Protein microarray .pptx

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1. SEMINAR ON - PROTEIN MICROARRAY PRESENTED BY MISBAUL HOQUE PHARMACOLOGY , M.PHARM SPER, JAMIA HAMDARD

2. OUTLINES INTRODUCTION COMPARISON WITH OTHER METHODS PRINCIPLE TYPES OF PROTEIN MICROARRAY APPLICATION FUTURE DIRECTION AND OPPORTUNITIES 2

3. INTRODUCTION Protein microarrays are a high-throughput technology used to analyze the expression levels and functional activity of thousands of proteins simultaneously. Protein microarrays consist of a solid support (such as a glass slide or a membrane) that has been coated with thousands of different proteins in a defined pattern or array. Each protein spot on the array represents a different protein, and multiple replicates of each protein are typically included on the array to ensure data reproducibility 3

4. Comparison with other methods (e.g., ELISA, Western blotting) Here are some of the key differences between these methods: Throughput: Protein microarrays allow for the simultaneous screening of hundreds or thousands of proteins in a single experiment, while ELISA and Western blotting typically only allow for the analysis of a few proteins at a time. Sensitivity: Protein microarrays can detect low abundance proteins with high sensitivity, while ELISA and Western blotting may have limited sensitivity for low abundance proteins. 4

5. Specificity: Protein microarrays can offer higher specificity by allowing for the screening of multiple proteins in a single experiment, while ELISA and Western blotting may have limitations in terms of cross-reactivity and specificity. Sample requirements: Protein microarrays require only small amounts of sample, while ELISA and Western blotting typically require larger amounts of sample. Cost: Protein microarrays can be cost-effective due to the high throughput and low sample requirements, while ELISA and Western blotting can be more expensive due to the need for multiple experiments or larger sample volumes. 5

6. PRINCIPLE Protein chip consists of a support surface such as glass slide, nitrocellulose, bead or microtiter plate to which array of capture proteins is bound. Probe molecules typically labeled with a fluorescent dye are added to the array. Any reaction between the probe and the immobilized protein emits a fluorescent signal and that is read by laser scanner. 6

7. Solid Supporting Material The most common supporting materials in use includes Aldehyde, carboxylic ester, nitrocellulose membrane, polystyrene, Agarose/polyacrylamide gel, hydrogel. An ideal surface for protein microarray fabrication has to be capable of Immobilizing proteins Preserving three-dimensional (3-D conformation of protein Should not change the chemical nature of the protein 7

8. The Probes on Chip A variety of materials can be immobilize on the protein chip based on the specific requirements. These include: Antibodies Antigens Aptamers (Nucleic Acid based ligands) Affibodies (small, robust proteins engineered to bind to a large number of target proteins or peptides with high affinity, imitating monoclonal antibodies, and are therefore a member of the family of antibody mimetics) Full length Proteins or their domains 8

9. Types of protein microarrays There are several types of protein microarrays, each with its own strengths and limitations. Here are some of the most common types 9

10. Analytical microarrays Analytical microarrays are typically used to profile a complex mixture of proteins in order to measure binding affinities and protein expression levels of the proteins in the mixture. In this technique, a library of antibodies, aptamers, or affibodies is arrayed on a glass microscope slide. The array is then probed with a protein solution. Antibody microarrays are the most common analytical microarray . These types of microarrays can be used to monitor differential expression profiles and for clinical diagnostics. 10

11. Functional protein microarrays Functional protein microarrays is different from analytical arrays. Functional protein arrays are composed of arrays containing full-length functional proteins or protein domains. These protein chips are used to study the biochemical activities of an entire proteome in a single experiment. They are used to study numerous protein interactions, such as protein-protein, protein-DNA, and protein-RNA interactions. Functional protein microarrays can be used to study a wide range of biological processes, including signal transduction, transcriptional regulation, and apoptosis. 11

12. Reverse-phase protein microarrays : Reverse-phase protein microarrays : reverse phase protein microarray (RPA). In RPA, cells are isolated from various tissues of interest and are lysed. The lysate is arrayed onto a nitrocellulose slide using a contact pin microarrayer. The slides are then probed with antibodies against the target protein of interest, and the antibodies are typically detected with chemiluminescent, fluorescent, or colorimetric assays. Reverse-phase protein microarrays can be used for biomarker discovery, drug target validation, and the study of protein expression patterns in disease. 12

13. Applications of protein microarrays in drug discovery Target identification and validation Lead optimization and screening Biomarker discovery and validation 13

14. Protein microarrays can be used to identify potential drug targets by screening large numbers of proteins simultaneously. This approach can be particularly useful for identifying targets in complex biological pathways or networks that are difficult to study using traditional methods. For example, a protein microarray may contain thousands of purified proteins, which can be probed with small molecule compounds, antibodies, or other molecules to identify proteins that interact with the probes. Proteins that show a strong interaction signal may represent potential drug targets . Target identification : 14

15. Target validation: et validation Protein microarrays can also be used to validate drug targets that have been identified using other methods. For example, a candidate target protein may be screened against a panel of other proteins on a microarray to assess its specificity and selectivity. The microarray may also contain variants or mutants of the target protein to test its function and identify potential binding sites for small molecules or antibodies. In addition, protein microarrays can be used to assess the effects of drugs or other compounds on the activity or expression of target proteins, which can provide valuable information for drug development. 15

16. Lead optimization: Protein microarrays can be used to optimize lead compounds that have been identified as potential drugs. For example, a microarray may contain variants or mutants of the target protein to test the binding affinity and selectivity of the lead compound. This approach can help identify structural modifications or functional groups that improve the potency or pharmacokinetic properties of the lead compound. 16

17. Lead screening : Protein microarrays can also be used to screen lead compounds for their ability to interact with target proteins or modulate their activity. This approach can be particularly useful for identifying compounds that act through non-traditional mechanisms or have multiple targets. For example, a microarray may contain multiple proteins involved in a biological pathway or network, and lead compounds can be screened against the array to identify those that have the desired effect on the pathway or network. 17

18. Biomarker discovery and validation: Biomarkers are biological molecules that can be used as indicators of disease or physiological states, and they play a critical role in the diagnosis, prognosis, and treatment of various conditions. 18

19. Biomarker discovery : Protein microarrays can be used to discover novel biomarkers by screening large numbers of proteins in a high-throughput manner. For example, a microarray may contain proteins from different biological pathways or networks that are relevant to a particular disease or condition. The microarray can be probed with biological samples, such as serum or tissue lysates, from patients with the disease or condition, as well as from healthy controls. Proteins that show differential expression or activity between the two groups may represent potential biomarkers for the disease or condition. 19

20. Biomarker validation Protein microarrays used to validate potential biomarkers that have been identified using other methods. For example, a candidate biomarker may be screened against a panel of other proteins on a microarray to assess its specificity and selectivity. The microarray may also contain variants or mutants of the candidate biomarker to test its function and identify potential binding partners or downstream effectors. In addition, protein microarrays can be used to assess the performance of potential biomarkers in large patient cohorts, which can provide valuable information for clinical translation. They can be used to identify novel biomarkers and validate potential biomarkers in a high-throughput and cost-effective manner, which can accelerate the development of diagnostics and therapeutics for various diseases and conditions. Biomarker validation : 20

21. Case studies/examples Examples of successful drug discovery using protein microarrays Discovery of a selective inhibitor of Bromodomain-containing protein 4 Identification of a biomarker for Alzheimer's disease over 900 human proteins to identify autoantibodies in the serum of patients with autoimmune disease. 21

22. Discovery of a selective inhibitor of BRD4: Bromodomain-containing protein 4 (BRD4) is a promising target for cancer therapy, but developing selective inhibitors has been challenging due to the high structural similarity of BRD4 with other bromodomain-containing proteins. Researchers used a protein microarray containing 42 human bromodomain proteins to screen for inhibitors that selectively bind to BRD4. They identified a compound that binds selectively to BRD4 and showed potent antiproliferative activity against multiple cancer cell lines. The compound has since been further optimized and is being developed as a potential cancer therapy 22

23. Identification of a biomarker for Alzheimer's disease: Researchers used a protein microarray containing over 9,000 proteins to screen for proteins that are differentially expressed in the brains of Alzheimer's disease patients compared to healthy controls. They identified a protein called REST, which is a transcriptional repressor that regulates neuronal gene expression. REST was found to be significantly decreased in the brains of Alzheimer's disease patients, and further studies showed that it may be a potential biomarker for the disease. 23

24. Development of a diagnostic test for autoimmune disease: Researchers used a protein microarray containing over 900 human proteins to identify autoantibodies in the serum of patients with autoimmune disease. They identified a panel of 11 autoantibodies that are highly specific for autoimmune disease, and developed a diagnostic test based on these autoantibodies. The test has been shown to have high sensitivity and specificity for autoimmune disease, and is being developed for clinical use . 24

25. Future directions and opportunities : Single-cell analysis: Recent advances in microarray technology and imaging techniques are enabling the development of protein microarrays for single-cell analysis. By allowing for the analysis of individual cells, these microarrays can provide new insights into cell signaling, heterogeneity, and function. Functional protein microarrays: Traditional protein microarrays typically only measure protein expression or binding, but functional protein microarrays are being developed that can measure enzymatic activity, protein-protein interactions, and other functional assays. These microarrays can enable high-throughput screening of potential drug targets, as well as the discovery of new protein functions and interactions. Multiplexed assays: Multiplexed assays that combine multiple types of protein analysis on a single microarray are being developed. These assays can enable more comprehensive analysis of protein expression, function, and interaction in a single experiment. 25

26. Integration with other technologies: Protein microarrays are being integrated with other technologies such as CRISPR/Cas9 gene editing, high-throughput sequencing, and mass spectrometry to enable more comprehensive analysis of protein function and interaction. Clinical applications: Protein microarrays are being developed for clinical applications such as diagnostic tests, patient stratification, and personalized medicine. By enabling high-throughput and cost-effective screening of large numbers of proteins, these microarrays can accelerate the development of new biomarkers and therapeutic targets. 26

27. REFERENCES 1. Wright C, Bergquist P, Hober S. "Protein microarrays for drug discovery". doi: 10.1517/17460441.2014.917207 2 . Heng Zhu   ,  Michael Snyder . “ Protein chip technology “ DOI:  10.1016/s1367-5931(02)00005-4 3.Christer Wingren Antibody-Based Proteomics PMID: 27686812 DOI: 10.1007/978-3-319-42316-6_11 4.Pablo San Segundo-Acosta , Ana Montero-Calle , Manuel Fuentes, Alberto Rábano , Mayte Villalba , Rodrigo Barderas ‘ Identification of Alzheimer's Disease Autoantibodies and Their Target Biomarkers by Phage Microarrays DOI:  10.1021/acs.jproteome.9b00258 5. www.frontiersin.org/articles/10.3389/fimmu.2021.645632/full 27