 In today's lecture we are going to talk about basics of mass spectrometry which could be used for characterizing identifying the proteins of interest after performing the immunoprecipitation base experiment. So, in the last lecture if you recall we talked about if you want to identify the potential interactors of a protein of interest you can do immunoprecipitation experiment and then identify the potential interactors using mass spectrometry. So, today we are going to have an application scientist who is going to talk to you about how to perform these experiments using mass spectrometers and then identify the possible proteins of interest using a software. So, let us have this lecture. Hi, good morning everyone. So, I am Pratip from Thermo Fisher Scientific. I am the application specialist for mass spectrometry for proteomics and biopharma. So, today till now you have done the IP experiments. So, immunoprecipitation you do the antibody precipitation. So, next from those proteins which have been precipitated how you identify the protein. Mainly we will focus on the mass spectrometry part. So, my topic is mass spectrometry analysis from IP experiment to protein identification. So, already you have done the this experiment. So, you have precipitated the protein and you have the protein. So, what is the next step? Next step either you start with this protein you can run a SDS page or in a solution you can digest the protein with some protease which for example, trypsin which cuts after arginine and lysine then it makes a peptides mixture. So, those peptide mixture is fractionate through LC and introduced to the mass spectrometry where you get the masses of those peptides. And now you search against some database using some search tools you identify those peptides ok. So, today we will discuss mostly on the mass spectrometry part. How you can analyze the data? How you can introduce the peptides into the mass and how can you analyze the data? So, before going to that for I am just giving you brief on the sample preparation. So, you have the IP proteins, proteins you have either you can run a SDS page normal SDS page or you can make a solution in solution digestion. So, in both cases you in case of in gel digestion you run the gel in SDS page then you cut the band of interest of your protein in protein of interest and cut the band then you digest that band extract. So, when you keep with the trypsin so protein get digested to peptides. So, when it will be in a peptide form it will come out from the gel bands and you extract those peptides and then further you proceed. So, in case of in solution digestion already thus protein in the solution. So, you have to do the reduction and alteration it is very important part otherwise what will happen there are disulfide linkage. Due to this disulfide linkage protein will not been open up all the parts are is not open up. So, you have to go through this reduction and alkylation DDT we are using is very common for a reduction it reduce the disulfides bond and because of reversible reaction you have to use some alkylation part. So, block that sulfide residues. So, you put iodoacetamide and those disulfide will be blocked by iodoacetamide and then you digest with some proteases. So, most common proteases is trypsin which cars after arginine analyzing. So, that masses basically important part is reduction and alkylation is very important otherwise you will not get 100 percent digestion. Next trypsin works in the basic pH. So, when you do the digestion procedure that has to be in the basic pH more than 8 pH ok and it has to be kept at 30 degree. So, these are the two criteria where trypsin works very well and then trypsin we all know trypsin cars after arginine analyzing then after digestion the clean up procedure. So, before injection to the injected to the mass spectrometry we have to clean up your samples. For ingel digestion it is fine the samples are comes pure, but in solution digestion. So, samples may contain some impurities some salts or some other impurities. So, that will block your MS analysis that is why the clean up procedure is very important. So, sample preparation that is why sample preparation is the most crucial part and if you done a proper sample preparation your 90 percent work is done. The next is the basically MS is basically a software driven. So, whatever you are setting the software it will do, but the sample preparation is the most crucial part for yours ok. Now, comes to the mass spectrometry. So, I am keeping basics of the mass spectrometry I am not going in very details on all the parts. So, what is mass spectrometry? Mass spectrometry basically is a production of ions that subsequently filtered or separated by m by z ok. So, where and we are getting a resulting mass spectrum of abundance of produced ion as a function of m by z. So, any mass spectra if you look into the mass spectra. So, x axis will be the m by z value mass by charge ratio and y axis will be the abundance either relative abundance or the absolute abundance ok. So, now sometimes if you run any samples in Maldi ok. So, Maldi is Maldi toft of is very common. So, that time you will sometimes you are saying your mass is coming x axis it comes as a mass m. So, whatever the m by z value is the m because in Maldi you are getting the plus 1 charge. So, always the m by z value will be equal to your mass, but in case of ESI electro spionation. So, that time you will get multiple charges that time m psi m by z value will not be the exact mass. So, do not be confused with this terminology ok. So, now we can simply explain the mass spectrometry in 4 letters gmsd. So, it has first is a generate. So, you have to be ionize the molecule. Once any molecule can be ionized it can be detected by mass spec. So, that is a simple way to identify. So, it has to be ionized properly the ionization method may be from solid to gaseous may be liquid to gaseous then it has to be moved properly. So, after ionization till the detector. So, that path should be the proper otherwise what will happen if any molecule touch in the wall that get discharged and it will not be detected in the mass spec. So, that path should be very proper then selection obviously, you have to separate the molecule because you have a mixture of masses. So, you have to filter which mass you want to identify you can control that one. And lastly there is a detector ok. So, you have to detect the molecule. So, let us see what are the components are there in the different sectors. So, first ion source ionization where the ionization happens. So, there are very different methods of ionization. It comes with the most popular is the ESI and Maldi you already heard about this electro spay ionization which is liquid to gaseous. So, you inject the sample in the solution format where from where you put the high voltage and that high voltage it will makes a ionization. Next is a Maldi matrix assisted laser desorption ionization. So, it is a solid form to gaseous form. What happen in Maldi you mix the sample with some matrix molecule spot on a plate may keep it for dry and then you shoot laser that laser gives the energy to ionization. So, from solid to gaseous space these are the two most soft ionization where your biomolecules will not degrade in during the ionization. Whatever the other mode of ionization fab, electron ionization, chemical ionization these are very hard ionization where already when you put your biomolecule it will fragment in the source. So, you will not get the exact mass of the molecule. So, it is applicable only in case of small molecules like chemical compounds and A P C I and A P P I atmospheric pressure chemical ionization these two are for non-polar molecules exclusively for non-polar molecules. Now come for the mass analyzer means where you separate your molecules most common everybody knows a quadruple toff instrument time of flight time of flight quadruple means it quadruple is not a analyzer I can say it is a filter. It can precisely filter your molecule which molecule you want ion traps. So, you can put your molecules in the you can trap your molecule inside a vessel and you can make detect that molecule. So, quadruple ion trap triple quartz these are very low resolution means here you cannot separate nearby masses, but on the other case F T I C R orbit trap toff or q top these are high resolution instrument where you can separate the nearby masses. So, in IIT Bombay we have orbit trap technology it is nothing, but a trapping of electron in a orbital motion. So, we put a high voltage and the ions are rotating around the molecule. So, we will come to those part and last is a detector. So, you have you require some detector the better part for orbit trap is orbit trap its itself acts as a analyzer as well as detector. So, you do not require a extra detector for orbit trap. Now we see how the mass spec works what we are doing in the mass spec after injection of sample. So, we inject a sample to mass spec through the mass analyzer and detector we get the m s 1 ok m s spectra we will get this m s spectra from this m s spectra we fragment we will select one of the peak and again we will do the fragmentation m s 2 and we get this fragmentation. Now, this fragmentation we will search against the database and we will find out what are the ions are there b and y ions and we get the database match we will get the peptides from the peptides we can say this protein is present in your sample ok. So, now, come to the nomenclature of identifying the ions this is a basically nomenclature how you can assign your peptides. So, this is example of a peptide of 4 amino acid R 1 R 2 R 3 R 4 ok. So, now, when you applied voltage to fragment the molecule in m s 2. So, this C alpha C C n and N C alpha these 3 bond can be broken. So, this any one of the broken ok. So, if it is C alpha C bond is broken then the N terminal side we will call the A ions the C terminal side we will call the X ion same way C n this is the peptide bond basically b and y ions and if it is N C alpha bond then it will call the C z ions ok. So, now, there are different kind of fragmentation different way of fragmentation C i d A C d E T d. So, different different fragmentation will give you different kind of ions. So, in case of C i d you will get only b and y ions. So, in case of E T d you will get C n z ions ok. Now, come to the facility here in IIT Bombay. So, it has a tribrid instrument orbitrap fusion with coupled with nano L C. So, why we require nano L C because we are doing the proteomics in proteomics sample is very small amount and it is very precious. So, we cannot run the normal HPLC where the flow rate is very high we have to run in a nano L C in a nano liter flow rate ok. Now, come to the symmetric diagram of orbitrap fusion. So, orbitrap fusion as I told it is a tribrid instrument it has quadruple it has ion trap and it has orbitrap ok. So, ions are inserted from here it gets filtered in the quadruple first then it goes to it comes to the ion routing multiple where the ion will store and it will tells you where to go because you have two detector ion trap and the orbitrap. So, from here either it will go to the ion trap or it will go to the orbitrap. So, then orbitrap it is orbitrap basically it is a high resolution it gives you 500 k maximum resolution and lastly the ion trap which is basically a dual cell ion trap you can store your ion and you can do the detection of ions and also you have a ETD option. So, in the next slide it will be clear for you we have a video how it works. Now, we are doing first the full MS MS 1 ok. So, now ions will generate here it will passes through ion transfer capillary inserting through that. So, after that here what happened ions trying to spread out because all the ions are very highly charged. So, it tries to spreading. So, you need a focusing. So, it will focus then it is called the bent flatter pole bending where the neutrals are removed because in when the ions are inserting to the instrument there are some neutrals also. So, you can with the high voltage voltage difference the new the charge molecule can bend, but the neutrals cannot bend. So, after that in the quadruple you can select the ions which one you want then it first comes to this ion routing multiple where it will first store here. So, here is the first stoppage where it will be decided either it will go to the orbit trap or it will go to the ion trap. So, now it will come to the orbit trap MS and then it is been detected in the orbit trap. So, now it is a full scan what happened in the MS MS. So, in case of MS MS only one molecule has been filtered from this mass only one molecule filtered and that gets fragmented and then it will be detected MS 2. So, same thing happen ions are going. So, inside quadruple the only one molecule will be there. So, other will be filtered out now that molecule go to this ion routing multiple and get fragmented. So, here it will fragmented in the ACD mode call it is nothing, but the collision induced with high energy. So, here after fragmenting here it goes to the ion trap where it is stored first and then trapped here and then it is get detected. So, it is detected here with the large surface area detected. So, it is a very parallel reaction basically parallel it is going on. So, when the first MS is going on here MS 1 at the same time the MS MS is going on simultaneously. So, it is a parallel both the detected is working. So, from one MS scan it can do 20 MS MS it can select 20 peaks from one MS scan and it can do the MS MS of 20. Now, come to the specification of fusion orbit trap. So, first is the mass accuracy mass accuracy is tells that is less than 1 ppm and for internal calibration and with less than 3 ppm with external calibration. So, what does it mean by external internal? So, every instrument requires a calibration. So, you have a standards you inject the standard. So, you have the mass of the standards and against that mass is exact mass you calibrate your instrument. So, that is the simple external calibration. Now, in case of internal calibration we fix one of the masses as a internal standard we fix that mass and do the again calibration. So, that in that time we will get the internal calibration. So, that is why when we do the internal calibration the calibration will be less less than 1 ppm. Now, the mass range. So, you can acquire the mass range from 50 to 6000 m by z not the mass. So, m by z. So, bigger the molecule the more the charge state and it will come inside the this range. But you cannot scan the total mass range 50 to 6000 in one shot. So, you can scan the first mass and the highest is the 15 x of the first mass. So, for example, if you start from 100 m by z you can scan up to 1500 m by z. So, if you want to scan the full mass range then you have to divide the scan range. So, start from 100 to 500 and 1500 to 6000. Then resolution you can get the maximum resolution of 500 k at 200 m by z scan speed 18 hertz per minute. So, fragmentation. So, you have different three different kind of fragmentation CID, ACD and ETD and there is a basically combination of any two ETD, ACD and ETD CID. So, in case of CID what it is basically collision induced distribution. So, what happen in that collision induced distribution you collide your you inject some neutral gases that will collide with your sample molecule and get fragmented that is CID. ACD is high energy collision induced distribution. So, this is nothing, but CID with higher energy we are putting higher energy to get better fragmentation. And ETD is a electron transfer dissociation what happened there you put a reagent molecule which ejects an anion. That anion will collide with the sample molecule and get fragmented that is called the ETD. So, according to your sample objective what do you want to do according to you have to select the fragmentation mode. Then polarity switching means it is not required for the proteins, but in case of small molecules if you do not know in which mode either it is a positive mode or negative mode it will ionize. So, you can do the both scan positive scan and the negative scan within a 1 second. So, you can run that one way MSN as it is a ion trap. So, you can do 10 MSN means MS MS 2 MS 3 MS 4 up to 10 theoretically isolation in quadruple you can go minimum 0.4 AMU means plus minus 0.2 Dalton's. So, the what that means means if you want to identify if you want to identify one molecular m by z of 100. So, you can scan between 99.8 to 100.2. So, that narrow window you can scan. So, now comes see some experiments where means how the instrument works in different workflows because we cannot show you in the instrument part. So, we have some animation where we can see how the instrument do the MS and MS MS. So, first experiment tells MS in OT orbit trap and MS MS doing the through the CID it will do in the ion trap. So, CID will be happened here. So, what happened first the ions will be detected here MS from that MS scan one molecule one at a time it will be filtered here and fragmented here. So, let us say so the first full MS will go. So, the first mass it will first it will stop here then it will comes to orbit trap when the full MS is going on we are doing the parallel. So, the second first parent ion has been selected here and it will be stop there first then it will go for the fragmentation in the HP cell when it is going fragmenting the second ion is waiting here. So, you are not losing any molecule as well as the time. So, it is a parallel reaction. So, when all the MS MS scan is MS 2 scan is over then only you will get the MS scan. So, at the end of the full scan you will get MS scan as well as MS MS scan. So, you are not losing any time as well as any molecules samples. So, like second experiment is the ACD on the first experiment we are doing in the CID what happened there in that case the fragmentation happens here. Now, in case of CID the fragmentation will happen here. So, now same way the full MS it will stop here then it will go to orbit trap for analyzing. Now, the first mass will comes here and get fragmented when it will be sending to the ion trap at the same time the second mass will come up. So, you are not the same way you are doing the parallel reactions. So, after all the MS scan MS 2 scan you will get the MS MS scan. Now, next is the ETD what happens in case of ETD. So, even the ETD fragmentation will happen in the ion trap also. So, first is the full MS in same way the full MS comes to orbit trap. Now, the first ion has been isolated in the quadruple and first stop here then it will injected to the ion trap. Now, I told in ETD you require a reagent which gives you the anion. So, that reagent will comes from here. So, it will inject a reagent it is reagent calls the fluorine thin. So, that is a cursive unit. So, it will it will gives you some anion it will make the anions and get fragmented that molecules. So, once the fragmentation is happen. So, it will give you the MS 2 radar as well as MS MS data. So, ETD is a very specialized case only when you are going for the PTM analysis. So, post transfer or motivation like phosphorylation or acetylation or glycosylation in those cases you have to use this ETD reaction. So, in the instrument the better part is that you already there are already the templates for all type of analysis. So, you do not have to start from the scratch. So, you just click on the which method you want to use either it is identification or it is a quantification or it is a PTM work. So, you have to select that method and run the method. So, there are already templates are made. Now, before going to the next step. So, LC is another big part because in the front end we are putting a LC. Why you require a LC for this kind of analysis? Because the peptide length may be from 300 to 2000 m by z. So, within the mass we cannot separate all the m by z's. We can separate the nearby masses by the resolution, but we cannot separate the in the range. So, we need to separate those peptides. So, that we put a LC in the front line where either with the basis of hydrophobicity or any other parameters. We separate those peptides and elude one by one and inject it to the mass peak. So, these are the different parameters we are using for setting up the LC run. Here the important part is the gradient. So, you have to choose a proper gradient. So, that gradient should be on the complexity of a sample. So, now here I am showing you 30 minutes gradient. So, it is work for a simple one protein like BSA or single protein. But when your complexity when you are working with the wholesale lysate that time you have to increase the gradient for 2 hours, 3 hours, 4 hours. So, write that like that you have to work on the gradient part not in the MS part. So, now come for the some data analysis part. For data analysis we have protein discoverer 2.2 that is a comprehensive and extendable software for qualitative and quantitative. So, you can identify as well as quantify the proteins. Here the quantification is a relative quantification not a absolute quantification. So, what it has basically it will have some different search engines like sequest, mascot, bionic, amanda. Out of this sequest is a free available. So, it will comes with the integrated with the protein discoverer. Other search engines mascot or bionic those has to be purchased it has the license. So, actually what this search engines works means how it works. So, when you have run a sample human sample you get the data MSMS data. So, now you tells the software search engine you extract the protein you say the database of human. So, the search engine will digest theoretically digest those protein from the human and make a theoretical mass list. And then the theoretical mass list will be compared with the experimental mass list. At the MS level first MS 1 level when it will match with the MS level then it will come for the MS 2 level. If it is match in the MS 2 level and go get those b and y ends to get the sequence then it will tells you that peptide is present. Then it will give you this protein peptide is present means this protein also be present there. So, it will measure determine radiative quantification. So, you can do radiative quantification either it is a level free or a level reaction like TMT, Cylac, I track. So, here is an window where you will see the first of is a whole cell lysate run for 120 minutes. We run a ddms2 method we do ot at MS and acd also in orbit trap MS 2 also detected in orbit trap we are not using ion trap here. It is a 120 minute 180 minutes run it is a whole cell lysate. So, first window it tells the TIC total ion chromatogram. So, like in HPLC you are getting UV chromatogram here we will get on the basis of total ions it will make a chromatogram. So, that is why it is called the total ion chromatogram. Second window it is called the BPC base peak chromatogram on the basis of highest intense speed for the each retention time ok. So, that is called the base peak chromatogram. Now, if you click anywhere you will get like this of MS spectra. This is the real MS spectra where x axis is the m by z y axis is the relative abundance and it will see different char states are there plus 2 plus 3 plus 4 because of ESI you will get the different char states ok. Now, when we search this data in PD with different with this 2 So, in PD you have 2 workflows one is the processing workflow where we search the data against the data base using the sequest and after getting the peptides those peptides may be a false positive or false negative. So, you have to validate those peptides. So, those has been done in the consensus step where we do the peptide validation as well as protein validations and we will ultimately get the data. So, here the string we are doing the validation at 1 percent FDI. So, what is that mean if you run the sample 99 times the protein will identify it may be in one time it not been identified. So, in such stringency we are detecting though validating those peptide and we are telling ok this peptide is present. So, then the report is very good for publishing. So, now what after search the data what is the report format. So, it comes like this. So, these are giving you the protein information what are the protein match and the sequence coverage how many peptides has been matched. So, here important thing is the unique peptides. So, unique we are looking for the unique peptides how many unique peptides we have identified. So, what that mean by unique peptide unique peptide is those peptides which are present only in those proteins not any other protein in the included in the database. So, where what database you are using in that database that peptide is not present in any other protein. So, we are looking for at least two or more unique peptides those kind of data will be the very good data ok. So, here we have identified almost 2200 5 5 protein groups. So, it comes like from that data set it has extracted this many MSMS spectra which is when we match against the database we are getting this many 23000 PSMs. And out of this PSMs we are getting this many 2255 protein groups. So, right now I am talking about the identification just how to do. So, fusion is not for identification it can do many works. So, right now we are doing the deep large analysis and the label free or label reaction to relative quantification. So, it can do the phosphoproteomics it can do the lipidomics it can do the metabolomics. So, you can use this instrument for different kind of analysis. Lastly this is a website PlanetOrbitrap.com where you will get all type of information of orbitrap in regarding application wise if you are working on proteomics. So, you will get all type of application notes published data or methods. So, you can register here and you will get the free updates ok. So, for the last two lectures you are now familiar that if you have a protein of interest which you want to dig deeper you want to further characterize that protein and want to understand the possible role of that protein an easy way of moving forward could be to do immunoprecipitation experiment followed by protein identification using mass spectrometry. In today's lecture you have seen how to take the complex which you have eluded out from the IP experiment and identify the potential interactors using mass spectrometry. There could be many softwares which could identify the potential proteins of interest. In fact, I would recommend you to use one of the open access software, Mascot to do the data analysis where you can easily identify the proteins of interest. There are many good softwares available even commercially one which we have shown today is proteome discoverer. In general I hope these two lectures have given you some basics and understanding about how to study the biomolecular interactions using immunoprecipitation followed by mass spectrometry based experiment. Thank you.