 Today's lecture and the next 2 lectures will be delivered by guest scientist Dr. Ramesh Omani. Dr. Omani is a scientist at the CSIR Indian Institute of Chemical Technology, Hyderabad. He focuses on identifying new potential biomarkers and understanding cell signaling mechanism driven by deregulated proteins associated with prostate cancer. Dr. Omani's group works on proteomics platforms for differential as well as functional proteomics. He is actively pursuing investigations to understand disease molecular mechanisms and drug-able targets specific to different solid tumors. Dr. Ramesh Omani is currently also working on many comprehensive projects which are related to identifying new chemical entities with anti-cancer and anti-tubour cooler potential using cell-based and target-based screening of the small molecule libraries. In today's lecture, Dr. Omani will talk to us the basics of reverse phase protein arrays which is a highly throughput proteomics platform for discovery, validation and clinical applications. Good afternoon to everyone. I am Dr. Ramesh Omani from Indian Institute of Chemical Technology, Hyderabad. I should thank Dr. Sanjeeva for inviting me to interact with you and share the technique how this reverse phase protein arrays are useful in the array platforms and also I would like to highlight how this RPPA platform is different from the protein array platforms. Many people have been using for quite a bit of time. So my lecture I do not have any hands-on training session to give you to perform the experiments but all the necessary steps you are doing same in this but exactly the sample to antibody is reverse that is it that is why it is called as reverse phase protein arrays. So I would try to stress where it is different from the routine array, routine protein arrays and what are the steps to be taken care to perform the arrays and how to do data analysis and also I will try to show some of our studies we have performed in the past how we use this technique and try to address the biologically relevant question in terms of clinical samples as well as in the biomarker validation as well. So all in all I would like to tell you that by end of my lecture so you should feel that it is imaginary how much you can imagine to use this technique into your lab research that is what I would like to say. So just imagine that how I can use this technique into my science or your PhD work or maybe your post-doctoral work or maybe any biological question to be asked to be addressed with the help of clinicians or with the help of researchers in the lab. So essentially here if you look at it here is a central dogma of molecular biology ok. So DNA to RNA, RNA to protein we all know that so all the essential functions to be done to be carried out by a cell stored its information in the form of DNA in the form of genes but those genes alone cannot do anything but it is the functions or the molecular functions of a cell are to be done by the products of the genes that is why. So if you look at the correlation between the gene to protein it is very poor if you look at the gene to RNA the correlation is only 60 percent that means 40 percent of genome information is already missing in the transcriptome level. When you look at the correlation between transcriptome to proteome level again the correlation is very very poor it is only 40 percent. So ultimately if you look back protein to genome the correlation is not more than 30 percent on average and also I should emphasize here the proteomics has been evolved and advance much more in this recent past to address many biologically relevant questions which are unanswerable by any of the genomics approach or transcriptomics approaches. Nevertheless and also it is important to mention here that mass spectrometer will tell about the abundance of the proteins ok. But advance mass spectrometry definitely tells you about the activation status of the proteins in terms of either phosphorylation or the post translation modification such as oscillation glycosylation and so on. So that is where it is very very important to look at the protein function really. Sometimes protein may express but it may keep quiet it says like a silent mutation in the gene it may keep quiet nothing to do. So ultimately if it is a functional protein then only it reflects in the disease state. If you look at here any illness or disease of genes integrated at the protein level that to at the protein functional level. So that is one of the reason you should perform the functional proteomics rather than the differential proteomics. If you look at any proteomics books available in the library or maybe in the internet see that proteomics is broadly divided into different areas like a differential proteomics and a structural proteomics and also the functional proteomics. So differential proteomics always tells about the upregulation downregulation. So what gene is also upregulated downregulated we have to see whether it is upregulation mean it is really influencing the disease or not. So that is where the functional proteomics came into picture and it gained the importance to work on this area further. There are many different approaches one can do functional proteomics. So particularly MS based approaches and interaction proteomics and protein microarrays. So here already you can see that we are moving towards the theme of our course work here. So in this case look at as Sanjeeva highlighted in the morning. So the basically how the concept has been evolved how the protein microarrays are evolved in the research field. So from the this concept has been evolved from DNA microarrays provided valuable platform for high throughput analysis of thousands of proteins simultaneously. We all know that the traditional biochemistry labs I did not have the nice cartoon to show here every time people used to clone one gene make a vector express it and wait what it happens what it will do the cell. That way they used to analyze one protein by one protein the traditional biochemistry labs. Now the once O mix has been evolved the thousands of proteins functions can be analyzed in a simultaneous manner. So that definitely or certainly can be done using this array platforms. So particularly I wanted to emphasize on the protein microarray this protein microarrays are also broadly classified into three different types analytical microarrays the functional microarrays and reverse phase microarrays. So based on the principle involved in this protein array technology. So this classification has been nomenclatured as on today. So particularly I do not want to emphasize on all of them today I will maybe in the next lectures I see in the program see they will cover all this analytical microarrays and functional microarrays that is the reason I would like to concentrate only on the reverse phase protein array array. And also all of you have done western blots in the lab. So to do one western blot at least you need a minimum 20 microgram protein and of course I have to measure about the housekeeping gene you need another 40 microgram protein another 20 microgram. So total all in all 40 microgram protein is required to measure whether the protein is up or down compared to one of the other samples. This case 40 microgram protein is a lot on today when the all proteomics platforms have evolved into such an advanced stage. So to avoid that definitely the technique what we are going to speak about today reverse phase protein arrays are the alternative to western blotting technique. So microarray development if you look at it relatively very young technology widely adopted by many researchers for many different applications and mainly this is used for gene discoveries but our clever proteomics community has been adopted to protein analysis as well. So when I started protein microarray projects I thought only array means it is a DNA array I did not know that protein array was something like that was there at all. So then I understood that in fact this array name is more exploited by the proteomics people than the genomics people that is what I always claim in any of the open platforms indeed. So nevertheless if you look at this picture this nice cartoon here there are different types of arrays. In the first cartoon you can see the same slide with the barcode and one glass slide printed with protein expressed libraries and if you can add the protein which is already pre-labeled to the glass slide if those proteins printed on the glass slide have the interactant partners in the lysate they will attract and immobilize them when you wash out. So other proteins will get washed away and those bound to that remain on the glass slide and we can visualize them. Then you can see that so particular protein X which is label bound to the protein Y on the glass slide that means they are interacting with each other then you can proceed to see that whether this interaction has any physiologically relevance in a disease state or non-disease state or not. The second way of approach is that again the glass slide so we all talks about glass slides today. So glass slide printed with an antibody or of course antigen also you can quote it in this case antibody and then add the lysate which is a pool of all the proteins which are expressed by the orpheum. Then those antigens which are specific to antibody captured by the captured antibody of course this protein is not label here that is why we need a detection antibody. So the detection antibody is can be any biotinulated antibody or any choice of your interest. So then you can detect the this protein which is immobilized which is captured by the captured antibody by looking at the signal here. So this signaling methods I will explain you what are the different methods can be used. And here that this is a real approach what we are going to talk next half an hour or 45 minutes. So here the lysates can be printed. So today morning Sinjiva explained that he is also printing a protein but it is a externally expressed pure protein in a each spot. But in this case we are going to print as such protein lysate take a cell lysate when you scrape them in a cell culture lab and slice them and spin down the supinated contains a pool of proteins that sample directly are going to spot here. After spotting then you add a primary antibody. Then primary antibody binds to the specific protein of interest protein in the spot and then the secondary antibody will detect the signal. So it is the same essentially you can call it as a dot blot or micro western blotting approach here ok. So then the last sample here you can make the peptides you can small peptides or a engineered protein like morning Sinjiva explained can spot them here and you can also take a complex mixture of proteins already pre-labeled add that mixture into the slide. So those proteins which have affinity towards these peptides or engineered proteins will bind. So more or less these two slides these two cartoons explain a similar approach but there is a little different here. You see that printed protein the full length protein is spotted here it is only peptide is spotted here. The difference is here you can also use this approach for epitope mapping. So you can make a fragmentation of the peptides. So those peptides can be printed separately and you can map on the which domain of the complete protein is binding to the binding partner. Maybe it is 100 amino acids you chop down into four different parts 25, 25, 25. So the interacting partner the pre-protein may bind to the peptide between 51 to 75. So how can we map that? So this is the approach can be used to map the really binding domain or the interacting domains between the two proteins. So you can see that this is a forward array where antibody is spotted and the protein A is captured by the antibody from the pool of lysate and the secondary antibody specific to protein A is used for the detection. So this is coupled to enzyme linked secondary antibody. If you look at here have a glass slide which is coated with a nitrocellulose membrane and is a pool of lysate along with your protein of interest. Here we have A to G proteins. So in a single spot it contains the protein of interest along with other proteins. Then you had a primary antibody against protein A. So it will bind to the protein A in the mixture. Then use a secondary antibody directly against the primary antibody you have used. So you can see that this red Y is now detecting the protein A. Here in this case red Y is capturing the protein A. That's the difference between forward array and reverse array. So once you have the primary antibody bound to the protein A after several washings you can use the detection antibody which is nothing but a secondary antibody labeled with specific visualization method then you can detect it. So this can be miniaturized into high throughput array by spotting many different number of samples. So this is basically the reverse phase protein array. This concept has been developed by these two great scientists, Petrikoin and Lance Lyota from USMD Anderson and they started to optimize this technique or they started to establish this technique at 1990 finally it came into the light in 2001 after proof of concept and so on. Further it is progressing. Now in 2015 people announced that this can go really into the clinic towards a personalized medicine. Now how? I will try to convince you that how it can be used in the personalized medicine and then obviously it requires lot of technically technical challenges. So this cartoon explains the spotting of the samples. It's a single lysate spot that means if you have 1000 samples. So 1000 samples will have 1000 spots here. Individual spot belongs to one sample. Remember that. One protein that's where always we think that one spot will contain one protein that is a forward approach. In reverse approach one spot will contain thousands of proteins from one sample. So once we spot it then we have a primary antibody to detect and then visualization after that array gives this kind of data then we have to proceed for data analysis. So advantage here is that in one spot requires only low volume of sample we spot only one nanoliter. Think about from one nanoliter you can quantify one protein. In western lot you need 20 micrograms that means it's 20,000 nanograms sorry 1 to 8 nanogram protein. Let's say 10 nanogram is required. So if you want 20,000 divided by. So you can quantify 2000 proteins from 20 microgram protein sample in this reverse phase protein array approach. So the total protein you will spot depending on the intensity or the protein of interest between 1 to 8 nanogram of total protein. Once you spot the array slide looks like here. Then of course to print such a high density array you need a sophisticated machine you cannot simply spot with pipette and this machine is really useful nowadays meant for reverse phase protein arrays which can print 50 replicate slides per run with 2000 samples per slide. And also why I am highlighting about this instrument I am not from definitely company thing is that when you sprint the first slide the same quantity should be printed on the last slide. So duration will be 3 hours. In the first slide with the one spot contains 1 nanogram 8 nanogram the last slide also should have 8 nanogram protein. So this machine has been designed in such a way that it will give a uniform printing of the slides. And also from one corner to other corner it should spot in a precisely the same amount of protein by maintaining the humidity and XYZ parameters inside the machine. So this machine capacity is very good as on today and main thing requirement as well as serious limitation in reverse phase protein arrays is the good quality antibodies. I am sure many of us might have used Santa Cruz antibodies when we do western blood we see 10 different bands which belongs to our target of protein interest we do not know. So these antibodies are very very limited and we cannot use them for reverse phase protein array approach that is where this reverse phase protein array is difficult to perform in protein laboratories. So how to there is a way and also there are multiple detection methods are available alkaline phosphate detection HRB detection chemiluminescence and also infrared imaging as well as site labeling and all. So in our approach in our lab previously we used infrared labeled secondary antibodies to visualize the primary antibody signals why? So IR signals always have a high dynamic range compared to chemiluminescence method or any alkaline phosphate method like a chromogenic substrate. So linearity means if the protein concentration is too high they will signal will get saturated very quickly. Once the signal is saturated you cannot get a proper quantification and proper quantifiable differences between control and experimental samples. So that is the reason to avoid that to have a broad range of detection range. So we used infrared imaging levels infrared even infrared labeled secondary dice. So then here I would like to highlight how the array looks like once you print can see that glass slide coated with nitrocellulose. This spotted lysates which can be recombinant proteins or the total protein lysates near is a detection antibody and near infrared dye labeled secondary antibody. It is a same simple western blotting approach explained to plus two standard student. Okay. Advantage single spot diameter is 300 micrometer that is why you can accommodate more number of samples on slide then you can perform the array and then you will get the signals like this. Here is a complete workflow I will walk through this complete workflow next one hour each step by step. First is tissues or cell lines what kind of samples can be analyzed what are the different types of methods to apply for sample preparation and then how to choose antibody. What are the different steps to be taken care while selecting the antibodies for RPMAs and how to avoid later on issues then spotting pattern spotting methods signal detection methods how different methods or advantages one on the other for detections and finally data analysis and what kind of data we see and I will show one or two examples how we generated the data how they are useful in literature. So, as you are aware that proteins can be printed in different manner on the chip and those could be used for many interesting applications we have discussed that you can print the cDNA and do the in vitro transcription translation to make the protein on the chip or what could do a laborious way of expressing and purifying the proteins of interest or you can have recommend antibodies to be printed or even you can have the reverse phase protein arrays where you can take the tissue lysates or cell lysates where you can probe with a specific antibodies and proteins of interest in addition to just having the purified protein or antibodies printed or even having cDNA to make chips like the cell free expression based arrays one could also try to probe directly a specific target of interest in reverse phase protein arrays as we have discussed today with Dr. Ramesh and Mani. So, I hope today you have learned about reverse phase protein arrays the basic workflows and how the array looks once it printed on the chip and the advantages of using RPPA. This talk will be continued in the next lecture where Dr. Umani will talk to us the entire workflow of reverse phase protein arrays in some more detail. Thank you.