Protein microarrays

1. Harini Chandra Affiliations Protein microarrays Protein microarrays, a concept that evolved from DNA microarrays, provide a valuable platform for high throughput analysis of thousands of proteins simultaneously.

2. Master Layout (Part 1) 1 This animation consists of 4 parts: Part 1 – Need for protein microarrays Part 2 – Protein expression & purification Part 3 - Array functionalization & printing Part 4 – Protein detection & analysis 2 Protein analysis Protein sample 3 Protein analysis Protein microarray 4 Several protein samples 5

3. Definitions of the components:Part 1 – Need for protein microarrays 1 1. Protein microarray: These are miniaturized arrays normally made of glass, polyacrylamide gel pads or microwells, onto which small quantities of many proteins are simultaneously immobilized. 2. Protein analysis: Proteins can be studied for their structural and functional aspects depending upon the requirement. Analysis procedures, until recently, involved long and tedious protocols that proved to be too difficult with an increasing number of protein samples. The analysis process was facilitated significantly with advent of protein microarrays. 2 3 4 5

4. Part 1, Step 1: 1 2 Protein sample 3 4 Action Description of the action Audio Narration Functional analysis of proteins is a time consuming process that requires many steps. Analysis of a single protein at a time would be a tedious and laborious process. (Please redraw all figures.) First show the protein sample followed by the clock on top. The arms on the clock must move slowly as each arrow keeps appearing followed by appearance of the green figure on the right and the clock above. The clock must be shown to tick such that the time changes from 3 to 8 as shown. 5

5. Part 1, Step 2: 1 2 Protein microarray 3 Protein microarrays allow analysis of thousands of samples simultaneously! 4 Action Description of the action Audio Narration Analysis of several protein samples will undoubtedly take a long time if they are run one at a time. Protein microarrays successfully overcame this hurdle by allowing analysis of several samples simultaneously. Multiple protein samples must be shown to appear one at a time as shown followed by constant ticking of the clock until it completes several circles. (Please redraw all figures.) First show several protein tubes appearing in sequence as shown in the animation followed by the hands in the clock above moving around continuously. Then the grey array figure on right must appear and hands of the clock must stop moving. 5

6. Master Layout (Part 2) 1 This animation consists of 4 parts: Part 1 – Need for protein microarrays Part 2 – Protein expression & purification Part 3 - Array functionalization & printing Part 4 – Protein detection & analysis Gene insert Protein expression 2 Gene of interest Heterologous host (e.g. Protein purification Expression vector 3 Chromatography 4 Purified protein of interest Protein purity tested SDS-PAGE 5 Biochemistry by A.L. Lehninger, 3rd edition (ebook)

7. Definitions of the components:Part 2 – Protein expression & purification 1 1. Expression vector: An expression vector or construct is used to introduce and express a particular gene of interest in a target cell. An efficient expression vector system must be capable of producing large quantities of the protein product. 2. Heterologous host: A system such as E. coli that will provide the required cellular machinery for transcription and translation of the gene of interest which is introduced by means of a suitable expression vector. 3. Gene of interest: The gene that codes for the desired target protein that needs to be printed on the surface of the microarray. 4. Gene insert: The gene sequence that is taken up by the plasmid vector for further expression is then referred to as an insert. 5. Protein expression: The host system carries out transcription and translation using its cellular machinery to express the protein of interest along with its own proteins as well. 2 3 4 5

8. Definitions of the components:Part 2 – Protein expression & purification 1 6. Protein purification: Since the target protein is expressed along with other proteins native to the host system, it is essential to purify the desired protein prior to printing on to the array surface. For this reason, the gene of interest is often fused with a convenient tag sequence such as His6 that will facilitate the purification process. 7. Chromatography: A convenient purification technique that separates proteins based on properties such as size, net charge or specific affinity towards a particular molecule or ligand. Several advancements have been made in chromatographic techniques that allow for accurate protein separation. 8. 2-D electrophoresis: This is another protein separation technique which is more commonly used for finer protein separation. It is a combination of isoelectric focusing in the first direction and SDS-PAGE in the second direction, thereby bringing about protein separation based on their isoelectric points (net charge) as well as their molecular weights. 9. Purified protein of interest: Once the purification is complete, the desired protein of interest is obtained which can then be used for spotting onto the array surface. 2 3 4 5

9. Part 2, Step 1: 1 Gene insert Protein expression machinery 2 Gene of interest Heterologous host Expression vector 3 Transcription Translation mRNA Expressed proteins 4 Action Description of the action Audio Narration The green arc must be incorporated in the multicolor circle. The second figure must then be zoomed into and animation below shown. The gene coding for the protein of interest is expressed in a suitable heterologous host system such as E. coli by means of expression vectors like plasmids. The host cell machinery is used for transcription and translation which results in a mixture of proteins consisting of the target protein along with other host proteins. First show the figure on the top left with the multicolor circle. Next, show the green arc being incorporated as part of this circle followed by appearance of the colored circles and brown figures. This must be zoomed into and the animation sequence shown below must be shown. 5

10. Part 2, Step 2: 1 His6 tagged protein Purified protein Protein of interest 2 Separation based on MW Ni-NTA coated beads Direction of migration 3 Chromatographic purification SDS-PAGE Protein of interest Unwanted proteins 4 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 red and blue 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 and purple objects must come down into the tube below and the ‘unwanted proteins’ label must appear. Finally the third column and tube must appear and the green object must come into the tube. The figure on the right must be shown with the 3 tubes and the blue slab. The arrow marks with labels must appear followed by colored spots. The green spot and tube must then be highlighted as shown. Since the protein of interest is expressed along with other proteins native to the host, it is essential to purify the target protein before it can be used for microarray applications. This can be done by chromatographic procedures to obtain the pure target protein. Protein purity is tested on SDS-PAGE gels. Tags like His6 are often fused with the protein of interest to facilitate the purification process due to its specific affinity towards nickel. 5 Biochemistry by A.L. Lehninger, 3rd edition (ebook)

11. Master Layout (Part 3) 1 This animation consists of 4 parts: Part 1 – Need for protein microarrays Part 2 – Protein expression & purification Part 3 - Array functionalization & printing Part 4 – Protein detection & analysis NH2 NH2 NH2 NH2 NH2 2 Array functionalization CHO CHO CHO CHO CHO CHO CHO CHO CHO CHO Si Si Si Si Si Si O O O O O O O O O O O O 3 4 Protein microarray Robotic array printing 5

12. Definitions of the components:Part 3 – Array functionalization & printing 1 1. Array functionalization : The microarray surface must be suitably derivatized with a chemical reagent that can react with the groups present on the protein surface in order to firmly immobilize them on the microarray. Functionalization is often done with silane derivatives as these react easily with the groups present on the protein. Aldehyde groups react with amine groups present on the protein to form Schiff’s base linkages which hold the protein firmly in place. 2. Robotic array printing: High-precision contact-printing robots are commonly used to spot nanoliter or picoliter quantities of protein solution onto the array surface. The proteins are usually mixed with glycerol solution in order to prevent them from evaporating. 2 3 4 5

13. Part 3, Step 1: 1 Commonly used array surfaces Glass Nitrocellulose Gold Hydrogel 2 3 4 Action Description of the action Audio Narration Commonly used array surfaces include glass, gold, nitrocellulose and hydrogels. While glass slides are easy to handle, available at low cost and can be used with existing scanning equipment, they show relatively low surface absorption and need to be derivatized with more reactive groups. They however continue to be used more extensively than the other array surfaces, which are significantly more expensive. Comparative features for commonly used array surfaces are demonstrated in this table. First the figures on top must appear followed by the table below. First show the parallelogram figures on top with their respective labels followed by the table below, one row at a time. 5

14. Part 3, Step 2: 1 Array functionalization 2 CHO CHO CHO CHO CHO Si Si Si O O O O O O 3 Protein microarray 4 Action Description of the action Audio Narration The hand must move as shown in animation and red cloud must appear. The spot must then be zoomed into and the figure above must appear. The array surface is functionalized with a suitable chemical reagent that will react with groups present on the protein surface. Aldehyde and silane derivatizations are commonly used as they interact well with amino groups present on the protein surface resulting in firm capture of the protein. (Please redraw all figures.) First show the parallelogram with spots. Then show the hand moving from left to right along the spots followed by appearance of the red cloud and disappearance of the hand. The spots are then zoomed into and the structures above must be shown. 5

15. Part 3, Step 3: 1 Robotic array printing Target protein NH2 NH2 NH2 NH2 NH2 Robotic arm Derivatized array surface 2 Aldehyde functionalization CHO CHO CHO CHO CHO Si Si Si O O O O O O 3 Silane derivatization 4 Action Description of the action Audio Narration The protein solution is printed on to the array surface in extremely small volumes by means of a robotic printing device that has small pins attached to it for this purpose. The slides are keptfor a suitable duration following the printing step to allow capture of the protein on to the array surface. The unreacted sites are then quenched by a blocking solution such as BSA which also prevents any non-specific protein binding in subsequent steps. The black robotic arm must move across the translucent surface after which the figure must be zoomed into. (Please redraw all figures.) First show the picture with the black machine arm moving across the translucent surface from bottom to top. Then zoom into the translucent surface to show the figure on the right. 5

16. Part 3, Step 4: 1 Types of arrays Reverse phase arrays Forward phase arrays Probe antibody Test lysate 2 Specific antigen binding Test lysate immobilized Capture antibody immobilized Direct labeling 3 4 Action Description of the action Audio Narration The dots must be zoomed into and the two figures above muse be shown as in the animation. There are two types of protein arrays that are commonly used. In forward phase arrays, the analyte of interest such as an antibody or aptamer is bound to the array surface and then probed by the test lysate which may contain the antigen of interest. In reverse phase arrays, however, the test cellular lysate is immobilized on the array surface and then probed using detection antibodies specific to the target of interest. First show the parallelogram below followed by zooming into the dots. Then show the figure on left top with the blue circles binding to the V shaped objects below. Similarly, then show the figure on right with the V shaped objects moving down and binding to the blue circles. 5

17. Master Layout (Part 4) 1 This animation consists of 4 parts: Part 1 – Need for protein microarrays Part 2 – Protein expression & purification Part 3 - Array functionalization & printing Part 4 – Protein detection & analysis Labelled target proteins Array slide 2 Capture antibodies Direct labeling Microarray scanner 3 Labelled secondary antibody 4 Capture antibodies Sandwich assay Data analysis 5

18. Definitions of the components:Part 4 – Protein detection & analysis 1 1. Direct labelling : Here, all the target proteins are labelled with a fluorescent or radioactive tag which allow direct detection upon binding to the immobilized capture antibody on the array surface. 2. Capture antibodies: The antibodies (or sometimes other molecules like aptamers) that have been immobilized on to the array surface for specific capture of a particular analyte. 3. Labelled target proteins: The target proteins are directly labelled by means of a fluorescent or radioactive tag which can be easily detected by scanning techniques or radioactivity measurement after the protein gets captured on to the array surface. 4. Sandwich assay: In this detection technique, a secondary antibody recognizing a different epitope on the same target analyte is labelled with a suitable tag and used for detection. 5. Labelled secondary antibody: An antibody that can recognize an epitope different from the one recognized by the capture antibody can be used for detection purposes. This secondary or detection antibody is labelled with a convenient fluorescent dye which is viewed in the microarray scanner upon antigen-antibody binding. 2 3 4 5

19. Definitions of the components:Part 4 – Protein detection & analysis 1 6. Microarray scanner: The microarray slide is scanned at wavelengths suitable to detect the fluorescently tagged proteins. The output from this scanner is generated on a connected computer device. 7. Data analysis: The protein array data can be analyzed by means of several available software to understand which proteins have been printed and subsequently detected. 2 3 4 5

20. Part 4, Step 1: 1 Detection techniques Labelled secondary antibody Labelled target proteins 2 Capture antibodies Capture antibodies 3 Direct labelling Sandwich assay 4 Action Description of the action Audio Narration As shown in animation. First show the figure on the left with the Y shaped objects on the grey surface. Then show the blue teardrop binding to the Y shaped object with colour of the sun changing from grey to orange. Then show the figure on the right and the red inverted Y shaped object binding to the blue teardrop with change in colour of the sun from grey to bright green. In the direct labelling detection technique, all the target proteins are labelled with a fluorescent or radioactive tag that facilitates easy detection upon binding to the immobilized capture antibody on the array surface. In the sandwich assay, however, a fluorescent tagged secondary antibody that recognizes a different epitope on the target antigen binds to it and is detected by means of the fluorescence. 5

21. Part 4, Step 2: 1 Microarray scanner 2 3 Data analysis 4 Action Description of the action Audio Narration The protein microarray is then scanned at in a microarray scanner that allows detection of the fluorescently labeled proteins or antibodies. The output from this scanner is then received by an appropriate software on which the data can be analyzed. The orange and green beams must be shown to appear instrument shown on the left. (Please redraw all figures.) First show the instrument on the left top followed by the orange and green beams coming out of it and the figures placed below. Then show the arrow mark and appearance of the computer screen on the right. 5

22. Interactivity option 1:Step No: 1 1 Certain well characterized proteins are printed on the array below with their corresponding query molecules shown on the left labeled with differently fluorescing dyes. Match the protein interacting pairs, Jun & Fos, p53 & MDM2, by dragging the query to the correct protein on the array surface in order to see the signal output. 2 p53 Jun 3 MDM2 Signal output Fos 4 Results Interacativity Type Options Boundary/limits Once the user matches the green shape to the orange dots, its color must change to blue and once the purple shape is matched with pink dots, its color must become red. Then the figure on the right must appear. User must drag and drop the figures given on the left to their corresponding coloured circles on the grey surface. Drag and drop. 5

23. Questionnaire 1 1. Reverse phase protein arrays have which of the following printed on their surface? Answers: a) Antibodies b) Aptamers c) Antigens d)‏ Affibodies 2. Which of the following array surfaces can be used for mass spectrometry applications? Answers: a) Hydrogel b) Gold c) Glass d)‏ Nitrocellulose 3. Match the following purification techniques with the principle used to separate them: a) Ion exchange chromatography i) Size b) Gel filtration ii) Specific interaction c) Affinity chromatography iii) Charge to mass ratio d) Native PAGE iv) Net charge Answers: a) a-ii, b-i, c-iii, d-iv b) a-iii, b-ii, c-i, d-iv c) a-iv, b-ii, c-iii, d-i d)‏ a-iv, b-i, c-ii, d-iii 2 3 4 5

24. Links for further reading Research papers: • Zhu, H., Snyder, M., Protein arrays and microarrays. Curr.Opin. Chem. Biol. 2001, 5, 40–45. • Brown, P. O., Botstein, D., Exploring the new world of the genome with DNA microarrays. Nat. Genet. 1999, 21, 33–37. • Predki PF: Functional protein microarrays: ripe for discovery. Curr Opin Chem Biol 2004, 8:8-13. • MacBeath G: Protein microarrays and proteomics. Nat Genet 2002, 32(Suppl):526-532. • Paweletz CP, Charboneau L, Bichsel VE, Simone NL, Chen T, Gillespie JW, Emmert-Buck MR, Roth MJ, Petricoin IE, Liotta LA: Reverse phase protein microarrays which capture disease progression show activation of pro-survival pathways at the cancer invasion front. Oncogene 2001, 20:1981-1989. • LaBaer, J., Ramachandran, N., Protein microarrays as tools for functional proteomics. Curr. Opin. Chem. Biol. 2005, 9,14–19. • Renberg, B. et al. Affibody Molecules in Protein Capture Microarrays: Evaluation of Multidomain Ligands and Different Detection Formats. J Prot. Res. 2007, 6, 171-179. • Pandey, A. & Mann, M. Proteomics to study genes and genomes. Nature 2000, 405, 837-846.