 Dr. Josh Liber has taught you various good concepts about planning for experiments for biomarker discovery. How a very novel protein microarray platform like nucleic acid, programmable protein array or NAPA can be utilized for many applications. Today he is giving his last lecture where he is going to talk to you about a case study in which how NAPA technology could be utilized for the functional studies. There are various type of modifications, post transition modification happens which makes protein functional which gives them different properties and which are very crucial to study. However, studying PTMs are not very straightforward, not very easy, there are variety of modification happens as you are aware like phosphorylation, glycosylation, acetylation and there are some newer forms like addition of AMP, AMP acylation, etc. All of those are very crucial for understanding a given cellular context. Supposedly we will also summarize all the various studies which have been covered during his section especially for the NAPA technology as well as the biomarker discovery program and other clinical applications. So let us listen to Dr. Josh Liber's lecture. Today since it is the last lecture of the series is focus a little bit more on sort of functional studies that we have been doing with the NAPA with a fairly heavy emphasis on our most recent story. And I just thought it would be useful to you know one of the things that I keep saying is that the proteins on the array are active and I think probably one of the best bits of evidence for that is when we test function of these proteins protein-protein interaction enzyme substrate activity that sort of thing we usually get it and so that is kind of how we look at it. So, the first one of the first stories that we looked at was ampillation. So, you are all familiar with this part of the pathway where you have ATP and the gamma phosphate on ATP is added to 3-inase serines or tyrosines on proteins in a process that we call phosphorylation and that is usually catalyzed by an enzyme called a kinase right. So, you are all you have seen that a million times by now. So it turns out that in some circumstances a slightly different but very related reaction occurs in which the AMP the adenosine plus the first phosphate get added to a protein. So, you get AMP 3-inase serine maybe and AMP tyrosine and that is called ampillation. So, you are taking the opposite half of the molecule and you are adding it to proteins and it turns out that this process is remarkably well conserved if you look through evolution. So, if you look at many, many bacteria and even in eukaryotic cells there are classes of enzymes called ampillators that will do this reaction and we do not fully understand what the biology of this interaction is, but one of the places where we see it the most often is when bacteria infect an individual and then the bacteria use this to modify host proteins. So, it is possible that pathogens have used this as a way to regulate expression in cells. So, the challenge with ampillation is that we do not really know what the targets are. For years people have been trying to study what are the targets that are being modified by these enzymes and when we began this work there was probably one bona fide target that we really knew about. There were two or three others that had been proposed, but not verified and the methodologies that people had used they had tried you know doing pull down experiments they had tried doing mass spectrometry experiments, they had done various chemical linkage experiments, but it was very hard to figure out what the targets were. So, we had a very ambitious postdoc in the lab Shabo Yu and he wanted to see if he could use the protein array as a way of discovering what were the targets of these enzymes. So, the approach that he took to do this was the idea would be you print an array, you express the proteins and you treat it with an ampillator that will add the amp group to the proteins and then you come back later and try to determine which proteins have the amp group on it. So, the initial approach that people thought would work would be you do this method you have the amp group and you come in with an antibody that recognizes the AMP here, but it turns out that the antibody did not work well it was really not very selective and it did not pick up what we wanted. And so, he came up with a different strategy that was very creative. So, the strategy he used is based on click chemistry, basically you take an alkyne group and an azide group these are two chemical groups and they are reactive species, but they are very selective reactive species and in the presence of copper they will they will form a chemical reaction that creates a covalent linkage and it requires copper for activity, but it is very selective. So, if you run a if you have the azide group on one protein in a cell isate and the alkyne group on the other protein even amongst millions of other proteins only those two will link and nothing else will. So, it is very selective. So, what he did was he worked with a group Howard Wong in New York who had made a modified version of ATP in which he put this alkyne group right here that is the alkyne group on an ATP that was linked to the sugar so it was part of the AMP molecule ok. And so, the idea then would be you take a protein array, you translate the proteins, you remove it turns out that one of the things that Shabo discovered was that to get this to work he had to remove all the DNA from the array. So, you all realize that we print plasmids on the array to make the proteins. Once the proteins are made you do not really need the DNA anymore and for a variety of reasons at times if you need to you can digest away the DNA with DNA's and you still have your proteins left on the array. So, that is what he did and then he added this alkyne modified ATP along with an ampilator that released a pyrophosphate and it added this modified AMP to whatever target proteins were there and now it is displaying this open alkyne group. He then came in with an azide linked to rhodamine which is a fluorescent marker added that to the array and added copper and that added the this it added it caused the covalent linkage and displayed the fluorescent tag. So, essentially he was marking the modified proteins with this azide and then he came in with rhodamine with alkyne and they came with rhodamine linked azide to find the proteins. So, then only proteins that are targets of the enzyme will light up. First thing he did was to make protein arrays here he shows you that he has the DNA remember we stain with a picogreen to look for DNA that confirms to be a good printing. Then he expressed the protein and captured it and then he digested all the DNA using a DNA's and so if he stains for DNA again it is completely gone ok and then and this is just showing you here is the DNA level before and DNA level after treatment. Then he tested with anti GST antibody and showed that he still had all the proteins. So, this is kind of useful to know in some circumstances when you are going to be working with a protein array like NAPPA if you don't want the DNA around let's say you are doing a transcription factor study or something like that you can digest the DNA away and you are still left with the proteins and it is still a perfectly good protein array alright and then this just shows that when he did two different array studies he got very reproducible results. Ok, so now he has got this whole protein array displaying protein no nucleic acid no DNA on the chip and he wants to then treat that array with an ampulator plus this alkyne modified ATP ok. So, here is the array if he treats it with modified alkyne ATP and buffer alone. So, you don't see anything that is good right you don't if there is no alkyne if there is no ampulator there you don't want to see a signal if you did then that would mean that you had contamination. VAPS is a well known ampulator and then IBPA FIC2 is also a well known ampulator. When he treats with those guys can you see that? All of a sudden a few not very many, but a few spots like these guys right here start lighting up that is kind of the result that you are hoping for right when you are a researcher in the lab when you are a graduate student in the lab and you see only a few spots light that is what really gets you excited because if everybody lit up then you know there was a lot of background and it probably didn't mean anything, but if only a few selective ones light up that is a sign that you really found something. So, this is what those spots look like and of course these are the identities of those spots you can see that they have very clean signals right and of course you don't see those signals over here on the control right. So, by the time he was done with these experiments and I am not going to walk you through all the studies he did of these experiments he this is what was known before he started that was the only known target and all of these targets in here were things that he uncovered by screening the arrays. So, he found a couple dozen more new targets for this when he actually looked at the targets and I don't think I have the slide to show you for that work, but he actually found that there was a sequence motif that was common to all the targets or at least most of the targets and so he was able to identify what it was that the amplilators were looking for when they modified proteins and a lot of the targets of these proteins turned out to be GTPase proteins. So, that is one example of how you can use the array to study for enzyme substrate type interactions. Another assay and I am only going to show one slide from this because it is still an early study is work that G. Cho and the lab has been doing and again I don't know hopefully you can see the dark spot there, the dark spot there, the dark spot there. He basically was looking for proteins on the array that autoacetylate and so in this case he was using an acetyl group that was labeled and then he was using a acetyl group that could be detected by an antibody and then he treated the array with acetyl CoA right there incubated the array and just allowed the proteins to acetylate themselves, washed away all the reactants and then stained with the antibody and identified proteins that autoacetylate. So, this is again a way to look for enzymatic activity on the array. Okay, so now I am going to switch gears and talk about yet a third application and I am going to spend a little bit more time on this one because I want to kind of walk you through what I think you want to do when you do these sorts of studies because one of the mistakes I see all the time as a journal editor and I can't tell you how often I see this pretty much every day I am rejecting at least two papers for this is people do one screen with a proteomics technology maybe it is a mass spec screen maybe it is a protein array screen they get the results then they write it up or they get the results they do some informatics and then they write it up and they don't follow up on any hypothesis they don't do any subsequent biology they just simply say here was my screen here is what I got enjoy it and you know for me that is not really what scientists should be doing they should be I using this tool to identify a hypothesis and then doing some kind of work to test that hypothesis you don't have to follow up every lead but you should follow up at least one or two so that by the end of your story you have shown something new that you didn't know before because that is really the goal of science and so we usually send those papers back to the authors and say this is good start too preliminary go back and solve a problem and come back to us when you have a bigger story okay so this is an app array and what we are going to do here is we are going to express proteins on the array so first here you see the array staying with pico green which by now you all know it means it is the amount of DNA and the fact that it is pretty even in its staining means that we did a pretty good job of printing and then here what we have done is express the proteins and then and then stain them with an anti flag antibody and the reason it is a flag antibody is in this particular circumstance these proteins which are all kinases happen to have the flag tag and not the GST tag and it is just a good point to remind you that we are not wedded to any one tag we have done NAPPA with mick tag with flag tag with GST tag with halo tag it is a technology that can be used a variety of different ways in this case we happen to have all the human kinases in the flag tag so we use the flag tag and this gives you a sense that the proteins are well expressed where they should be right okay so now the question we wanted to ask was are these proteins phosphorylated so we took this array and if we don't treat it with if we treat it with buffer and just but no ATP and you stain it with an anti phosphotyrosine antibody none of the proteins light up so that means that after if you strip the kinase if you strip the proteins with phosphatase to remove the phosphates and stain with anti phosphotyrosine antibody you won't see any the proteins won't have phosphates that no surprise the question was were they were these proteins active and so if we added back ATP to the array and just incubate the array with ATP now some of the proteins are lighting up all of these proteins are autophosphorylating right because just by adding ATP to the protein on the array they're they're phosphorylating themselves on tyrosine okay so that that's really good evidence that all of these proteins are enzymatically active on the surface of the trip okay and it was evidence to us that we had the possibility at least of of exploring now the function of these proteins in the array setting so one question that comes to mind is can you inhibit this activity using drugs so the first experiment that that Fernanda did this was my this was a postdoc in the lab at the time where she took a broad spectrum kinase inhibitor called Storosporin and Storosporin inhibits most kinases and she increased the dose of Storosporin on the array and so here's no ATP here's full ATP and then here's increasing amounts of Storosporin and as you can see the kinase activity is headed is decreasing due to the drug it's not completely wiped out but it's significantly inhibited so that means that the enzymes are behaving as we expect them to a more interesting question is can you selectively inhibit kinases so can you use a kinase inhibitor that knocks out one kinase but not another kinase and will it also behave on the array right and that's this experiment here so many of you are familiar with this drug imatinib imatinib is the same thing as Glevec Glevec is the was the first selective drug inhibitor chemical inhibitor ever used to treat a targeted pathway in cancer so I mentioned the other day that Herceptin was the first targeted pathway that was an antibody this was the first compound this is Brian Drucker's work he you know essentially invented this molecule that selectively knocks out the BCR able protein the people with type of CML get this translocated enzyme that links the BCR gene to the the able kinase and it activates a kinase and it becomes an oncogene that turns on that creates cancer and using imatinib you can put people into remission in fact there are long-term survivors now with that disease who've been treated with imatinib and who have never gotten their cancer back so it's it's a pretty promising compound all right so what I want you to look at first is this protein in the green circle a green square and you can see that this is the TNK2 kinase and notice that no matter how much drug we add it's still active so the drug is not inhibiting TNK2 but if you look at BCR able here it is here now it goes down a little bit now it's much down and now it's completely down so the drug is selectively knocking down BCR able but it's not knocking down TNK2 similarly if you look at able which is right down here you can see that the able is also decreased okay so so on the array these proteins are behaving exactly like you want them to so we did a number of studies like this to convince ourselves that the array platform was behaving as we expected and then we then decided now can we discover something new with that so we started treating the array with other kinase inhibitors and what we looked for in particular was were these kinase inhibitors ever hitting a kinase that we didn't expect them to hit and one of the first ones that Fernanda found was this one so a Brutinib is a drug that's used to treat an enzyme called BTK or Brutin's tyrosine kinase BTK is an important enzyme kinase in the B cell pathway and it plays a role in a lot of B cell cancers so mental cell lymphoma for example relies on BTK and a Brutinib has turned out to be a very useful drug in treating those patients it inhibits the BTK it essentially stops the growth of that tumor and it's well tolerated by patients not a lot of side effects so we asked you know does the Brutinib inhibit anything else so the first thing I'm going to point out to you is that and that is able one able one was the example in the last slide and you saw that able one was inhibited by a matinib but able one is not inhibited by by a Brutinib you can see that the signal is the same in all four spots okay so then we asked well is it working for BTK which is the one it's supposed to work for and that's in red and sure enough there's BTK it's going down it's going down even more it's going down even more so in this case even though it's not affecting this this kinase it is affecting that kinase and then what Fernanda noticed by carefully reviewing these slides was this guy down here strong signal over here weaker signal here weaker signal there and we could sit weaker still over here and lo and behold that protein turned out to be herb B before that was pretty exciting because if you think about it the herb family right so EGFR receptor herb B2 her to new those those are two of the most prominently known oncogenes in all of cancer studies right there are very very successful drugs against both EGFR and and herb B2 and now we found a drug against herb B4 there wasn't a lot of data on herb B4 and so that's why we decided to kind of pursue this story a little bit so the first question we wanted to ask was could could this drug inhibit cell growth so the first thing we had to look for was cells that had herb B4 in it okay now the next thing we thought about was what about artifact what what potential confounders could screw us up so what else do we know about BTK as a drug what does it normally inhibit a Brutonib so it the main target the reason it was invented was to target what kinase BTK right so if I put it if I use it in a cell and it has BTK in it then the reviewers are going to look at me and say well how do you know it's due to the herb B4 it's probably due to the herb BTK so our first thought was we need a cell line that has a lot of herb B4 and no BTK right okay so it takes a little time to you have this when you do your experiments you have to think about them a little bit and make sure you're doing them in a logical way so that's what we did we searched all the CCLE is a is a website that has thousands of cell lines and their gene expression labeled and we scanned all those data specifically searching for proteins that had high levels of herb B4 and low levels of BTK and then we ordered a bunch of them and then the first thing we did was confirm by Western plot that these cell lines were as advertised so here we show here's our positive control that has BTK all these cell lines have no BTK so that we've taken care of that that that's not going to be a confounder and then all of them have varying levels of herb B4 so the protein is definitely present and then this is just a loading control okay so now we know that we have some cell lines that have both and the first question we want to ask is will BTK will a Brutonib inhibit these cell lines because our hypothesis now based on our protein array study is that a Brutonib inhibits herb B4 and we think that that might in some way inhibit some cell lines that rely on herb B4 for their cell growth keeping in mind that no one's ever really shown that before so we treated all these cell lines with with the Brutonib and what we saw was a range of activity so some of them this is relative when I say relative cell viability what that means is cell growth plus minus drug so if it's 100% it means that with drug it's the same as without drug if it's if it's down around 25% that means that with drug it's inhibited by 75% and so you could see that some of these cell lines out here were significantly inhibited by a Matt nib a Brutonib and these guys not so much so these guys look like they're resistant to drug these guys look like they're sensitive to drug you still with me okay so then we did a dose response curve which is the logical thing to do next and that's what you get so adding increasing amounts of drug you see increasing inhibition of cell growth so that looks like a Brutonib is inhibiting a cell line that has high erby before and no BTK so you can't argue that this is due to BTK okay and then and then we did a couple of key controls we looked at cell lines that were either erby before negative or BTK negative and sure enough neither of these cells were responsive to drug okay so let's so let's think a little bit about the erby before pathway because one of the first questions that comes to mind is okay maybe you've added a drug that you think you've shown inhibits erby before on your protein array how do you know that it's really inhibiting it in these cells in a way that affects the cell pathway the the biochemical pathway by erby before because you have to show that right you can't just say well okay inhibits in vitro but I don't know what happens in vivo so let's look at the pathway there are two pathways for erby before there's a growth growth survival pathway over here and there's a proliferation pathway over here they're they're kind of similar erby before also has an alternate pathway depending on the the splice form that you use but you can see that it signals directly is erby before like the other eGFR receptors is a dimer there are this is neregulin binding here there are other ligands that bind to this this protein it sends signals through the RAS pathway the RAS ERC pathway it also sends signals to the PI3 kinase AKT pathway via mTOR so these kind of look very similar so if you were going to think about key parts of these pathways to test you might look at these guys AKT it's a well-known oncogene that that drives cell division and then Mech and ERC which are also oncogenes and play a role in signaling through the transcription factors like the June Foss and so on all three of these proteins AKT Mech and ERC get phosphorylated when they're active and we have good antibodies for those phosphoforms so a good simple test would be when we inhibit the cells with ibrutinib do we see a reduction in the phosphoform of these proteins which are downstream of erby before right and I wouldn't be telling you this if we didn't do that experiment and that's the result so here you see that first of all erby before itself is less phosphorylated with drug so that tells you right there that it's already that that it itself is being inhibited by drug and then here you see phospho mech is going down not dramatically phosphoric is definitely going down and also AKT is certainly going down so and keep in mind that the level of AKT is the same so the protein is still there it's just not as phosphorylated same as true of ERC same as true of mech although this is a little bit bigger than that I think but you get the idea right so so when we treat with the mat when we treat with a brute nib it inhibits the act the downstream pathway of of erby before okay so now what's the next objection we're going to get from the reviewers so we've shown that it inhibits the kinase we've shown that it turns off the bio chemical signaling pathway right we've shown that it's reliant on erby before expression in cells and and and it doesn't matter the if BTK is not there what else do we have to worry about what about other members of the of the the her erby B2 family or erby B family right so those proteins are all very similar there's a very good chance that a brute nib could also inhibit erby B2 could inhibit it could inhibit EGFR and so one of the objections the reviewer might make is well how do you know that it's specifically through the erby B4 pathway and not through these other members of the of the erby B family because after all they're really well known we know their cancer proteins right so so that was what we have occurred to us you know it might it might inhibit erby B Sark family members some of these proteins in the literature had been listed by someone somewhere as being inhibited by erby by imatinib a brute nib so I keep confusing those although the data were not very strong so and then some of these had this cysteine residue which is in the binding pocket where the drug seems to bind and so it's possible that because they have that cysteine like BTK like erby B4 they too might be inhibited so the question we want to ask was if we you know could EGFR or erby B2 be contributing to this improvement response could it be that this is all due to these guys and not erby B4 right so so how would you rule that out knock down right so you would take out you take these guys out right and still see if you get the effect right so that's what we did so we we did shRNA here you can see that this is the level of EGFR using a during shRNA that knocks down EGFR we could significantly reduce EGFR notice that these shRNAs do not affect erby B2 or erby B4 and similarly we had some erby B2 shRNAs that knocked out erby B2 did not affect EGFR did not affect erby B4 and yet and yet despite that we despite knocking down EGFR or knocking down erby B2 we still saw the drug effect the drug still inhibited the cells so now we can say that they're sensitive to a brute nib even if you inhibit EGFR or even if you inhibit erby B2 okay so so then then the question was alright I'm still not convinced how do you know that not can you prove to me that knocking down erby B4 is really going to stop cell growth so we're going to a lot of extreme here because people have tried to study erby B4 a lot and they haven't the people have tested it superficially but they haven't really spent a lot of time on it but so far the data have not suggested it was an oncogene so that's why we wanted to spend some time on it so so we wanted to ask the question can we knock down erby B4 without the drug how would you go about that shRNA right so that's what we did so here here is in one of our cell lines 522 this is one of the sensitive cell lines here's an erby B4 shRNA clearly knocking down protein right and and and if you knock down the protein cell division gets reduced so just knocking down erby 4 is sufficient to give you cell inhibition here's another cell line knocked down erby B4 once again you see reduced cell growth here's yet another cell line knock it down once again you see reduced cell growth so knocking down erby B4 by itself using three different shRNAs in each case is sufficient to knock down cell growth so we felt like we had pretty significantly demonstrated that this cell growth inhibition was due to erby B4 inhibition and this is just to show you that when you knock down erby B4 it doesn't affect EGFR erby B2 so that's yet another possible criticism as well okay you're knocking down erby B4 maybe you're also affecting EGFR erby B2 but we can show you that we actually couldn't detect your EGFR in these cells but if you knock down you don't see any change in erby B2 no change in erby B2 no change in EGFR no change no change so this was really due to EGFR erby B4 yeah there aren't any good drugs right now that we know of I mean obviously we were we think we found one of the first erby B4 inhibitor you could know you could knock down you could block the activity of EGFR or you could have done that you could have done that yeah that would have been another way to do we actually just did it genetically which was easy but you we could have gotten the drugs right and then of course the other way to take out erby B4 would be to do CRISPR but that's a very involved process and we didn't really need to go that long okay so then kind of the last piece of this piece of this puzzle was asking the question does this matter at all in biology right would it matter in an animal and so we took we took these cells created tumors out of these cell grew tumors out of these cells in mice and then either treated them with a brute nib or no a brute nib and and and you can see the effect so this is tumor growth without a brute nib and this is tumor growth with a brute nib right so clearly even in animals this is working and notice that if you take if you take one of the resistant cell lines it's not a brute nib responsible that the use of a brute nib doesn't really affect it at all the difference is really over here where where where we had a sensitive tumor okay so now you remember when I started all this and I showed you a bunch of cell lines several of the cell lines were sensitive to the drug but quite a few of them were not sensitive to the drug so the question then becomes well how come what why are the non-sensitive cell lines resistant right what what makes them resistant and I think to me that was the crux of the matter because historically early before inhibition had not been a successful cancer or early before had not been an obvious oncogene and I think part of that reason is because there's a lot of resistant cells and so when it when people did expect experiments they sometimes stumbled on these resistant tumors and they saw no difference as they decided didn't matter at all all right so so we looked at the tumors that we had and we looked to see if we could figure out what was different between the sensitive tumors and the resistant tumors the responsive tumors are in blue right and then the resistant tumors are in red and so looking at this at erby b4 levels you really couldn't see anything different from erby b4 even phospho erby b4 not really an obvious difference here's a resistant cell line with very strong phospho erby b4 here's a sensitive cell line with really strong phospho erby b4 not not an obvious correlation there and we looked at two different phosphorylation sites on erby b4 so it didn't correlate with abundance or phosphorylation we also looked to see if the state of the of the eGFR erby b2 or erby b3 could also have an effect and we looked at their phosphorylation levels and once again you know we could spend some time on this I won't bother comparing responsive to non responsive there was no obvious difference so we were left with this you know unsatisfying situation of having resistant cells and sensitive cells but not really understanding the difference so we thought we would do a gene expression profile in these cells so we looked at we had these two groups we had three cells that were sensitive and four that were resistant so we put them through gene expression and asked is there anything in their gene expression that would correlate specifically with responsiveness could we find a difference right so that's what you're looking at here you these are the sensitive cells I get that right sensitive cells resistant cells and right we ran that did did we did them but we did them in untreated cells we did that because we kind of wanted to know what was at that baseline we also did the treated ones but that gets more complicated we did high-seq sequencing you know assemble them into genes identify which genes responsive and then compared them compared these results among this population to data that had where these cell lines have been treated by have been looked at in the CCLE data so we had our own RNA-seq data and we also looked at the CCLE data I'm not going to go through all of the informatics that we did to kind of sort this out but in the end what we did was look at which pathways which gene pathways were best correlated with responsiveness this is a diagram of gene expression that shows a difference between sensitive versus resistant so red means it drives resistance and blue means it drives sensitivity and the bigger the dot the more the effect okay or actually the more the p-value I should say and so what you can see is there's so here's the smart a4 for example but we were focusing right here on wind 5a and DKK1 and the reason these two really stood out for us is these are opposing proteins this guy inhibits that guy so they are directly in opposition and they're acting in opposition this guy drives resistance this guy drives sensitivity and so they were among the 10 most predictive genes and we decided to follow them a little bit more closely the one of the question was can you take a non-responsive cell and make it sensitive by blocking wind 5a so our mall our model is that wind 5a drives resistance so the question was could you could you change that and so Femina did this work she took she took the cells she used a wind 5a sh h RNA she tried several of them and she found a couple right there that worked pretty well she created cell lines with knocked down wind 5a and showed that in fact now they are a little bit sensitive to the drug right so this is using scramble sh RNA and then these two are both the target s RNA so so you can make the cells sensitive by doing that and she did that in two different cell line backgrounds just heroic right and so then the then the flip question is also present so here we made the cells more sensitive so the question is can we take a sensitive cell and make it resistant by giving it wind 5a right and so she did so yeah so she did that experiment it turns out if you take cells that produce a lot of wind 5a if you take their media just the cell called your media you can there's a lot of wind 5a in the media the proteins right there and this is the evidence for that and so if she treats the cells a sensitive cell line with with wind 5a so this is with wind 5a and this is without you can see that they are much less sensitive to drug so in 5a does what we predicted it does right so that's all I have on that story but it kind of illustrates for you kind of what what you hope that your proteomic studies will do right they will open up a new idea a new possibility in this case they indicated a new drug that targets a protein that wasn't previously thought to be related to cancer allowed us to explore that that protein as a possible cancer protein and and kind of pick apart a little bit of a story that we think reflects back on the biology and the disease which is what we're really trying to do right our real goal here is to understand disease it's not just us to use a technology it's to use that technology to study something. So in conclusions you have learnt about how to perform functional studies and especially AMP salation assays using NAPA technology as I mentioned is studying PTM's are not straightforward you need very sensitive technologies you need very careful assay design to really try to capture how a post transition for modification happens themselves as a result NAPA technology very elegantly offers you a very novel platform to look at high throughput manner how the PTM's can be studied you also studied about high throughput screening on human studies as well as the one-step autoacetylation on NAPA arrays today you were also introduced to the non-selective kinase inhibition on arrays and how NAPA technology could be employed for performing such assays very easily you are exposed to the concept of identification of drug targets using NAPA technology in the continuation of trying to give you the feel of how protein microarrays and the technology associated with microarrays could be utilized for different applications in next few lectures we are going to talk to you about different type of array platforms and different clinical applications how this could be utilized for other biologically relevant problems you will see how to perform a protein microarray experiment in the laboratory settings where directly from my proteomics laboratory some of my senior PhD students will show you the various assay that steps performed in doing microarray based experiments it will definitely give you much better idea about this technology as well as the intricacies involved in doing the experiments in the laboratory settings in case if you are planning to apply these technologies in your own research I think these exposures are very valuable and really needed to take your understanding to the actual experiments and try to employ that in your own work so in the upcoming lectures we will use different types of microarray chips for these experiments as well as some demonstrations will be given to try to convey you the protocols involved in doing these experiments although the basic principles and the workflow almost remain same whether you use the in vitro transcription translation based protein arrays like NAPPA or you used purified protein arrays like UPROT which will be also showing or you use reverse field arrays variety of these array platforms the starting materials could be different but ideally you will see the workflows remains very similar but depending on what the objective is you are looking at a very specific potential interactor you are looking at protein modification you are looking at a biomarker even a globulin protein or you are looking at some sort of inhibitor assay accordingly your experimental design has to be changed and you have to thoughtfully carefully think about what should be my best controls for giving me answers or the right answers to address these questions so you learn about some of these aspects more in the upcoming lectures and I hope you will be then very confident about how to use this one of the very promising technologies for variety of discovery and functional studies in your own work thank you