Applications of protein microarrays

1. Harini Chandra Affiliations Applications of protein microarrays Protein microarrays have found wide applications for discovery and functional proteomic studies. They allow rapid analysis of thousands of proteins simultaneously.

2. Master Layout (Application 1) 1 This animation consists of 3 parts: Application 1 - Antigen-antibody interactions Application 2 - Biomarker detection Application 3 - Protein interaction studies Detection of antigen-antibody interactions at various concentrations using antigen and antibody microarrays. 2 Labelled antibodies Labelled antigens ` 3 Antibody array Antigen array Poly-L-lysine coated slide 2 3 1 2 3 1 4 Varying antibody concentrations Varying antigen concentrations 6 4 6 5 4 5 5 Haab B, Dunham M, Brown P: Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions. Genome Biol 2001, 2 (2).

3. Definitions of the components:Application 1 -Antigen-antibody interactions 1 1. Antibody array: An array onto which 114 different antibodies were spotted, for detection of varying concentrations of antigens from a protein mixture. In this studyauthors made use of six such arrays for detection of six unique antigen mixtures. 2. Poly-L-lysine coated slide: The antibodies were printed on to a glass microscopic slide coated with poly-L-lysine for effective capture on to the surface. 3. Labelled antigens: Six different concentrations of Cy3 or Cy5 labelled antigens were detected by means of the antibody arrays. 4. Antigen array: Six arrays of 116 different antigens were spotted, 6-12 times each, for detection of the corresponding antibodies. Varying concentrations of antibody solutions were used. 5. Labelled antibodies: Antibodies labelled with Cy3 or Cy5 dyes were used for probing the antigen arrays. 2 3 4 5

4. Application 1, Step 1: 1 Labelled antigens 2 3 Poly-L-lysine coated slide Antibody array 4 Action Description of the action Audio Narration Haab et al. (2001) printed six arrays of 114 different antibodies on to poly-L-lysine coated glass slides using a robotic arrayer. These were used to analyze interactions in six unique antigen mixtures ranging in concentration from 1.6 mg/mL to 1.6 ng/mL. The antigens were tagged with Cy3 and Cy5 fluorescent labels. The brown shapes must bind to the black surface followed by selective binding of the green circles as shown. First show the black parallelogram on the left with its labels. Then show the brown Y shaped objects binding to this surface. Then show the various colored shapes with their flags. The green circles alone must bind to the Y shaped bodies below as shown in animation. 5

5. Application 1, Step 2: 1 Washing solution 2 Unbound antigens removed 3 Specific Ag-Ab binding Antibody array Antigen-antibody interactions 4 Action Description of the action Audio Narration First show the black parallelogram with the bound green circles and other unbound, flagged shapes. Then show the blue cloud appearing on top. This must wash away all the coloured, flagged shapes except the green circles which must remain bound as shown on the right. The blue cloud must appear on top of the black parallelogram and other flagged shapes above. Once the antigen-antibody binding reaction was complete, excess unbound antigens were washed off using phosphate buffered saline and water at room temperature. 5

6. Application 1, Step 3: 1 Microarray scanner 2 Array image 3 Varying antigen concentrations 4 2 3 1 Action Description of the action Audio Narration (Please redraw all figures) First show the picture of the ‘scanner’ followed by a beam of light coming out as shown and falling on the plate below. Then show the arrow to the computer followed by the ‘array image’. Once the excess antigens were washed off, the bound antigen-antibody interactions were detected by means of a microarray scanner at wavelengths of 532 nm and 635 nm. The authors found that such microarrays of antibodies could detect their corresponding antigens at concentrations as low as 1 ng/mL.. The picture on top left must be shown followed by the beam of light and the parallelogram below. 5 6 4 5

7. Application 1, Step 4: 1 Antigen mixture Labelled detection antibodies 2 ` ` 3 Glass microscopic slide Antigen array Specific Ag-Ab binding 4 Action Description of the action Audio Narration The blue and red colored shapes must attach the to grey surface below followed by binding of the orange inverted Y shapes to the blue figures. First show the black surface followed by the blue and red shaped above which must move down and attach themselves to the surface below. Then show the inverted Y brown and orange shapes and show only the orange ones binding to the blue shapes below. In a complimentary experiment, the authors generated six antigen arrays having 116 different antigens , which they probed with Cy3 or Cy5 labeled antibodies of varying concentrations. 5

8. Application 1, Step 5: 1 Washing solution 2 Unbound antibodies removed ` ` 3 Antigen-antibody array Specific Ag-Ab binding 4 Action Description of the action Audio Narration Show the blue cloud appearing over the grey array of shapes and then removing only the brown shapes with it. First show the grey parallelogram with the inverted orange Y shapes bound to the blue shapes below and other unbound shapes. Then show the blue cloud appearing and removing with it only the brown inverted Ys and leaving the orange Ys bound to the blue shapes below. The antigen-antibody binding reaction was allowed to go to completion and excess unbound antibody was washed away using PBS and water at room temperature. 5

9. Application 1, Step 6: 1 Microarray scanner 2 Array image ` 3 Varying antibody concentrations 4 2 3 Action Description of the action Audio Narration 1 Show the scanner picture with the green beam coming out from it and falling on the black surface below, as shown. (Please redraw all figures) First show the picture of the scanner followed by the green beam appearing from it and falling on the black surface with bound figures on it. Then show the arrow to the computer followed by the ‘array image’. The microarray slides were scanned at 532 nm and 635 nm. It was found that these antigen arrays allowed detection and quantitation of antibodies down to absolute concentrations of 100 pg/mL. These detection limits can further be improved by using high affinity and purity antibodies, thereby showing great promise for high throughput and sensitive clinical applications. 5 6 4 5

10. Master Layout (Application 2) 1 This animation consists of 3 parts: Application 1 - Antigen-antibody interactions Application 2 - Biomarker detection Application 3 - Protein interaction studies Identification of potential diagnostic markers for infection from Neisseriameningitidis. 2 Protein expression & purification N. meningitidis genes Cloned genes E. coli host cell 3 Array screening Array spotting Purified bacterial proteins Patient sera 4 Detection & analysis 5 Steller, S. et al. Bacterial protein microarrays for identification of new potential diagnostic markers for Neisseria meningitidis infections. Proteomics 2005, 5, 2048-2055.

11. Definitions of the components:Application 2 – Biomarker detection 1 1. E. coli host cell: The genes of interest from N. Meningitidiswereexpressed through suitable vectors such as plasmids in a host system like E. coli, which provides the required cellular machinery for transcription and translation. 2. N. meningitidis genes: The authors amplified 102 phase-variable genes of serogroup B strain MC58 by PCR and then cloned them for expression in E. coli. 3. Cloned genes: The genes that have been inserted into the plasmid vector and are ready for expression into proteins by the host system. 4. Protein expression & purification: Once the genes have been taken up by the expression vector, they are transcribed and translated into their corresponding proteins by the cellular machinery of the host. These proteins are then purified by chromatographic or electrophoretic separation procedures to obtain the pure proteins for array spotting. 5. Purified bacterial proteins: The authors were able to express and purify 67 of the proteins of N. meningitidis, representing 66% of the genes. 2 3 4 5

12. Definitions of the components:Application 2 – Biomarker detection 1 6. Array spotting: The purified proteins were spotted on to the coated glass microarray slides using a robotic arrayer to generate pure protein arrays. 7. Array screening: The protein microarrays were then probed for markers using sera from patients suffering from meningitis, a highly contagious disease that causes epidemic outbreaks. 8. Patient sera: The authors tested 20 male and female convalescent patients as well as sera from healthy controls. 9. Detection & analysis: The protein microarrays slides were scanned using a microarray scanner and then analyzed using a suitable software. 2 3 4 5

13. Application 2, Step 1: 1 Transcription & translation machinery Cloned genes N. meningitidis genes E. coli host cell 2 3 Expressed bacterial proteins 4 Action Description of the action Audio Narration Steller et al. (2005) amplified and subcloned 102 genes from N. meningitidis for expression in E. coli. Clones were grown for 16h at 37oC in antibiotic containing medium, following which protein expression was induced by addition of isopropyl-b-D-thiogalactoside. First show the grey circle in the rounded rectangle followed by the coloured arcs. Then show these entering the rectangle and forming a circle as shown in the next figure. Then show the coloured ovals appearing and the rest of the figures as in the animation followed by the coloured protein figures below. As shown in animation. 5

14. Application 2, Step 2: 1 His6 tagged proteins Purified proteins 2 Separation based on MW Expressed bacterial proteins Ni-NTA coated beads Direction of migration 3 SDS-PAGE Chromatographic purification 4 Unwanted E. coli proteins Array printing Proteins of interest from N. meningitidis Action Description of the action Audio Narration As shown in the animation. (Please redraw all figures.) First show the mixture in the can on top along with the first column. Then show the mixture in the can being poured into the column followed by appearance of the flask on top. The grey objects must then come out of the column into the tube below. Next the second column and tube must appear and from this the yellow, blue & red objects must come down into the tube below. Finally the third column and tube must appear and the green & purple object must come into the tube followed by the label below. The figure on the right must be shown with the 3 tubes and the blue slab. The arrow mark with labels must appear followed by colored spots. The 2nd & 3rd tubes & spots must then be highlighted as shown. The cells were harvested four hours after induction and their proteins purified based on specific Ni-NTA binding followed by analysis on SDS-PAGE. The 67 purified proteins obtained were then printed on nitrocellulose coated glass slides using a robotic arrayer. 5 Biochemistry by A.L. Lehninger, 3rd edition (ebook)

15. Application 2, Step 3: 1 Microarray scanner Labelled secondary antibodies 2 Patient sera Detection 3 47 immunogenic proteins detected of which OpaV (NMB0442)showed response in 11 patients. Protein microarray 4 Action Description of the action Audio Narration (Please redraw all figures) First show the grey parallelogram followed by binding of the yellow cloud with its inverted Y shapes to its surface. This is followed by binding of the purple inverted Ys to the green ones and then appearance of the picture of the scanner as shown followed by comp & the right-most figure. As shown in animation. The array was probed with sera from 20 convalescent patients by incubating it overnight at 4oC. The array was then washed with PBS and detection carried out by means of Cy5 labeled secondary antibody. Excess detection antibody was washed off and the array then dried and scanned. Authors detected 47 immunogenic proteins, one of which showed response in 11 of the patients. Protein microarrays have been successfully used for detection of several other disease biomarkers like cancer, autoimmune disorders as well as for diseases like Q-fever and other viral infections. 5

16. Master Layout (Application 3) 1 This animation consists of 3 parts: Application 1 - Antigen-antibody interactions Application 1 - Biomarker detection Application 3 - Protein interaction studies Biotinylated calmodulin Array printing 2 Phosphoinositides 3 His6 tagged yeast proteins Nickel-coated slide Array signal 4 5 Zhu, H. et al. Global analysis of protein activities using proteome chips. Science 2001, 293:2101-2105.

17. Definitions of the components:Application 3 – Protein interaction studies 1 1. Array printing: Authors cloned and overexpressed 5800 open reading frames of yeast. These proteins were purified and spotted at high spatial density on to microarray slides to give a yeast proteome microarray. 2. Nickel coated slide: The microscopic slide surface was coated with nickel in order to facilitate better capture of the His6 tagged proteins. The group had initially used aldehyde functionalized slides which reacted with the amine groups of proteins but observed superior signals with nickel coated slides. 3. His6 tagged yeast proteins: The yeast proteins were fused with His6 tags in order to enable capture on to the nickel-coated array surface. 4. Biotinylated calmodulin: The authors probed the bound yeast proteins for protein-protein interactions using biotinylated calmodulin in the presence of calcium. Six known calmodulin targets were identified in addition to 33 additional potential partners. 5. Phosphoinositides (PI): The proteome chips were also probed for protein-lipid interactions using five types of PI liposomes, each containing phosphatidyl choline. 6. Array signal: The protein-protein and protein-lipid interactions are detected using a microarray scanner and signal generated. 2 3 4 5

18. Application 3, Step 1: 1 Anti-GST antibodies Array printing 2 3 His6 tagged yeast proteins Whole proteome array Nickel-coated slide Signals observed for more than 93.5% protein samples! Detection signal output 4 Action Description of the action Audio Narration Zhu et al. (2001) generated a yeast whole proteome array by expressing 5800 purified proteins on a single nickel coated slide. The chips were probed with anti-GST antibodies to determine the reproducibility of protein immobilization. More than 93.5% of protein samples were found to give significant signals and over 90% contained 10 to 950 fg of protein. First show the grey surface and then the picture of the robotic arm. This must move across the slide with appearance of spots as shown. Next, the purple coloured inverted Y shapes must bind to the spots with appearance of the square with red dots below as shown. As shown in animation. 5

19. Application 3, Step 2: 1 Cy3-streptavidin Phosphoinositides Biotinylated calmodulin 2 3 Protein-protein interaction studies Protein-lipid interaction studies 4 Action Description of the action Audio Narration As shown in animation. First show the surface with spots on the left. Then show binding of the blue inverted U shaped objects to the upper half of the spots as shown. Then show the surface on the left and binding of the purple inverted Vs to the bottom half o f the spots. Next, the green inverted Vs with stars must bind to the blue and purple shapes. To understand the potential applications of such whole proteome arrays, the authors screened the immobilized proteins for protein-protein and protein-lipid interactions. Theyused biotinylated calmodulin in presence of calcium and phosphoinositide liposomes respectively. Detection was carried out using Cy3 labeled streptavidin. 5

20. Application 3, Step 3: 1 Signal output – green spots indicate either calmodulin or phosphoinositide binding proteins. Microarray scanner 2 3 6 known calmodulin targets and 33 potential targets were identified in this study, which could provide more information about the role of calmodulin in cellular processes. The PI liposomes identified 150 protein targets, of which 52 were uncharacterized proteins. 45 of the proteins were found to be membrane associated. 4 Action Description of the action Audio Narration (Please redraw all figures) Show the picture of the scanner with the light beam coming out of it and illuminating the surface below. Then show arrow to the comp followed by appearance of the black rectangle with spots on the right and the text below. 6 known calmodulin targets and 33 potential partners were identified with 14 of these proteins possessing a consensus sequence. The PI liposomes were able to identify 150 protein targets, of which 45 were found to be membrane associated, predicted to have membrane spanning regions. This study testified the tremendous potential of whole proteome arrays in identifying new protein targets. As shown in animation. 5

21. Interactivity option 1:Step No:1 1 The advent of protein microarrays has been marked by the work done by MacBeath and Schreiber (2000) who made use of protein arrays to study three well-characterized protein-protein interactions namely i) protein G-IgG, ii) p50- IkBa and iii) FKBP12-rapamycin binding domain-FKBP12 in presence of rapamycin. Match these interactions on the array below in order to get the signal output. 2 IgG FKBP12 + Rapamycin FKBP12 binding domain-FKBP12 3 Protein G-IgG IkBa p50- IkBa FKBP12-rapamycin binding domain Protein G p50 4 Results Interacativity Type Options Boundary/limits Once the user matches the shape correctly, the glowing spots on the black surface shown on the right must appear. User must drag and drop the figures given on the left to their corresponding shapes given on the grey surface. Drag and drop. 5 MacBeath, G., Schreiber, S. L., Printing proteins as microarrays for high-throughput function determination. Science 2000, 289, 1760–1763.

22. Interactivity option 2:Step No:1 1 Miller et al. (2003) robotically spotted 184 unique antibodies on polyacrylamide based hydrogels and poly-l-lysine coated glass slides, which they probed with sera from prostate cancer patients as well as healthy controls. Shown below are the results obtained for the hydrogel slides which were found to give better signals. 2 Prostate cancer patient sera Control group sera 3 Polyacrlyamide-hydrogel slides Villin IgM a 1-antichymotrypsin IgG von Willebrand Factor 4 Five proteins were shown to have significantly different expression levels in prostate cancer patient samples compared to the control group. vWF was found to be elevated and the remaining four were lower relative to the control group. 5 Miller JC, Zhou H, Kwekel J, Cavallo R, Burke J, Butler EB, Teh BS, Haab BB: Antibody microarray profiling of human prostate cancer sera: antibody screening and identification of potential biomarkers. Proteomics 2003, 3:56-63.

23. Interactivity option 2:Step No:2 1 Which of the following test group array patterns corresponds to the results described above? (Red indicates elevated expression while green indicates reduced expression compared to normal levels) 2 B) A) 3 C) D) 4 Results Interacativity Type Options Boundary/limits Option A is the correct answer. If user chooses this, a popup must appear saying ‘Correct answer’ else it must say ‘incorrect’. User must be allowed to choose one of the four options. Choose the correct option. 5

24. Questionnaire 1 1. What absolute concentration of antibodies could be detected using antigen microarrays by Haab et al. (2001)? Answers: a) 90 pg/mL b) 1 ng/mLc) 100 pg/mL d)‏ 100 ng/mL 2. Microarray slides coated with nickel interact with which of the following fusion domains? Answers: a) GST b) His6 c) Biotin d)‏Avidin 3. p50 interacts with which of the following? Answers: a) p53 b) Protein G c) IgGd)‏IkBa 4. FKBP12 binding domain does not give any signal on interaction with FKBP12 due to absence of which of the following molecules? Answers: a) Calcium b) Rapamycin c) Nickel d)‏ Biotin 5. How many of the 67 proteins of N. meningitidis were found to be immunogenic? Answers:a) 47 b) 67 c) 11 d)‏33 2 3 4 5

25. Links for further reading Research papers: • Sreekumar A, Nyati MK, Varambally S, Barrette TR, Ghosh D, Lawrence TS, Chinnaiyan AM: Profiling of cancer cells using protein microarrays: discovery of novel radiation-regulatedproteins. Cancer Res 2001, 61:7585-7593. • Ehrlich, J. R., Qin, S., Liu, B. C. S. The ‘reverse capture’ autoantibody microarray: a native antigen-based platform for autoantibody profiling. Nat protoc. 2006, 1(1), 452-460. • Nishizuka S, Charboneau L, Young L, Major S, Reinhold WC, Waltham M, Kouros-Mehr H, Bussey KJ, Lee JK, Espina V et al.: Proteomic profiling of the NCI-60 cancer cell lines using new high-density reverse-phase lysate microarrays. Proc Natl Acad Sci USA 2003, 100:14229-14234. • Carbayo, M. S., Socci, N. D., Lozano, J. J., Haab, B. B., Cardo, C. C., Profiling bladder cancer using targeted antibody arrays. Am. J. Pathology 2006, 168, 93–103. • Chan, S. M., Ermann, J., Su, L., Fathman, C. G., Utz, P. J. Protein microarrays for multiplex analysis of signal transduction pathways. Nature medicine 2004, 10(12), 1390-1396.