 After doing a discovery based proteomics experiment, let us assume you have identified a protein of interest for which you do not know what is the possible role of that protein. You want to characterize that protein in much more detail, so possible way of doing that experiment could be you can do immunoprecipitation and identify that this protein of interest interacts with which other proteins. So, protein interaction networks could at least tell you the possible role that what that protein may have. In this slide immunoprecipitation followed by mass spectrometry have become a very powerful technique to do the protein interaction analysis. In today's lecture, we will have a demonstration by a research scholar who will show you how to perform immunoprecipitation mass spectrometry based experiment. Raisin or a bead support and we will conjugate our antibodies with it and those antibodies are definitely depends on your experiment, but they will be specific to a particular protein right and that protein you can trap on your antibody and then that protein can act as a bait and on it many of its interacting partners can get bound to right. So, what we will do is we will separate this whole complex. So, we will be doing a complex zone study or an interactome study using mass spectrometry. Though immunoprecipitation predominantly also the co immunoprecipitation is used in even in western blocks where you can use one protein and see whether it is interacting with another protein and you can use an cross antibody and check it. So, this also is a is basically how IP is conventionally used, but here we will be doing it how we will study it using like we in western block you can identify maybe one interacting partner, but using mass spectrometry you can identify a plethora of proteins from your complex. So, thereby I will be talking about understanding protein complexes and their interacting partners using IPMS. So, basically this is the workflow, so we will have a bead support majorly people use Cepherose beads, people use agarose beads also with protein A or protein G immobilized because protein A or G will bind to the FC region of the antibody. So, you will conjugate your antibody and then you will incubate this. So, in the protocol you will incubate your bead antibody conjugate for overnight, so that from your lysate you can trap the protein of interest. So, we will call this protein of interest which is bound to your antibody as the bait and then this bait will actually help you trap all the proteins the interacting partners of this protein. So, in we will take a case study today that is stat 3. So, stat 3 as you know is a part of the jack stat pathway and what if if you were interested to see that how which proteins are there potential interactors of stat 3. So, we will take an anti stat antibody and we will conjugate it with the bead and then stat 3 will let us assume that this yellow dot is stat 3. So, stat 3 will bind to the antibody and then all the interacting partners of stat 3 will we are expecting that it will come and bind to stat 3 and then what we can do. So, this is only one of the ways that you can do IP there are many ways to do it. So, people also directly use the antibody on the lysate and then they trap the complex. So, that can also be done. So, there are modifications of it, but in this case we will have the whole complex home like this and then we can run a simple SDS space, we can excise the bands and do an ingelization and give it to mass spectrometry analysis. So, what will happen you can exactly know which molecular regions you are looking at and you can go back to the database and you can see at your identified proteins on the mass spec and you can know how relevant are your hits whether at all their interacting partners of stat 3, but at least rather than doing a global proteomics like many of you are familiar with wherein you are identifying everything at least now you have just the way you can enrich your PTM you can enrich your sample to look at a particular complex right. So, this is the overall methodology there is an alternate way to do it in you can use this complex and use a buffer like something that has high pH like a glycine buffer what it will do this interaction will break the bead and the antibody interaction will break off and then you can do an in solution digestion and that you can subject to mass spectrometry analysis. So, overall this is the simple workflow of IPMS. So, protocol in brief what we do is first of all we will use the bead slurry and we will decant of the buffer. So, then the antibody mixture is incubated at 4 degrees for 1 to 4 hours. So, this is to ensure that the antibodies properly bound to the bead because that is very important that is what will pull. So, you are sort of doing a pull down essay we can also say that right and then you will centrifuge to get rid of any like things that are not bound. So, unbound things will be you will get rid of it and then you will wash the mixture using IP wash buffer. So, basically it is trace NACL and all those buffers they will keep your protein intact. And then, but at any point you must remember that for this to be successful you should not disrupt the interaction. So, all your steps you need to be very gentle you need not to you should not use something like a very harsh chemical like urea or anything that can denature your protein or even disrupt your interaction. So, that is something that you should keep in mind and then this lysate bead antibody conjugate this mixture you can keep for with a rotary gentle agitation at 4 degrees for overnight binding. So, now again it depends on your experiment you can plan it according if you know like if you have already verified the response using a western blot you will know how much of it of the protein of interest is in your sample. So, accordingly you can design if you if you think that it is too abundant and maybe you might not go for an overnight incubation. So, these things can be optimized. So, further the next day so it basically IP takes like a consecutive three days. So, you can use like a couple of days to get this done. So, then in the next day you will again wash it. So, again you are washing so as to not have those things that are just weekly bound or maybe as a carry over you just want to be sure that you are identifying only the complex of your interest and then you will remove it you will do an elution. So, again this step is optional you can directly load the whole thing in the gel. So, whatever it is it will come up and also you can use the whole complex and do an in gel in solutionization after the elution step. So, applications of IPMS so IPMS is very widely you know is being used now because conventional IP methods have also led to understanding of interactions protein-protein interactions. So, integrating with mass spectrometry just takes it to another level because now you you can do functional essays using mass spec as well and you are people there are many people who do targeted validation of proteins peptides using targeted proteomics for them as well. Their traditional biologist often raises a question that have you monitored the you know have you done a western laws have you done an Eliza. So, for them to if you have someone who wants to address both these crowds you can definitely use this technique. So, one of the preliminary studies that have come up is this by Mathias Olin group. So, they have given a proposal by which you can validate antibodies. So, here by on now onwards I will be talking about three major applications of IPMS. So, there are much many, but in this scope of lecture so application one of the applications I will be talking about is validation of antibodies using immunocapture technique. So, immunocapture is again IPMS because you are capturing the antibody of your interest followed by a mass spectrometry. Secondly, we will be talking briefly about an identifying protein-protein interaction that is interactomics. This is what the this is the demo session that you will be going for. And the third thing is enriching residues that are less in abundance and undergo iron suppression like phosphorylation or even ubiquitinylation and many other PTMS. So, because of that it is often very difficult to you know even if the if the post translational modification is very complex like a ubiquitinylation it is not easy to identify using a global proteomics approach. So, we can also use IPMS for enriching those residues like we can enrich the residues in we can ubiquitinylate the proteins and see which are the residues getting ubiquitinylated. We can use a tyrosine specific antibody which can bind to the tyrosine residues tyrosine phosphorylated residues and then we can do a mass spectrometry. So, thereby there are many things that you can do with this technique. And to the first application. So, in this proposal for validation of antibodies Mathias Olin group what they have proposed is that immuno capture followed by mass spectrometry is a very nice way to know whether your antibody specific or not. So, what they are aiming for is if your antibody is very specific to your target. So, if you bind it to a bead conjugate and then you do an immuno capture it should bind only to the protein of your intramus. If you do the washing if you follow the whole IP protocol what should you get is only your protein of interest and maybe a few interacting partners right. But so they have devised this statement that is an antibody can be considered specific if the top three peptides are derived from the expected target protein. So, if you are looking at an antibody. So, you have three antibodies say from vendor A's, AAPCAM, CST, Sigma. So, you want to know which one works the best for your experiment. So, before probably you are going to do something very you might use something very expensive or you have some sample which is very precious. So, before you proceed you might want to check how specific your antibodies. So, you can do an immuno capture and you can just see whether the top three peptides that are coming up after mass spectrometry are of the protein of your interest in this case stat 3. So, if that is coming you can be confident that whichever antibodies more specific will give higher number of peptides or the top hits would be from the protein of interest. So, in this way they have proposed that for validating an antibody they have proposed a panel of many studies and one of the studies they have proposed is immuno capture. So, the second application is studying protein-protein interaction or the typical interactomics. So, as you can see for any physiological process that you are studying be it in plants humans cancer or infectious disease. So, you often find out your list of targets does not involve one protein you always have something else along with it may be a transcriptional factor may be a protein or another family of proteins. So, it is never in isolation. So, exactly this is what we can achieve by doing an interactomic study in it. So, we will get to see the PPIs or protein-protein interactors. So, in this way we can map the whole interactome using IPMS. So, in the first slide I had mentioned that the protein of interest can be used as a bit in which the various interactors will come and bind to the bit and so, you can identify the direct interactors. So, if protein A is interacting with protein B. So, stat 3 is direct interactor say June or whatever binds to stat. So, now even you can identify proteins that are bound to B or June or whatever it is. So, you are not only identifying the direct interactors, but you are also identifying the co you know indirect interactors. Thereby you can map it and you can form a map of the interactome. Of course, this alone might not be the ultimate experiment because there are hundreds of other things that there can be some molecules that are weakly bound or the washing might not be efficient. So, you need to verify whatever interactors you are getting through alternative techniques, but this can definitely give you a big list of proteins which are potential interactors. So, the application 3 I spoke about is how to study low abundant residues using IPMS. So, again taking one of the case studies, so in we can enrich our samples in the residue that we are interested to look into. So, just the way phosphoproteomic study can be done using TiO2, but you would be aware of the TiO2 technique, it only enriches serine and trionine. You would hardly see any tyrosine residues because tyrosine residues A are very less in the whole proteome and B they are very transient in nature. Sometimes they are phosphorylated, sometimes they are not phosphorylated, so it is hard to get. So, what you can do is you can you can take an antibody, you can pull the tyrosine phosphorylated residues and you can perform an MS to be very confident that you are getting the tyrosine phosphorylated residues. Similarly, in one of the studies what they did is, so they studied ubiquitinilation using IPMS. So, this is the study by an Annie et al. So, they have done peptide level amino affinity enrichment and enhancing the ubiquitinilation site identification on individual proteins. So as you can see, first of all they have monitored the response or the ubiquitinilation using an anti-ubiquitin protein. So one of the steps before you proceed for IPMS, you should check the response or the number of residues present or the protein of interest using a western blood. And secondly, you can do your IP experiment followed by so you can use the antibody and then the bait protein and then do an IP and then you can run in the gel and then you can make fractions of it and directly subjected to mass spectrometry. So in this case what they did is, so ubiquitinilation involves glycine to lysine residues. So the ubiquitin attached to the lysine residues of the substrate protein. So what they have done is, they have enriched the sample using this KGG stretch. So they have actually tried to only enrich samples that have that yeah ubiquitinilation and then what they have seen is, they have seen in even in the mass spectrometry they can identify the sites in which the ubiquitinilation, so they can see the stretches of K and G. Thereby they can be confident that these are the, they have identified the proteins that are enriched in ubiquitinilation. So now today's case study, we will be doing a small IP experiment. Of course the whole stretch of experiments is beyond the scope of this course. But what we will see is, we will see that those small steps wherein you conjugate the bead with the antibody followed by the centrifugation and of course the lysate and antibody bead conjugation, how it happens in the live session. So what we will do is, we will, we have split this experiment into three stages. So stage one is, we have verified the stat 3 is present in the tissue lysate that we are probing with. So presence of stat 3 was verified using the western blot. And then the protein is present, we can have two inferences. Number one that we have stat 3 in the sample because it is coming up in the western blot and number two the antibody is very specific because it has not given multiple bands. So then we have moved on to the IP experiment wherein we have done, we have immobilized stat 3, like we have enriched, strapped stat 3 on the antibody and then we have done the whole IP experiment and we have excised the band and it has been subjected to mass spectrometry. So the stage 3 of the experiment is identification of the proteins, specifically the ones that has been enriched through IP. So we are not taking the whole sample, we are just taking those bands that have come specific and we are subjecting it to mass spectrometry. So you will get a list of proteins and you can then do bioinformatic analysis. So you can do a scoring based on string, you can string DB which is an open access software. There are other softwares as well and you can come up with a score and you can do other experiments to see that how the list of protein you are getting from mass spectrometer, how closely they are the interactors of your interest. And thereby you can form an interactome map. We will demonstrate to this technique in detail as well as provide you the brief protocol how to perform these experiments. So let us have this demonstration session. Hello students. So in continuation with the lecture where we learnt the theoretical aspects of IPMS that is immunoaffinity and immunoprecipitation based pull down followed by mass spectrometry. Today we will see what are the steps in which way we can perform this experiment and how to do that for your biological problem. So first of all just to brief you a little bit about the steps. So majorly in the immunoprecipitation based mass spectrometry we will be doing the pull down of protein of whose interacting partners you are interested to look into. So for example let's say protein say S108N. So you want to learn what are the interacting partners of S108N. So for that first of all you need to have an antibody which is specific to S108N. So for that we need the antibody of the protein of interest whose interacting partners you will be finding out. So here first of all we will take the bead. So this is protein A or protein G beads you can take. So basically the protein A beads have the protein A which is derived from bacteria and it is highly specific and it binds to the FC region of the antibody. In that way we will conjugate the antibody of interest with the bead that is present in your tube. So first of all we will prepare an bead antibody conjugate and then because the antibody is specific to the protein of interest we will try to pull down that protein from your biological sample. So we will perform the experiment in three steps. Number one we will prepare the antibody bead conjugate. Number two we will be doing the pre clearing of the lysate. So for example you want to look into a specific cell line or patient derived tissue or tumor and you want to isolate the protein of interest. So for that we will do the we will first take the lysate of your sample of interest. So this lysate contains thousands of proteins and you do not want to have a very high background. So first we need to do a step which is called the pre clearing of lysate. And then the last step is using the antibody and bead conjugation the mixture where the antibody is already bound to the bead and using this pre clearing lysate which is the lysate which is clear of all the noise or all the background proteins that we will incubate overnight and this is the way we will actually try to get all the interacting partners together in a big complex which we will subsequently run in a SDS gel and then we will do the gel excision followed by the mass spectrometry. So as you can see we have the beads here. So first we have to add washed buffer in these beads. So usually what we will do is we will usually use the pre chilled washed buffer. So this buffer is already chilled. Why this is done is because the beads need to get like activated prior to the binding with the antibody. So the once you put the so you just give it a little bit of a tap and once you put the beads and the buffer so the beads will swell after some time and then you can perform a centrifugation step followed by which we can put the antibody. So we will just use we will just place it in a container and we will gently rock it for some time so that the buffer is uniformly distributed between the beads. So once it is mixed properly you can also again do a gentle tap and do an inversion so that there is nothing like there are no beads that are not in touch with the buffer and then we can do a centrifugation. So now that your beads are activated you can get rid of the washed buffer put some fresh washed buffer and in this and in this you can now add the antibody. So usually 2 to 5 microgram of antibody is usually used but then it depends on the protein of interest and how specific your antibody is. You can use monoclonal antibody as well as polycronal antibody. So for example if you are trying to pull down a protein which has a many of the family members like for example if you are pulling down something like anxin so there are many anxin proteins that are present in the human or mammalian tissues anxin A1, A6, A2 so no matter how specific your antibody is these all all the members of these protein will definitely cross react. So according to the protein that you want to select you need to optimize how much time you want to like keep this binding solution so you can go either for an overnight binding at 4 degrees or you can do a binding for 2 to 4 hours depending on your experiment. So now what we will do is we will add the antibody to the activated beads. So now the bead is already activated using the wash solution to which we will add the antibody. So after adding the antibody it is not advised that you do a very vigorous shaking or mixing so you just gently tap it so that everything is properly mixed and then you again place it in a container and put it on a locker. So either you can leave it for 2 to 4 hours or you can do this step overnight. Similarly for the lysate you can do a same step wherein you incubate instead of the antibody you incubate the lysate with the bead. So here there is already a pre-cleared lysate and after this step is done your beads are already containing the antibody of your interest we will now put the pre-cleared lysate into the antibody bead conjugate. So here the beads have your antibody and now we will add the pre-cleared lysate. Again you can give a gentle tap and then put it back in the container and place it back on the locker. After the incubation you can see that now the beads which are conjugated with the antibody will bind to the protein of your interest from the lysate from the pre-cleared lysate. So after this what will happen is you will already have the antibody bound to the protein of your interest and ideally from the lysate all are the proteins which are interacting with the protein of your interest will stick to it and form a complex. So what we will do is we will discard the flow through we will give it a brief spin we will store the flow through in a separate tube and now we will perform washing. Then why do we have to do washing because if you want to reduce the background that is if you if you consider this mixture it has lots of proteins which are also binding to the antibody bead conjugate which is also which could be not very strong interactors of your protein. So the moment we add buffer we will give it a gentle mix so what will happen is all these loosely bound proteins will get washed away. So you will usually we perform a washing for like 2 to 5 times again depending on the protein of your interest and your specificity of the antibody you can optimize these parameters. So we will gently mix the buffer with the complex and then we will give it a gentle stir and then again do the centrifugation. After giving a gentle spin will again store the flow through in the separate tube set will be repeated for 2 to 3 times you can even go up to 5 times depending on the specificity of your antibody and then what we will do is so now we only need to look at the bead and the conjugated complex. So what we will do is there are two ways to go about it so either you can use an illusion buffer which has a lesser pH that is you can use Trisglycin as a buffer. So what it will do is it will disrupt the bonds now it will disrupt the linkage and it will elute out the complex from the bead. So we will use the illusion buffer and again give it a gentle tap after that we will again put it in the rocker and leave it for few minutes. Make sure this is not too harsh like you don't do it do a vigorous mixing or shaking or even in this rotator because the you don't want to lose the interacting partners that have already bound to the protein of your interest. So with this now we are sure that we have got rid of all the background that might interfere with our study and now we have only the antibody bound to the protein of interest and the interacting partners after the illusion step even the bond between the antibody and the proteins of interest starts getting disrupted. So what we will do is we will again use a little bit of IP buffer and will perform a spin. Once this is done you have to take the supernatant and put it in a fresh vial. So this supernatant now contains your protein of interest as well as all its interacting partners. So there can be two ways by which you can go for the protein identification. Number one you can go for the insolution digestion of this elute. Number two you can perform an ingel digestion. So here you can see when you run the protein in a gel you will get different bands and now you don't know what is what. So what happens is various proteins that are present in the mixture will get separated in the SDS. So what we will do is we will do the ingel digestion. So we will cut the gel and we will make small small pieces depending on the number of fractions you want to do. For example I will cut this lane into four pieces. So this you can see there is a prominent band here. This looks like the protein of interest and probably many of the proteins that have become formed a complex with it. So this will cut into further smaller fractions will take the band that has been excised from the gel and will perform an ingel digestion. So here this you can see this small band may have hundreds of proteins which are potentially the interacting partners of the protein of interest against which you had used the antibody. So after this ingel digestion would be done and you will elute the peptides out. So this is the digested peptide and now we will inject it for mass spectrometric analysis. To summarize our entire experiment we have now performed immuno precipitation of a protein of interest from mammalian cells. So we have taken the cell lysate and we have pre-cleared it using beads and similarly in the another section of it what we have done is we have conjugated an antibody specific to the protein with the bead. So both of these steps were done and then the mixture of the antibody bead conjugate was incubated with the lysate. The protein of interest gets captured by the antibody and all its interacting partners also binds to that protein. So that we can get a big complex or a complexome or we can get the interactome that is present in the cell lysate. So after that what we did is we ran the sample in the gel and we excise the bands and this bands would now cut into small fractions and were digested. After digestion we have separated the peptide and this peptide would now be injected into the mass spectrometry to see what are the proteins, what are the other proteins that have interacted with our protein of interest. The advantage of coupling a very traditional method like immunoprecipitation with something advanced like mass spectrometry is that you get best of the both worlds that is while antibody based detection can detect even a very low abundant protein mass spec can help you identify that protein as well as several other proteins which are potentially the interacting partners of the protein of interest. Therefore IPMS is a very promising approach to look into mechanistic details of how a cell performs, how a signaling cascade performs and hence it is emerging as a very prominent tool in the interactome studies. So as you saw I already briefed you about the experimental workflow of IPMS that is how we perform the immunoprecipitation using the antibody. After the process is done we also saw that we can perform ingel digestion as well as we can go for an insolution digestion of the precipitated complex and now when that is done what we have to do is we have to now submit the samples for mass spectrometry analysis. So now we will be injecting our sample in the mass spectrometer. So now the sample will be run using the LC gradients and the mass spectrometer and we will be possibly able to identify the interacting partners of our protein of interest against which the antibody was used. Therefore IPMS gives a very nice approach to identify not only just one protein but also its interacting partners and not only that one of the advantages it has over traditional immunoprecipitation approaches while immunoprecipitation mostly is followed by a western blot in which another counter antibody is used to see whether you can see the protein of interest binding to other interacting partners there are issues related to antibody specificity and cross reactivity. In this case whatever has been pulled down by the antibody and whatever has been bound to the the bait or that is the protein of interest so the bait and the praise can be characterized very confidently using the mass spectrometry and in the next lecture you will learn in detail how this approach works. Thank you. Hope now you are all familiar with the entire workflow of immunoprecipitation followed by mass spectrometry based experiment. You may have realized that it is a good way to characterize protein complexes and their response to regulatory mechanisms. The IP experiment allows hundreds of proteins to be identified in a single experiment however it is also important to keep in mind that a majority of proteins identified in this experiment may also be non-specific binders. And now you need to do further investigation about from the possible list of interactors how many of those could be the real or direct interactors or how many could be just the sticky proteins who are coming along with the interactors of interest. Researchers are now looking ways to improve the accuracy of identifying these interactors which could be drawn from the IP experiment. However the way mass spectrometry technologies have now become more robust and easy to operate it is now not very difficult to perform immunoprecipitation followed by mass spectrometry experiments. We will give you some basics of mass spectrometry and how to perform these experiments in next class. Thank you.