![Protein Acetylation—
•Histone acetylation and deacetylation, which are catalyzed by histone
acetyltransferases (HATs) and histone deacetylases (HDACs),
respectively, are emerging as critical regulators of chromatin structure and
transcription. It has been hypothesized that many HATs and HDACs might
also modify non-histone substrates.
• For example, the core enzyme, Esa1, of the essential nucleosome
acetyltransferase of H4 (NuA4) complex, is the only essential HAT in yeast,
which strongly suggested that it may target additional non-histone proteins
that are crucial for cell to survive.
•To identify non- histone substrates of the NuA4 complex, Lin et al. (2009)
established and performed acetylation reactions on yeast proteome
microarrays using the NuA4 complex in the presence of [14C]-Acetyl-CoA
as a donor.
• Surprisingly, 91 proteins were found to be readily acetylated by the NuA4
complex on the array](https://image.slidesharecdn.com/presentationprtnmicroarray-180310145420/85/PROTEIN-MICROARRAYS-39-320.jpg)
PROTEIN MICROARRAYS
- 1. ANN MARY MATHEW BGS051705 MSc. IST YEAR GENOMIC SCIENCE CENTRAL UNIVERSITY OF KERALA PROTEIN MICROARRAYS
- 2. •Microarray technology is a term that refers to the miniaturization of thousands of assays on one small plate that contain small amounts of purified proteins in a high density format. •A protein microarray (protein chip)is a high throughput method used to track the interactions & activities of proteins,& to determine their functions,& determining function on a large scale. •They allow simultaneous determination of a great variety of analytes from small amounts of samples within a single experiment.
- 3. HISTORY •Ambient analyte iimmunoassay by Roger Ekins in 1989. •DNA microarray – mRNA expression levels of thousands of genes in parallel. •Proteins are the major driving force in almost all cellular processes. Therefore, protein microarrays were developed as a high-throughput tool to overcome the limitation of DNA microarrays and to provide a direct platform for protein function analyses. •Immunoassays, the first form of protein microarray, take advantage of highly specific antigen-antibody recognition to build a protein detection system. The expansion of the capability of conventional immunoassays to antibody arrays enabled a parallel and multiple detection system using a small amount of sample (Haab, 2005; Kopf and Zharhary, 2007). •Around the same time, another type of protein microarray was
- 4. PROTEIN MICROARRAY ANALYTICAL FUNCTIONAL Functional protein microarrays are made by spotting all of the proteins encoded by an organism and therefore are useful for characterization of protein functions, such as protein-protein binding, biochemical activity, enzyme-substrate relationships, and immune response
- 5. More recently, a so-called reverse-phase array was developed, providing an alternative format to the analytical microarrays in which tissue/cell lysates (or fractionated lysates) are used to form such an array . PROTEIN MICROARRAY ANALYTICAL REVERSE PHASED FUNCTIONAL
- 7. Fabrication of protein microarray Protein microarrays are typically prepared by immobilizing proteins onto a microscope slide using a standard contact spotter or non contact microarray. PROTEIN PRODUCTION Recombinant antibodies(phage display) •Using antibody-fragment encoding genes (VH and VL) and bacteriophage capsid gene fusion, this technology enables sets of human antibody libraries to be stored in prokaryotic systems where they can be readily expressed by phage infection. • Prokaryotic expression systems promise large-scale antibody production in short time periods. In addition, this system generates antibody fragments lacking the Fc domain rather than intact IgG, eliminating nonspecific binding to the Fc receptor. •The recombinant antibodies are expressed and displayed in the phage capsid, and then purified using column chromatography.
- 8. • Recently, other methods have been developed to generate recombinant antibodies in eukaryotic expression systems (i.e., yeast display) or even in vitro environments (i.e., mRNA display, ribosome display), which provide additional advantages for recombinant antibody fabrication. •Fabrication of functional protein microarrays faces an even bigger challenge due to the need for large amounts of highly purified proteins. •. To overcome these hurdles, high-throughput protein purification protocols have been developed using both Saccharomyces cerevisae (yeast) and E. coli protein expression systems. • Using a batch purification protocol in a 96-well format, >4000 recombinant proteins can be overexpressed and purified in yeast or E. coli (Jeong et al., 2012). •Because it is challenging for most labs to purify large numbers of proteins, Angenendt et al. (2006) developed an alternative technology in protein chip fabrication, dubbed nucleic acid- programmable protein array (NAPPA).
- 9. • Spotting plasmid DNAs with capture antibodies allows for the generation of a protein microarray via simultaneous in situ transcription/translation reactions and protein immobilization on the printed slides. •A significant benefit of this approach is that the printed template DNA microarray can be stored for a long time, and the resulting protein microarrays are always freshly made. •This method allows the generation of up to 13,000 protein spots on one slide without laborious cloning and expression vectors. • However, such arrays have not flourished due to low protein yield and difficulties in producing large proteins (e.g., >60 kD). • For reverse-phase protein microarrays proper samples should be isolated from cell culture; frozen, ethanol-fixed, or paraffin-embedded tissue or laser captured microdissections of cell populations from certain tissues (Charboneau et al., 2002; Espina et al., 2007)
- 10. •A variety of slide surfaces can be used. Popular types include aldehyde- and epoxy-derivatized glass surfaces for random attachment through amines , nitrocellulose , or gel-coated slides and nickel-coated slides for affinity attachment of His6-tagged proteins.
- 11. Different methods of arraying the proteins:- •Robotic method •Ink jetting method •Piezoelectric spotting •Photoliyhography. In these methods,robotic is contact microarray method while the other three are non contact microarray methods.
- 12. DETECTION label- dependent fluorescent dyes • narrow excitation and emission spectra • Cy3, Cy5 Enzymes • horseradish peroxidase Radioisotops • 32P, 33P, and 14C liposomes label-free Surface plasmon resonance spectroscopy (SPR) imaging optical ellipsometry (OE) reflectometric interference spectroscopy (RIFS) oblique-incidence reflectivity difference (OIRD) Atomic force microscopy (AFM)
- 13. • Rolling circle amplification (RCA) and tyramide signal amplification (TSA) have also been developed to detect low abundance proteins
- 15. Analytical Protein Microarray • The first model to demonstrate the application of antibody arrays was the “analyte-labeled” assay format. In this format, proteins are detected after antibody capture using direct protein labeling . •Some limitations have to be considered because this method lacks specificity in protein target labeling and has poor sensitivity for low abundance proteins. Moreover, targeted protein labeling may lead to the epitope destruction due to some chemical reaction.
- 16. •Another model of antibody array provides higher sensitivity using the “sandwich” assay format. This format employs two different antibodies to detect the targeted protein . • One antibody, called the capture antibody, immobilizes the targeted protein on the solid phase, while the other antibody, called the reporter or detection antibody, generates a signal for the detection system. Using two antibodies significantly increases the specificity and sensitivity of the “sandwich” assay format, even at femtomolar levels . •These assays offer a multiplexed format of the original Enzyme- linked Immunosorbent Assay (ELISA).
- 17. •Analytical microarrays(or antibody microarrays) have antibodies arrays on solid surface,and are used to detect proteins in biological samples. •Often a second is used to detect a protein that is captured by the antibody attached to the solid phase,in a principle similar to that of sandwich immunoassay, in which the first antibody is spotted on the array and then a captured antigen on the chip is detected with a second antibody that recognises a different part of antigen. •Analytical protein arrays can be used to monitor protein expression levels or for bio- marker identification, clinical diagnosis, or environmental/ food safety analysis.
- 20. LIMITATIONS •Antibodies are the most popular protein capture reagents, although their affinity and/or specificity can vary dramatically . • Many antibodies may cross-react with proteins other than their expected target proteins when tested on functional protein microarrays, especially when multiple analyte detection is employed. •The need for highly specific antibodies has become a major challenge in analytical protein microarrays because nonspecific binding will lead to large numbers of false positive result. •Another challenge comes from producing a large number of antibodies in a high-throughput fashion. Recombinant
- 21. Functional Protein Microarray •Also known as target protein array.With functional protein microarrays purified recombinant protein are immobilised onto the solid phase. •Functional protein microarrays have recently been applied to many aspects of discovery based biology,including protein- protein,protein- lipid,protein-DNA,protein-drug,&protein – peptide iinteractions. •These can be used to identify enzyme substrates.
- 22. •These can also be used to detect antibodies in a biological specimen to profile an immune response. •The first use of functional protein microarrays was demonstrated by Zhu et al. (2001) to determine the substrate specificity of protein kinases in yeast. • Protein microarrays enable us to study many post- translational modifications (i.e., phosphorylation, acetylation, ubiquitylation, S- nitrosylation) in a large-scale fashion, which is critical for understanding cellular protein synthesis and function.
- 24. Reverse-Phase Protein Microarray •Involves complex samples, such as tissue lysates.cells are isolated from various tissues of interest and lysed.The lysate is arranged onto the microarray & probed with antibodies against the target protein of interest.These antibodies are typically detected with chemiluminescent,fluoresent or colorimetric assays. • This type of microarrays was first established by Paweletz and colleagues to monitor histological changes in prostate cancer patients. Using this method, they successfully detected microscopic transition stages of pro-survival checkpoint protein in three different stages of prostate cancer: normal prostate epithelium, prostate intraepithelial neoplasia, and invasive prostate cancer. •The high degree of sensitivity, precision and linearity achieved by reverse-phase protein microarrays enabled this method to quantify the phosphorylation status of some proteins (such as Akt and ERK) in these samples; phosphorylation was statistically correlated
- 26. •RPAs allow for the determination of the presence of altered proteins or other agents that may be the result of disease. •Specifically, post translationl modifications,which are typically altered as a result of disease can be detected using RPAs. • Harnessing this sophisticated technology, Ciaccio et al. (2010) profiled EGF receptor signaling dynamics using micro-western arrays (MWA), which combine western blotting and reverse- phase protein microarrays to produce better sensitivity by separation of whole lysate sample components. •This method allowed them to precisely measure 91 phosphosites of 67 proteins at 6 different time points with five EGF concentrations in A431 human carcinoma cells to analyze the dynamic profile of EGF receptor concentrations•A significant drawback of this approach, however, is that it is highly dependent on the availability and specificity of commercially produced antibodies. Because of this bias, it is has limited applications.
- 28. APPLICATIONS:- There are five major areas where protein arrays are being applied:diagnostics,proteomics,protein functional analysis,antibody characterisation & treatment. Diagnostics involves the detection of antigens & antibodies in blood samples;to discover new disease biomarkers;the monitoring of disease states & responses to therapy in personalised medicine;the monitoring of environment & food. Proteomics pertains to protein expression profilling i.e;which proteins are expressed in the lysate of a part of cell
- 29. Protein functional analysis is the identification of protein-protein interactions,protein- phospholipid interactions,small molecule targets,enzymatic substrates & receptor ligands. Antibody characterization is characterizing cross reactivity,specificity & mapping epitopes. Treatment development involves the development of antigen- specific therapies for autoimunity,cancer & allergies;the identification of small molecule targets that could potentially be used as new drugs.
- 30. Applications in Basic Research •Development of new assays An obvious advantage of functional protein microarrays is their ability to provide a flexible platform that can characterize a wide range of biochemical properties of spotted proteins. To date, these assays have been successfully developed to detect various types of protein binding properties, such as protein-protein, protein-DNA, protein-RNA, protein-lipid, protein-drug, and protein-glycan interactions. And identify substrates of various classes of enzymes, such as protein kinases, ubiquitin/SUMO E3 ligases, and acetyltransferases, to name a few.
- 32. Detection of Protein-Binding Properties Protein-protein interaction •Zhu and Snyder (2001) developed the first proteome microarray composed of ~5800 recombinant yeast proteins (>85% of the yeast proteome) and identified binding partners of calmodulin and phosphatidylinositides (PIPs). •They first incubated the microarrays with biotinylated bovine calmodulin and discovered 39 new calmodulin binding partners. •In addition, using liposomes as a carrier for various PIPs, they identified more than 150 binding proteins, >50% of which were known membrane-associated proteins.
- 33. Protein-Peptide Interaction— •MacBeath and colleagues fabricated protein domain microarrays to investigate protein-peptide interactions that might play an important role in signaling in a semi-quantitative fashion. •They constructed an array by printing 159 human Src homology 2 (SH2) and phosphotyrosine binding (PTB) domains on the aldehyde- modified glass substrates and incubated the arrays with 61 peptides representing tyrosine phosphorylation sites on the four ErbB receptors. •Eight concentrations of each peptide (10 nM to 5 mM) were tested in the assay, allowing semi-quantitative measurement of the binding affinity of each peptide to its protein ligand
- 34. Protein-DNA Interaction— •Protein microarrays have also been applied extensively and successfully to characterize protein-DNA interactions (PDIs). •In an earlier study, Snyder and colleagues screened for novel DNA- binding proteins by probing yeast proteome microarrays with fluorescently labeled yeast genomic DNA . • Of the ~200 positive proteins, half were not previously known to bind to DNA. By focusing on a single yeast gene, ARG5,6 , encoding two enzymes involved in arginine biosynthesis, they discovered that its protein product bound to a specific DNA motif and associated with specific nuclear and mitochondrial loci in vivo .
- 35. Protein-Small Molecule Interaction— •Discovering new drug molecules and drug targets is another field in which protein microarrays have shown its potential. •For example, Huang et al. (2004) incubated biotinylated small-molecule inhibitors of rapamycin (SMIRs) on the yeast proteome microarrays, and obtained the binding profiles of the SMIRs across the entire yeast proteome. • They identified candidate target proteins of the SMIRs, including Tep1p, a homolog of the mammalian PTEN tumor suppressor, and Ybr077cp (Nir1p), a protein of previously unknown function, both of which are validated to associate with PI(3,4)P2, suggesting a novel mechanism by which phosphatidylinositides might modulate the target-of-rapamycin pathway
- 36. Protein-Glycan Interaction— •Protein glycosylation, a general posttranslational modification of proteins involved in cell membrane formation, dictates the proper conformation of many membrane proteins, retains stability on some secreted glycoproteins, and plays a role in cell-cell adhesion. Profiling monoclonal antibody specificity— •Antibodies are widely applied for many purposes in proteomic studies. Because of their specificity, monoclonal antibodies (MAb) are a better option compared to polyclonal antibodies for most applications. •Jeong et al. (2012) combined immunization with live human cells and microarray-based analysis to develop a rapid identification method of monospecific monoclonal antibody (mMAb). • Because a human proteome microarray composed of ~17,000 individually purified full-length human proteins was used in the monoclonal antibody binding assays, antibodies that only recognized a single antigen on the microarrays could be identified as highly specific mMAbs
- 37. Protein posttranslational modification Protein phosphorylation— •Protein phosphorylation plays a central role in almost all aspects of cellular processes. •The application of protein microarray technology to protein phosphorylation was first demonstrated by Zhu et al. (2000). They immobilized 17 different substrates on a nanowell protein microarray, followed by individual kinase assays with almost all of the yeast kinases (119/122). •This approach allowed them to determine the substrate specificity of the yeast kinome and identify new tyrosine phosphorylation activity.
- 38. Protein Ubiquitylation— •Ubiquitylation is one of the most prevalent PTMs and controls almost all types of cellular events in eukaryotes. •To establish a protein microarray-based approach for identification of ubiquitin E3 ligase substrates, Lu et al. (2008) developed an assay for yeast proteome microarrays that uses a HECT-domain E3 ligase, Rsp5, in combination with the E1 and E2 enzymes. •More than 90 new substrates were identified, eight of which were validated as in vivo substrates of Rsp5. Further in vivo characterization of two substrates, Sla1 and Rnr2, demonstrated that Rsp5-dependent ubiquitylation affects either posttranslational processing of the substrate or subcellular localization.
- 39. Protein Acetylation— •Histone acetylation and deacetylation, which are catalyzed by histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively, are emerging as critical regulators of chromatin structure and transcription. It has been hypothesized that many HATs and HDACs might also modify non-histone substrates. • For example, the core enzyme, Esa1, of the essential nucleosome acetyltransferase of H4 (NuA4) complex, is the only essential HAT in yeast, which strongly suggested that it may target additional non-histone proteins that are crucial for cell to survive. •To identify non- histone substrates of the NuA4 complex, Lin et al. (2009) established and performed acetylation reactions on yeast proteome microarrays using the NuA4 complex in the presence of [14C]-Acetyl-CoA as a donor. • Surprisingly, 91 proteins were found to be readily acetylated by the NuA4 complex on the array
- 40. S-Nitrosylation— •S-nitrosylation is independent of enzyme catalysis but an important PTM that affects a wide range of proteins involved in many cellular processes. • Recently, Foster et al. (2009) developed a protein microarray-based approach to detect proteins reactive to S-nitrosothiol (SNO), the donor of NO+ in S-nitrosylation, and to investigate determinants of S-nitrosylation (Foster et al., 2009). •S-nitrosocysteine (CysNO), a highly reactive SNO, was added to a yeast proteome microarray, and the nitrosylated proteins were then detected using a modified biotin switch technique. • The top 300 proteins with the highest relative signal intensity were further analyzed, and the results revealed that proteins with active-site Cys thiols residing at N-termini of alpha-helices or within catalytic loops were particularly prominent
- 41. Applications in Clinical Research Extending the applications in basic research, protein microarrays have proven highly useful in clinical research because the development of almost all diseases is related to protein function and interaction. In addition, the protein microarray format can be directly employed to develop highly sensitive and specific diagnostic and detection tools Host-microbe interactions •A new trend in the field of host-pathogen interactions is to use host protein microarrays to survey relationships between a pathogenic factor of interest and the host proteome. •This idea is particularly suited for studying host-virus interactions because, after entering the host cells, the viral genome and proteins are in direct physical contact with the host components, whereby a pathogen can hijack/exploit the host pathways and machineries for its own replication. This approach would alleviate the problems associated with RNAi-based screening in identifying direct host target
- 42. •Pathogen entry and infection in the host cell are a series of processes that abuse protein pathways and interactions. •Li et al. (2011) demonstrated the use of protein microarrays to study conserved serine/threonine kinase of herpesvirus that play an important role in their replication in human cells. • They identified shared substrates of the conserved kinases from herpes simplex virus, human cytomegalovirus, EBV, and Kaposi’s sarcoma- associated herpesvirus using human proteomic chips. • From this study, they found that the histone acetyltransferase TIP60, an upstream regulator of the DNA damage response pathway, was essential for herpesvirus replication. •This finding is promising for broad-spectrum anti-viral development.
- 43. Biomarker identification •Biomarkers are a crucial tool in expeditious detection of infection or diagnosis of autoimmune diseases. In most cases, production of antibodies against pathogens or autoantibodies in blood serum is correlated with infection or occurrence of autoimmune diseases. •Therefore, to find a powerful biomarker, protein microarrays can be used to directly detect antibodies that statistically correlate with the corresponding disease in a patient’s serum. •Zhu et al. (2006) developed the first viral protein microarray to detect biomarkers for severe acute respiratory syndrome (SARS).
- 44. •This array, which consisted of all SARS coronavirus (SARS-CoV) proteins, as well as proteins of five additional coronaviruses, could readily distinguish serum samples as SARS-positive or SARS-negative with 94% accuracy compared to the traditional ELISA method. •More recently, human protein microarrays have been widely used to identify biomarkers for a variety of autoimmune diseases •In additions, there have been a series of studies that employed pathogen protein microarrays to profile serological responses following infection. For examples, protein microarrays have been developed in bacteria and viruses for biomarker identification in various infectious diseases
- 45. Future Prospects Protein microarrays have become one of the most powerful tools in proteomic studies and can be applied with many different purposes. High-throughput processing has become the trend due to cost reduction and high productivity of results. Given a high-throughput and parallel system, protein microarrays will speed up new findings in protein interactions for basic research as well as clinical research purposes. Reduction of sample volume usage is one important factor that demonstrates the superiority of this technology compared to other techniques. This factor is especially important for clinical research that uses precious samples, such as human serum samples. In addition, the high sensitivity and specificity of protein microarrays provides a powerful tool in quantifying and profiling proteins.
- 46. •Despite many successful applications of protein microarrays, limitations of this technology still leave many challenges to be overcome. •The large-scale production of high-quality antibodies using recombinant platforms is still hard to be applied due to the complexity of expression and purification procedures. •Another consideration is to perform high-throughput detection without sample labeling. Label-free detection systems should be the future of protein microarrays. • In conclusion, although this technique still needs to be explored, it would not be surprising if after several years, this technique is the leading technology in proteomic and diagnostic fields.
- 47. Reference •Reymond Sutandy et al. Overview of Protein Microarrays ,Curr Protoc Protein Sci . 2013 April ; 0 27: Unit–27.1. doi:10.1002/0471140864.ps2701s72.. •Haab BB. Antibody arrays in cancer research. Molecular & Cellular Proteomics. 2005; 4:377–383. [PubMed: 15671041) •Kopf E, Zharhary D. Antibody arrays—An emerging tool in cancer proteomics. The International Journal of Biochemistry & Cell Biology. 2007; 39:1305–1317. •Jeong JS, Jiang L, Albino E, Marrero J, Rho HS, Hu J, Hu S, Vera C, Bayron-Poueymiroy D, Rivera- Pacheco ZA, Ramos L, Torres-Castro C, Qian J, Bonaventura J, Boeke JD, Yap WY, Pino I, Eichinger DJ, Zhu H, Blackshaw S. Rapid identification of monospecific monoclonal antibodies using a human proteome microarray. Molecular & Cellular Proteomics. 2012 mcp.O111.01625. •Angenendt P, Kreutzberger J, Glökler J, Hoheisel JD. Generation of high density protein microarrays by cell-free in situ expression of unpurified PCR products. Molecular & Cellular Proteomics. 2006; 5:1658–1666. [PubMed: