 In the previous lecture, you obtained a glimpse of the principle of different label-free technologies and their possible applications. In today's lecture, we are going to talk about the recent advancements in the field of label-free techniques and how one could integrate different technology platforms. Imagine that we have talked about mass spectrometers, we have talked about SPR, we have talked about BLI. Can we start comparing these technologies? Can we start integrating these technology platforms? This is what we are going to talk in today's lecture where I am going to talk to you about SPR imaging, one of the latest high throughput platform which can do similar thing like SPR but much more in high throughput manner. Integration of SPR with mass spectrometers for identification of protein interactions and some of the pros and cons and comparison of two major technology platforms for label-free biosensors like SPR and BioLayer Interferometry. So my today's lecture, I hope is going to further unfold the applications of label-free technologies in analyzing different molecular interactions. So let us continue with my lecture which I have delivered in this workshop. So SPR-I, SPR imaging is one of the high-special resolution platform which allows for high throughput analysis of various type of biomolecular binding interactions that you want to measure. Here you can intentionally that you are using imaging platforms. So imagine that you had the gold chip which was you are using for SPR but in SPR gold chips you have the very small region where the gold part is there rather if you have the full chip which is having the gold and then now you are using imaging to image the entire surface. And then that image is going to generate some sort of pseudo image like a microarray which will show you that you know each one of those features which you have printed on the chip will give you an image which looks like a microarray but it is not actually microarray. So that kind of concept was used can we image the whole chip rather than you know exciting on a specific angle only which what you use in the SPR case. So you are radiating by the light imaging on the detector arrays you are using some sort of CCD devices which are not so costly. And then this particular platform showed potential to combine with the microarray based technology because anything now which you can do in high throughput way for the whole chip is very much compatible with the microarrays. So this is where you know one of the image I am showing with one of the experiment I was doing in Josh lab earlier when we were developing some SPR imaging based platform on the early prototypes. So as I mentioned to you you are seeing something here just the pseudo image it is not the actual you know you are not measuring fluorescence these are some of the pseudo image obtained from this kind of platforms which shows you compatibility with SPR imaging. So what happens here conceptually we have this prism we have this gold chip but now here we are exploring the full chip potential to scan the whole surface here and then by generating this SPR image again the concept is same we are still looking for reflection angle we are measuring the percentage reflectivity change when you have too many spots printed on the whole chip surface you need to find out some common angle which can be best angle for doing this SPR experiment. And that is how you have to play with the conditions to find out what can be that best angle which can be fixed for the whole chip surface to do those kind of assays. Once you find that and again that may not be the best and most accurate angle it is a 19 you have decided or 20 you have decided you may have to play with more to find out what can be the best angle to freeze for the whole chip to be scanned. Nevertheless the idea here is to generate the pseudo image of whatever is printed on the chip without adding any kind of fluorescence labels you are just generating the image on the gold chip. I am showing you now one of the workflow which was done several years ago which was on a prototype to give you feel of you know how these kind of experiments can be done. So this is you know prism here and this is a chip full of the microarray kind of chip where you can add all the you know protein feature which you want to analyze and there are certain holes on this you know on both the sides. And these holes you can now combine with the flow cell from which your liquid can come and pass which you want to test out. And then now you can screen the whole chip surface on the SPR kind of setup and it is you know just reminds probably Josh that you know one of the old setups we had the prototypes where now you can test eight antibodies for example or eight different proteins in very much to format like you know you are developing a new instrument setup and these kind of you know solutions are coming in you have the gold chip everything is you know passing from those and then you know is going back in the washing containers. So these kind of platforms could be used and it result into these images which are the pseudo images as I mentioned to you it shows you these kind of spots. Now all these red green what is shown here these are actual spots but along with them whatever you see the gray color is the just background. So software are artificially add some blue color dots these spots could be used for subtracting the background. So in this way now you are you are ready to test a lot of molecules unlike the four what you can do just by using the listening technologies. And these are known as the regions of interest otherwise in any kind of micro experiment any kind of SPR imaging experiment you have to ensure that you know what you are measuring is the right signal and not just kind of you know that particular feature what intensity it has. So you have to take into consideration the intensity of these spots in combination with the background what is there. So therefore for each of these spots we had four of the differences spots. So if you subtract the average of these four from this particular one and same you do for every spot then probably you are only measuring what is printed on each of the features right. Then you have removed all kind of side effects coming from those chips. And then now you can see that how much difference it could make. For example this was the actual spot which is showing you the binding. And now this is something coming from your blue spot because you know buffer is flowing on the whole chip. So there is some sort of you know bulk effect you will see. Now if you subtract this then your shape got changed your sensor ground looks little different now right. So these are the kind of thing which can make the your curse very different. If you are just not using normalization or if you are using those background subtraction which is very crucial. One should really normalize these features on the chip surface and becomes more critical here because you have many spots printed on the long chip and imagine that you know when the liquid is flowing from one side and reaching to the other side it will take some time. And you are you know you are start seeing binding for some molecule and then eventually you will see for every other molecule on the chip right. So then they will show the different type of time at which the binding started. But ideally you want to compare all of them together. So then you have to normalize again them so that now everything whatever you see so it was coming from you can see the intensity that different looking here for different angles. Now you have normalize the SPR curves and everything is now looking same then now you have a opportunity to compare their signals. So the SPR signals which is we talked about sensor ground they are the one actually if your eyes are pretty expert and tuned to look at a data immediately start telling you a lot of information. The SPR data just you know it shows you the different curves will tell you that you know what is happening for the kinetics how much concentration I can see for those proteins what is the surface capacity, molecular weight etc. And many times you know these kind of curves which are showing you the just the difference in the on rate and off rate can be very powerful because imagine that you know you are working towards developing different drugs. And objective for you know those drug development probably you might have quoted several examples. If you want to really make that drug work very fast right you want immediate effects for you know any accidental condition any kind of you know headache kind of things you want just very quick response. So your on rate you are looking at that time versus your off rate becomes very different right. So you want immediate action to happen something has to go bind very quickly and then if you are looking at you know the sleep response you are looking at long-term response for a given particular drug then you are thinking that okay let it goes off slowly you know you do not want it to just you know show a very quick binding and release you are thinking about it should go very long so that you have more effect for that particular molecule. So both of these conditions probably can give you the same KD value it will show you same kind of response overall. But on rate and off rate could be different and those can be so detrimental or useful for your kind of drug discovery you know portfolio which you want to develop. So therefore these kind of you know curves are very crucial as you are watching and there are many companies who work with us who do the testing in our labs in our facility who are trying to look at many of their you know the biosimilar products and some of these kind of testing they do. We can see from their experiments that you know just looking at the shapes of these curves of sensorogram can make them feel excited or not so excited because immediately it tells them it is working or not working. So all these kind of stuff is just visually you are seeing of course you need to do better fitting of these curves to find out the actual values for concentration or looking at the KD values but you know many times these experiments gives you lot of visual feel of doing those analysis. But as I said you know if you want to really get the proper values you have to do good cropping you have to do good fitting in this case for example as I mentioned your response timing is different because you have different molecules printed on the whole gold surface where you have some starting right now and some starting later part so then your response units are shown differently here but now you are normalizing all of them and then you want to see comparison of their responses throughout the whole experiment. So therefore data processing becomes very crucial but advantage of using SPR imaging is like high density microarrays where you are using less of the analytes which you want to test. In the same kind of sample which you have available especially the drug molecules which are very low in quantity or some of the clinical samples I think you have opportunity over there to not test out on hundreds of features simultaneously right with SPR imaging platforms. So that is something you know time saving cost saving of course and going to be much more reproducible experiment and then within the same type of condition which you provide now you can study the behavior of lot of molecules under the same condition which is very critical. If you are varying the you know chip to chip there will be some variation and the day to day temperature variation lot of things even you are doing under controlled condition but if you split hundred analyte testing on 25 chips versus doing on one will have huge difference. So buffer temperature many variables are there and if they are on the same spot at least you are you can neglect them you can normalize them you have a better ways of having the controls to negate those kind of you know negative effects from coming from those molecules. All right so latest popular technologies one of them is Bayakor T200 is one of them offered by G-Healthcare. There are many type of chips which are coming from these particular type of technologies where you know how you want to immobilize your molecules sometime you have different type of carboxy methyl based surfaces sometime nickel NTA kind of chips can be very useful for you if you have histag based proteins. So you can select the right kind of chips for your use and accordingly you can make use of them. Bayakor T200 is right now one of the industry leading technology which is pretty much GLP approved so whatever data you obtain here can be very much be sent for FDA or other kind of approvals and that's a lot of industries are looking for these kind of technology platforms. Nevertheless you know these kind of technologies are giving you very precise data but they're very costly you know considering the how much cost it takes to do the experiments. So many times if you are in the discovery development mode and you have hundreds of you know compounds or libraries to screen this may not be the best platform to start with because you are limited with the you know how much cost you have to screen that many large number of compounds. So new technology platforms like one which I'm showing you here which is biolayer interferometry based platform can be very powerful for first round of screening. It may not have that sensitive on-date off-rate and those kind of KD values which you want to obtain but it will definitely tell you binding happening yes no in a very quick format using your solutions what you have for different type of molecules to test out and here they're looking at the interference pattern. So on this sensor there are some you know these antibodies which you want to test out they're immobilized and then they go inside the like ELISA plate and then they those sensors are going to go and test out your your binding using the interference change which is going to happen. So this something is we have a follow-up lecture so I'm not talking much detail but I wanted to give you the feel that you know in which way you can move forward from different type of platforms. So a good thought can be you know from the microarray experiments you have got leads let's say you know 200 molecules which is of your interest now you can go to BLI to narrow it down further now you can use probably be a code to test out you know how many of those you want to know whether the exact on it offered right okay so there are many label free platforms which are possible I'm restricting right now the applications of those mainly based on the SPR right now because that's what something which we have currently available at IT Bombay and I'm also running a core facility which is offering services for many type of applications for internal users and external users and the kind of you know this particular distribution but what it shows to you that we are doing all kind of applications here looking at even from protein fiber interactions protein liposomes protein protein various organic molecules and one you know interesting experiment came when somebody wanted to print bacteriophage virus on the chip and test with the E. coli whether those abiding can be seen or not so you know all kind of thoughts you can have what you want to measure on the SPR and of course popular ones includes protein peptides nanoparticles concentration analysis something which we developed a new essay and optimization of many new applications so that's the ongoing thing we always want to see that what more we can obtain from these instrument platforms so I'm now kind of you know flashing it through couple of application slides with just an intention that what all things can be done on these platforms these are not my results just coming from our facility whichever people are running the experiments but what it shows to you that you know variety of things can be done for example one of the chemistry faculty lab was looking at one of the Kellexeran molecule and copper binding and to do that there was no chip which was available for us to do immobilization so we gave them the bare gold chip the small part of the chip and then they did the immobilization themselves of those and after doing those particular this is a step which are shown to you so sometime you may not have ready made ships available for the kind of functionalization which you want to follow and that's when you can use the bare ship where you can try out your own immobilization chemistry which could be then used to further do the SPR experiment on bare core chip so that's what we have some bare gold chips as well so this one showing you two different experiments the KD values obtained are pretty close and that was at least you know interesting experiment for us to try out because it was not the ready made chip available for it and still immobilization was pretty reproducible another lab from electrical engineering their intention is to have some benchmarks so let's say you know bare core technology the benchmark available in the in the field if you can measure you know an interaction and generate some KD from bare core and now if you develop another biosensor and you can show that you know I can generate same kind of KD then probably you are talking you know really robust platform development right so even for any new technology development you need to have some sort of you know benchmark development so therefore what they wanted to do they wanted to just look at the myoglobin protein binding with the anti-myoglobin protein and in fact they're looking at also the serum now so when we try to do this thing we were able to pretty much get good concentration of this myoglobin protein and that's something which made them very happy because now they can test their own biosensors and see the benchmarking how close these values are another experiment is from the bio labs here in the protein carbohydrate interaction which is one of the very common thing which we see many people want to look at local binding protein and different type of carbohydrates binding to the proteins and again you know why I'm showing your duplicates because it's become very crucial to give us the confidence that different experiments are giving the same KD values again I'm not talking the biology of it probably I should not talk is others work but I'm just giving you the feel of variety of applications which are possible by doing SPR kind of technologies now one of the industry sample which of course I cannot disclose which was trying to compare that what data we obtain on beacore how that you know similar or dissimilar when they are running to their parent company somewhere in Switzerland and and they had that kind of numbers and the you know KDs in their mind and they wanted to compare the things here and those molecules show pretty close binding based on these beacore experiments we also have some faculty from chemistry who are looking at protein is small organic compound their interactions in this case one of the unknown protein which is not known to us 27 KD protein was immobilized and then we are looking at how small organic compounds can bind to them so again many of these time the KDs are very close and that is giving us you know good confidence that these experiments are you know in duplicates are working well one interest which we had in our lab was to measure some of the known protein concentration in the serum for example from our discovery approach of using a diage and coitraic based platforms couple of protein we identified which looks good to us and as a potential biomarkers so we thought can we measure the level of those proteins in the serum using SPR and to do that you know some new assays were developed which was you know which we termed as a concentration free calibration analysis which was a CFCA analysis where you know different type of you know patients from this is one of the malaria project when we had patients suffering from either a phalcipin or vivax malaria or dengue fever and we wanted to measure the level of one of the proteins serum iodide A and we wanted to see that you know how that protein concentration is different in different type of conditions and because we already had idea for this protein from our other data set discovery data set so we felt pretty confident that you know we can now measure and this is what you need you know you cannot do validation on on large number of samples using mass pack or other type of technologies so then you have to come down to either ELISA or these kind of label free approaches which could do screening of you know the same molecules in a very high throughput manner so this was a good experiment for us to to show that you know we can actually measure the concentration of this protein of interest to us again you know sometime you have to develop these assays in the beginning so you need pure protein it's good idea to try out something if you are planning to do this kind of experiments with the known proteins for which you have the purified proteins available build the confidence and the say conditions from those then you can apply for some unknown compounds as well all right so i'm not showing you some data actual data from two different type of technology platforms so we compared for a couple of proteins both BLI platform and by a koti 200 platform for a few experiments because you know the cost wise they are very different but sometime the you know looking at the acceptability wise things are different and the kind of GLP compliance wise only one platform is more compatible so can we use this you know at least the BLI platform as the first level of screening with high confidence so in this case we use anti beta microglobulin and this antibody and the protein interaction so using SPR and BLI we tried binding for various time points analysis which we did different concentration series came pretty close actually you know this is showing you 20 the overall kd is almost 3 e to the power minus 9 and it is also 2.11 e to the power minus 0 9 these things just imagine was done just you know in a like this kind of instrument what is being brought here not available in my lab that time so not in the best optimized conditions but still the way i can see there's not huge variation just the measuring these kind of you know the abundant molecule bindings then we looked at some of the you know compounds of interest for a given project when we had some drug protocol to look for their binding and again things are pretty close you know coming from the two platforms available we also tested you know another drug which is shown here curcumin and prumbagan which we are testing for some bacterial protein against those and we are seeing you know the changes that are pretty close coming from both the platforms so you know what i want to convey you that you know many times just don't just rush for one technology platform think about if you have to test out many compounds what can be the cost for doing that what can be the variations of doing those experiments start you know using those platforms which can narrow down your your leads and hits then do the final experiments on something which is really you know acceptable and of course if you have huge amount of you know funds available for doing that project then you can do everything on the same platform but otherwise you can mix and match and choose which technologies can give you what sometime you know a good experiment may involve every technology which we are you know studying in different workshops right now here starting from you know discovery from my my spec based stuff and microarray based stuff you know take those leads forward and then validate using these kind of label free platforms so everything you know has its own utility and you have to pick the technologies in the right context all right so now over the period but you would have realized from various lectures given by Josh that there is so much need for having integration of new technologies and development of new technologies and that's the coupling different existing platform becomes very crucial in Josh lab we thought couple of years ago and many people are still working on developing those technologies of coupling Napa technology with SPRE waging based platforms the initial experiments done by Manuel Funtis and then I joined the lab where actually you know these are you know quite old Napa chips 2006 2008 that kind of timing so the glass lights are showing these kind of signals which I'm sure by now you'll have seen it has improved tremendously and then the same features printed on the gold slide are showing much better much more intense signals so first of all the objective was to to see whether Napa concept can work on gold or not because so far it was all done on the on the glass slides and first time it was you know planned whether we can mimic the same thing on the gold surface but looking at the 3D view then AFM images it was I think you know apparent that probably Napa could work on the gold surface as well but the traditional Napa chemistry which you are aware of having you know many components in the master mix right which included you know BSA BS3 cross linker you had the capture antibody and of course your clone of interest the serine of that having GST tag so all of them were actually making the whole mass too much which you are adding on a given chip surface right so the initial experiment when it was started the baseline when you are printing so much mass on the gold chip and in the SPR kind of platforms you are only measuring that how much binding you can see happening right so to begin with itself for the baseline itself is going off the response unit so here you have literally no room to find out how much binding you can observe later on because with the buffer alone the baseline itself is very high which is passing beyond the response unit of the instrument which is the SPR imaging platform so I talked to you about you know briefly about the technology which is SPR imaging you have been exposed for Napa arrays think about the combining the two to harness the both the power so if you add the entire GST antibody and you want to measure the binding of entire GST with the GST protein then probably you have no room here to see the binding where the binding is happening because it's already passing beyond the response unit of the equipment so the next thing was whether the same Napa chemistry can work here probably not so then there were you know a newer approaches came which was one of them was Coil-Coil chemistry of using E-Coil and K-Coil can our clones may have you know E-Coil and the chip may have the K-Coil and using those particular type of chemistry now if you chip you know print your all the clones using the same Napa concept but now you have the K-Coil peptide the capture peptide and each of the clone contains E-Coil tag so you are not talking about peptide-peptide interactions here and it's a very you know strong interaction not having so much material printed on the chip surface so you have you know no you know the same issue what you have seen earlier probably could be resolved by using the new chemistry of Napa so now if you had the self-re-expression system on the chip itself and imagine everything now you're doing on the gold slide earlier what are you doing on the glass slide but Napa chemistry is modified with the E-Coil and K-Coil peptides so now if the proteins are being formed with the you know this interaction of E-Coil and K-Coil peptides the haptamards being formed now you will have the tight interaction you can see these proteins synthesized on the chip surface and which could be then detected and used for the further interaction studies so now the baseline which was seen here earlier now the same baseline is much lower here with the new chemistry right so the old chemistry what's in the new chemistry and now you can have much more room for measuring any kind of binding responses you can change different concentration and you can still see how much binding you can observe so this case is not only entire GST which was injected for testing how much GST proteins are being synthesized so several experiments were done in this case initially you know various type of proteins were tested for expression first of all whether on the gold chip with the new chemistry can we detect the protein expression so every protein has the GST tags so in this case you know handful of 8 or so protein were tested out and you can see there all of them are showing binding but is there the real binding or the bulk effect you have to negate those possibilities so another experiment was done where sequential antibodies were injected starting from anti-P53 and type 4 and type June antibody on the same chip and now if you can see the triplicate spots are showing binding in response to those particular antibody which you are injecting then probably this binding is not bulk effect you are what you are measuring is protein specific antibodies you are using so protein specific responses you are observing right in case of GST it can it can be something you know everything is showing your response so probably may not be very confident thing that it is proteins are always being expressed but if you are using a specific antibody for P53, 4 or June then probably you have a specific type of you know measurements which you can make here and then you know some protein interactions were tested using June and FOS as a pair FOS was injected and June was you know printed as the cDNA on the chip and there was some binding observed similarly MDM 2 and P53 were tested out and then you know those kind of things showed some binding so you know a next experiment which was planned which was more kind of high throughput experiment and which was very challenging to really do that thing on the prototypes is what we had available where we have you know from one of the companies in Cambridge that time gave lot of clones of P53 mutants and these are all hot spot mutants of you know different P53 regions to test out you know how MDM 2 protein it binds to the wild type of P53 versus a different type of mutant forms of it and our this is as a cartoon images to show you we are trying to look at 144 spots binding how that can be measured simultaneously once you do the experiment it was very you know first time high throughput type of experiment being performed 144 spots binding you are trying to observe simultaneously but it was very complicated because there was no software available that time which could do normalization which could process the data and there was you know lot of issues especially we were also finding that you know probably there was some sort of bulk if it we could observe even in the control spots there is some sort of you know binding which one could be observed so for the next steps of these kind of technology development which is even that time we were seeing that even for Napa chemistry there are you know the halotech chemistry which you are seeing now is showing much robust signal so could we now try your the same thing on the you know SPRI platform using the revised Napa chemistry and that is where now people in the George lab are following up the work which they are building a new type of SPRI platforms where halotech based chemistry could be used with the chloroalkanes and then probably that will have much more robust signals which is what is required for the low abundant proteins for the clinical sample context so this is how the technology you know development takes place this is one of the approach you can appreciate could be used for high throughput screening which I am sure eventually will come to the market another thing is which you can think about coupling the surface plasmon resonance technology with the mass spectrometry based platform because just imagine that you know SPRI having you know its own unique property then mass spec can identify the proteins very effectively so if you are just doing a testing of a known protein with a known potential interactor on SPRI this is what you do usually right you have a known protein which you immobilize and you are floating one of the potential known protein to find out the interaction so then you are not doing much justice to your actual experiment and the technology because you are already knowing the things so much and then you are saying these two are should be binding but now you are I am confident they are actually binding this is what you do on SPRI right what can be actual power of the technology that you have a unknown protein printed and now you are passing your you know a lot of compounds and you know you can see are they binding or not right and then can I discover something new which is binding but what is that new thing which is binding which you have no control which have no idea so then eventually you want to identify that particular binding with the mass spec so but you know in real situation if you have done mass spec you would realize you need enough of the peptides which is required for detection of those peptides and the SPRI experiment you are doing is things that you know just very low concentration that is not sufficient for you to give you enough peptide for doing the experiments so it is of course not a novel thought it is just you know a practical concept which we want to to employ to really find out the unknown interactors but to detect them using mass spec but even the practical concept can become very difficult when you do the actual experiments so the concepts are you know very straightforward we are employing one of the SPRI technology and one of the mass spec you know technology two together are trying to get the best of the two technologies to identify the unknown interactors in this case again we started as a proof of concept which are two micro globulin experiment and then use all the four flow cell because you need lot of analyte to bind which you can recover and then use for the mass spec testing so all the four flow cells were immobilized and now when you are passing your beta micro globulin protein then you are enriching them and we actually you know use it pretty high concentration 30 micro micro per ml flow rate was used 5 micro per minute all the four flow cells were employed after doing you know series of cycles so we did that for maybe almost 20 hours or so several rounds on all the four flow cells with the intention that you know enough of the protein should bind beta micro globulin should bind on the anti beta micro globulin and then can we chop off that particular bound protein beta micro globulin then get peptide is selected out of those proteins and then finally we candidate them using mass spec that was the objective so after doing mass spec sexes are not so great but still we could see eight peptides coming out of the same protein of interest at the top hit beta 2 micro globulin you can see here as a first hit of mass spec these are the kind of chromatogram which we saw so then we tried on another protein which was of the you know physiological interest lipoprotein E and intention was again to you know look for their binding this time we could see four distinct peptides so at least you know good news was and this one we are using QTOF platforms not the latest orbit technologies so we could see the right protein and it detects the right peptides which was good news but of course many times you do not have opportunity to run all four flow cells for so many round of cycles to really enrich your protein so how low we can go is what we have to now optimize using mass spec based platforms but this can be something which is useful for biological experiment when you really want to identify the unknown interactants where you see binding but you do not know the protein of interest so you can employ both SPR and MS platforms all right it's SPR MS you know now we have at least tested with five protein pairs which looks more confident we are kind of evaluating those technology to see limit of detection what could be best used and some of these could be you know heavily useful for the clinical sale especially serum type of samples you have a known protein of interest and you want to see you know where it binds with other interactants so lot of ligand fishing applications can be you know thought of build on this application which is what we are testing out and some of the GE application scientists are also working with this so this particular technology having lot of applications including it could be used for the PTM kind of analysis as well as the inhibitor screening analysis but you know now we have to really use the best of the mass spec power to go to the lowest detection limit with the minimal run cycle with the minimal repeat cycle then only I think this can make some impact in the actual samples of interest all right so in summary I think I have given you probably a broad overview of various interactomic field you know in which way the dynamic molecules to really understand them you need a gamut of technologies you cannot just rely on one that I have studied in this course and I can now start going using for every of my application you have to very open to look for various type of platforms which are available to you I am sure with overall you are probably convinced that you know there is lot of potential of using these technologies label free technologies and the different platforms available starting from you know looking at conductance based principle reflectance based principles interference based principles many of those could be utilized you can choose which platform can be useful for your kind of application but you know I think you have to really open to think about what should be your experimental strategy starting from where you should start from discovery workflow to reaching to the validation phase and then for for you know finally you want to translate those finding to the you know actual products so you have to follow you know a good pipeline which involves very much you know all kind of technologies which you are studying in this course as well which are being taught in the other courses in the proteomics field right now now you are able to understand the utility of label free technologies in the field of interactomics and also integration of novel technology platforms especially SPR imaging SPR with mass spectrometers as well as other technology platforms in the proteomics areas which could be utilized for multiplexing for these interaction studies that can even lead to identification of novel interacting partners or establishing with confidence the high throughput screening for the drugs in a given context the experiments that were discussed in this lecture would have also provided you an insight into wide applications of label free techniques it will definitely encourage you to think about how to plan these experiments using label free biosynthesis especially for your own experiments and I must say whether you are going to work in the areas of proteomics in depth or not but you will at some stage need to study protein-protein interaction or protein drug interactions in the upcoming lectures we will also discuss about coupling of immunoprecipitation with mass spectrometry to detect and identify the interacting partners and the molecules of your interest I hope these novel technology platforms which we are discussing in the last two lectures and in the upcoming lecture will make you much more aware of available technology platforms for your applications thank you