Application 2, Step 2:
Protein targeting and digestion
Proteolytic enzymes
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4
Action
5
The blue objects
(noodle-like) must
bind to the red
surface features
on the yellow
circles as shown.
Description of the action
Audio Narration
(Please redraw all figures.)
The target lectin proteins bound selectively to the
First show the blue, noodle-shaped objects carbohydrate ligands on the surface of the gold
moving and binding with the red surface
nanoparticles. Proteolytic enzymes were then
features present on each of the yellow
added for digestion of the bound target proteins.
circles as shown in the animation. Then
show appearance of the brown & white
circles on top followed by their motion
downward next to the yellow circles.
1 Application 2, Step 3:
Protein digestion
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Free peptide fragments
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Bound
fragments
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Action
5
Show the blue
objects being cut
into small
fragment pieces
as shown.
Description of the action
(Please redraw all figures.)
The blue objects must be cut into
small pieces by the white and
brown circles , some of which
must remain on the yellow circle
surface while others become free,
as shown.
Audio Narration
The enzymes cleaved the bound proteins into
peptide fragments, some of which remained bound
to the NP while others went into solution.
1 Application 2, Step 4:
Centrifugation and detection
MS Spectra
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Supernatant
Pellet
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MALDI-TOF-TOF-MS
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Action
5
Show a spinning
motion of the
contents on the
left followed by
the animation as
shown on the
right.
Description of the action
(Please redraw all figures.)
First show the figure on the left and its
spinning motion followed by the labels.
Then show the blue line and yellow
circle group moving as shown in the
animation followed by appearance of
the graphs above.
Audio Narration
The authors carried out direct on-probe MS analysis
using MALDI-TOF-TOF following separation of the
nanoparticles by centrifugation. A clean MS profile
was observed with no significant peaks being
observed due to the NPs, indicating the tremendous
potential of nanoprobe-based affinity mass
spectrometry.
Master Layout (Application 3)
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This animation consists of 3 parts:
Use of functionalized SWNT as multicolor
Application 1 – Immunological studies
Application 2 – Protein interaction studies Raman labels for multiplexed protein
Application 3 – Biomarker detection detection.
12C
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13C
Detection of biomarkers for
Wegener’s granulomatosis using
single walled nanotubes (SWNT)
Raman labels.
GaH-SWNT
Mouse IgG
Human IgG
Mouse IgG
Human IgG
GaH-IgG-SWNT
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GaM-SWNT
Anti-proteinase 3
Multiplexed antigen detection
Gold coated
array surface
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Serum proteins
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Other human IgGs
Raman intensity
Proteinase-3
Anti-human IgG
13C SWNT
12C SWNT
Anti-mouse IgG
Raman shift (cm–1)
Chen, Z., Tabakman, S. M., Goodwin, A. P., Kattah, M. G., Daranciang, D., Wang, X., Zhang, G., Li, X., Liu, Z., Utz, P. J., Jiang,
K., Fan, S., Dai, H., 2008. Nat Biotechnol. 26, 1285-1292.
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Definitions of the components:
Application 3 – Biomarker detection
1. Gold coated array surface: The surface used for immobilization of the
antigen of interest followed by detection using a Raman active agent
(single walled carbon nanotubes, in this case) is coated with gold in order
to enhance the SERS effect and provide better signal output.
2. Proteinase 3: This is a clinically relevant biomarker used in diagnosis
of the human autoimmune disease Wegener’s granulomatosis. The
antigen is immobilized onto the gold-coated array surface and probed with
serum sample containing the corresponding autoantibody.
3. Anti-proteinase 3: Autoantibody against the antigen proteinase-3,
which is directly implicated in pathogenesis of the disease and is used to
detect antigen bound to the array surface.
4. GaH-IgG-SWNT: The goat-anti-human antibody against the antiproteinase 3 antibody that is used for detection of antigen-antibody
interaction by combining it with a Raman active SWNT.
5. Serum proteins: Other proteins that may be present in the serum
sample used to probe for the antigen of interest.
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Definitions of the components:
Application 3 – Biomarker detection
6. Multiplexed antigen detection: Differentially labelled SWNT Raman
tags can be used for simultaneous detection of two types of IgGs.
7. 12C-GaM-SWNT: Goat-anti-mouse antibody that has been coupled with
12C SWNT Raman label and binds specifically to mouse IgGs present on
the array surface.
8. 13C-GaH-SWNT: Goat-anti-human antibody that has been coupled with
13C SWNT Raman label thereby producing a different colour label when it
binds specifically to human IgGs.
1 Application 3, Step 1:
GaH-IgG-SWNT
Anti-proteinase 3
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Serum sample
Proteinase-3
Gold coated
array surface
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Serum proteins
Action
The blue
inverted V must
bind to the
green spot and
the pink
inverted V must
bind to the blue
V.
Other human IgGs
Description of the action
(Please redraw all figures.)
First show the yellow parallelogram
with its label. Then show the green
spots followed by the inverted V
shaped objects and triangles and the
green cloud. Then show the pink
inverted V attached to the rectangle
binding to the inverted blue V as
shown.
Audio Narration
The proteinase 3 antigens were captured on a goldcoated microarray surface. Chen et al. (2008).
successfully detected autoantibodies in human serum
against proteinase 3, a clinically important biomarker
for diagnosis of the autoimmune disorder Wegener’s
granulomatosis. SWNT conjugated to GaH-IgG were
used for detection of antigen-antibody binding.
1 Application 3, Step 2:
Multiplexed antigen detection
GaM-SWNT
13C
GaH-SWNT
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Mouse IgG
Human IgG
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Mouse IgG
Human IgG
Raman intensity
12C
Anti-human IgG
13C SWNT
12C SWNT
Anti-mouse IgG
Raman shift (cm–1)
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Action Description of the action
5
The red and
blue inverted
Vs must bind to
the circles as
shown with
simultaneous
appearance of
the graph.
(Please redraw all figures.)
First show the pale purple parallelogram
followed by the circles and their labels. Then
show appearance of the inverted blue and red
Vs and axes of the graph. Then show binding
of the blue and red Vs to the circles as shown
along with simultaneous appearance of the
graph on the right.
Audio Narration
Multiplexed detection of more than one antigen was
successfully achieved by using multicolored Raman labels.
Anti-human and anti-mouse IgGs were bound to 13C and 12C
SWNTs respectively. Rapid, protein detection was possible
through excitation with 785 nm laser followed by comparing
the Raman scattering intensity at their respective maxima
which showed very less cross-reactivity.
1 Interactivity option 1:Step No:1
Semiconductor quantum dots have been successfully used for in vivo molecular imaging and active
tumor targeting. Luminescent QDs encapsulated with a triblock copolymer and tumor targeting
ligand were used for in vivo targeting of human prostate cancer. Click on the quantum dots to view
their movement during tumor targeting.
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Normal blood
vessel
QD capping
ligand topo
Tumor angiogenic
vessel
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PEG
4
Tumor targeting
ligand
Interacativity Type
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Click on the image
of dots. (Please
redraw all figures.)
Triblockcopolymer
coating
Options
Boundary/limits
User should be
allowed to click on
the group of dots
shown on the left
of the image
above.
Gao, X., Cui, Y., Levenson, R. M., Chung. L. W., Nie. S., 2004. Nat. Biotechnol. 22, 969-976.
Results
The dots must
move and
distribute
themselves as
shown in the image
in the next slide.
1 Interactivity option 1:Step No:2
Endothelial cells
Tumor cells
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Normal blood
vessel
Tumor angiogenic
vessel
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The QD probes were found to accumulate selectively at the
tumor cells due to antibody binding to the specific cancer cell
surface biomarkers, as well as enhanced permeability and
retention at the tumor sites.
1 Interactivity option 2:Step No:1
Silicon nanowires can be used as sensitvie nanosensors for the label-free detection of protein-small
molecule interactions. Wang et al. (2005) made use of SiNW-FET devices for characterizing the
concentration-dependent inhibition of ATP binding by Gleevec (STI-571) to the enzyme Abl, a
tyrosine kinase enzyme whose activity is responsible for chronic myelogenous leukemia (CML).
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ATP
Drag the green or yellow small
molecules into their enzyme
binding pocket (active site) to view
the corresponding reactions.
Gleevec
P
Phosphorylated
protein product
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Abl enzyme
SiNW
Inhibition of
phosphorylation
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Interacativity Type
Drag and drop.
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Options
The user should be
allowed to drag either the
green or yellow diamond
shapes into the
corresponding pocket in
the blue shape below.
Boundary/limits
Results
If the user drags the green
diamonds into the blue pocket, the
first reaction arrow must appear
along with the product on the
right. If the yellow diamonds are
selected, then the second reaction
arrow must appear as shown.
Wang, W. U., Chen, C., Lin, K. H., Fang, Y., Lieber, C. M., 2005. Proc. Natl. Acad. Sci. USA. 102, 3208-3212.
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Questionnaire
1. Raman labelled SWNTs were used to detect which of the following diseases in the animation
described earlier?
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Answers: a) Breast cancer b) Prostate cancer c) Wegener’s granulomatosis d) Cystic fibrosis
2. The role of gold coating on an array surface is to
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Answers: a) Improve immobilization b) Enhance SERS effect c) Improve antigen-antibody
interaction d) To reduce the refractive index
3. Which of the following principles forms the basis for use of silicon nanowires in detection
studies?
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Answers: a) Change in refractive index b) Change in wavelength of scattered light c) Variation
in temperature d) Changes in conductance
4. Gleevec has been successfully used for treatment of chronic myelogenous leukemia by
inhibition of which of the following enzyme?
Answers: a) Abl kinase b) Amylase c) Peptidyl transferase d) Glucokinase
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Links for further reading
Research papers:

Drouvalakis, K. A., Bangsaruntip, S., Hueber, W., Kozar, L. G., Utz, P. J., Dai, H., 2008. Biosens
Bioelectron. 23, 1413-1421.

EI-Sayed, I. H., Huang, X., EI-Sayed, M. A., 2005. Nano Lett. 5, 829-834.

Geho, D., Lahar, N., Gurnani, P., Huebschman, M., Herrmann, P., Espina, V., Shi, A., Wulfkuhle, J.,
Garner, H., Petricoin, E., 3rd, Liotta, L. A., Rosenblatt, K. P., 2005. Bioconjug Chem. 16, 559-566.

Gokarna, A., Jin, L. H., Hwang, J. S., Cho, Y. H., Lim, Y. T., Chung, B. H., Youn, S. H., Choi, D. S., Lim,
J. H., 2008. Proteomics 8, 1809–1818.

Nam, J. M., Thaxton, C. S., Mirkin, C. A., 2003. Science 301, 1884-1886.

Okuno, J., Maehashi, K., Kerman, K., Takamura, Y., Matsumoto, K., Tamiya, E., 2007. Biosens
Bioelectron. 22, 2377-2381.

Patolsky, F., Zheng, G., Hayden, O., Lakadamyali, M., Zhuang, X., Lieber, C. M., 2004. Proc. Natl. Acad.
Sci. USA. 101, 14017-14022.

Ray, S., Chandra, H., Srivastava, S. 2010. Biosens Bioelectron, doi:10.1016/j.bios.2010.04.010