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Applications of nanotechniques in proteomics

Harini Chandra Affiliations. Applications of nanotechniques in proteomics.

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Applications of nanotechniques in proteomics

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  1. Harini Chandra Affiliations 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.

  2. Master Layout (Application 1) 1 This animation consists of 3 parts: Application 1 – Immunological studies Application 2 – Protein interaction studies Application 3 – Biomarker detection Use of aptamer functionalized silicon nanowire-field effect transistors for detection of vascular endothelial growth factor (VEGF). 2 Silicon nanowire CONH COOH CONH COOH 3 VEGF sample injection Anti-VEGF RNA aptamer Current Detection 4 Gate voltage 5 Zheng, G., Patolsky, F., Cui, Y., Wang, W. U., Lieber, C. M., 2005. Nat Biotechnol. 23, 1294-1301.

  3. Definitions of the components:Application 1 – Immunological studies 1 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. 4. Detection: Changes in current due to localized charge transfers caused by binding events are monitored continuously. 2 3 4 5

  4. Application 1, Step 1: 1 3’ Silicon nanowire 2 NH O O Anti-VEGF RNA aptamer NH 3 COOH CONH COOH CONH Si O O 4 Action Description of the action 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. (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. The pink strip must be zoomed into and figure on the right shown. 5

  5. Application 1, Step 2: 1 VEGF sample injection 2 3 Current CONH CONH Gate voltage 4 Action Description of the action 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. (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. Liquid must be shown to come out of the injection followed by appearance of the graph. 5

  6. Master Layout (Application 2) 1 This animation consists of 3 parts: Application 1 – Immunological studies Application 2 – Protein interaction study Application 3 – Biomarker detection Use of carbohydrate encapsulated gold nanoparticles as affinity probes for protein separation and on-probe detection by mass spectroscopy. Proteolytic digestion Protein mixture 2 Binding of target protein Carbohydrate encapsulated gold nanoparticle Centrifugation 3 4 Unbound protein Bound protein (pellet) MS analysis MALDI-TOF-TOF-MS 5 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.

  7. Definitions of the components:Application 2 – Protein interaction study 1 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. 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. 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. 2 3 4 5

  8. Application 2, Step 1: 1 Nanoparticle encapsulation & addition of protein mixture Protein mixture 2 Carbohydrate ligand 3 Gold nanoparticle 4 Action Description of the action 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. (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. The yellow circles must appear followed by their red surface features. The protein mixture must then move down as shown. 5

  9. Application 2, Step 2: 1 Protein targeting and digestion Proteolytic enzymes 2 3 4 Action Description of the action Audio Narration The target lectin proteins bound selectively to the carbohydrate ligands on the surface of the gold nanoparticles. Proteolytic enzymes were then added for digestion of the bound target proteins. (Please redraw all figures.) First show the blue, noodle-shaped objects moving and binding with the red surface features present on each of the yellow 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. The blue objects (noodle-like) must bind to the red surface features on the yellow circles as shown. 5

  10. Application 2, Step 3: 1 Protein digestion 2 Free peptide fragments 3 Bound fragments 4 Action Description of the action Audio Narration The enzymes cleaved the bound proteins into peptide fragments, some of which remained bound to the NP while others went into solution. Show the blue objects being cut into small fragment pieces as shown. (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. 5

  11. Application 2, Step 4: 1 Centrifugation and detection MS Spectra 2 Supernatant Pellet 3 MALDI-TOF-TOF-MS 4 Action Description of the action 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. (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. Show a spinning motion of the contents on the left followed by the animation as shown on the right. 5

  12. Master Layout (Application 3) 1 This animation consists of 3 parts: Application 1 – Immunological studies Application 2 – Protein interaction studies Application 3 – Biomarker detection Use of functionalized SWNT as multicolor Raman labels for multiplexed protein detection. 12C GaM-SWNT 13CGaH-SWNT Mouse IgG Detection of biomarkers for Wegener’s granulomatosis using single walled nanotubes (SWNT) Raman labels. 2 Human IgG Mouse IgG Human IgG GaH-IgG-SWNT Multiplexed antigen detection Anti-proteinase 3 3 Anti-human IgG Proteinase-3 13CSWNT 12C SWNT Anti-mouse IgG Raman intensity Gold coated array surface 4 Raman shift (cm–1) Serum proteins Other human IgGs 5 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.

  13. Definitions of the components:Application 3 – Biomarker detection 1 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 anti-proteinase 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. 2 3 4 5

  14. Definitions of the components:Application 3 – Biomarker detection 1 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. 2 3 4 5

  15. Application 3, Step 1: 1 GaH-IgG-SWNT Anti-proteinase 3 2 Serum sample Proteinase-3 Gold coated array surface 3 Serum proteins Other human IgGs 4 Action Description of the action Audio Narration The proteinase 3 antigens were captured on a gold-coated 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. (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. The blue inverted V must bind to the green spot and the pink inverted V must bind to the blue V. 5

  16. Application 3, Step 2: 1 Multiplexed antigen detection 12C GaM-SWNT 13CGaH-SWNT 2 Anti-human IgG 13CSWNT 12C SWNT Mouse IgG Anti-mouse IgG Raman intensity Human IgG Mouse IgG 3 Human IgG Raman shift (cm–1) 4 Action Description of the action 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. (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. The red and blue inverted Vs must bind to the circles as shown with simultaneous appearance of the graph. 5

  17. Interactivity option 1:Step No:1 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. QD capping ligandtopo 2 PEG Normal blood vessel Tumor angiogenic vessel Triblock- copolymer coating Tumor -targeting ligand 3 4 Results Interacativity Type Options Boundary/limits The dots must move and distribute themselves as shown in the image in the next slide. Click on the image of dots. (Please redraw all figures.) User should be allowed to click on the group of dots shown on the left of the image above. 5 Gao, X., Cui, Y., Levenson, R. M., Chung. L. W., Nie. S., 2004. Nat. Biotechnol. 22, 969-976.

  18. Interactivity option 1:Step No:2 1 2 Normal blood vessel Tumor angiogenic vessel 3 Endothelial cells Tumor cells 4 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. 5

  19. Interactivity option 2:Step No:1 1 Silicon nanowires can be used as sensitvienanosensors 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). Drag the green or yellow small molecules into their enzyme binding pocket (active site) to view the corresponding reactions. 2 P ATP Gleevec Phosphorylated protein product 3 Abl enzyme SiNW Inhibition of phosphorylation 4 Results Interacativity Type Options Boundary/limits 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. Drag and drop. The user should be allowed to drag either the green or yellow diamond shapes into the corresponding pocket in the blue shape below. 5 Wang, W. U., Chen, C., Lin, K. H., Fang, Y., Lieber, C. M., 2005. Proc. Natl. Acad. Sci. USA. 102, 3208-3212.

  20. Questionnaire 1 1. Raman labelled SWNTs were used to detect which of the following diseases in the animation described earlier? 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 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? 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) Ablkinase b) Amylase c) Peptidyltransferase d)‏Glucokinase 2 3 4 5

  21. 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

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