BCMB Weeks 7-13 Lecture Notes PDF

SPEAKER 0 OK, Time to make a start. Looks like we're down on a few numbers as week seven cuts and midway through the semester. Uh, also suspect that there's a a few people who, uh, did the proteins in cells unit last year and had three lectures from me. Four lectures from me on this topic. Um, so t...

SPEAKER 0 OK, Time to make a start. Looks like we're down on a few numbers as week seven cuts and midway through the semester. Uh, also suspect that there's a a few people who, uh, did the proteins in cells unit last year and had three lectures from me. Four lectures from me on this topic. Um, so that might be a reason as well, but we definitely need to look at those numbers as we move forward. So now we're starting the block on specific post translational modifications. That's a block of seven lectures in total. And today, uh, what I'm gonna do is discuss kind of the biochemistry behind the post translational modification of phosphorylation. And so we'll talk a little bit about signal transduction. As I said, it'll probably be a little bit of, uh, rapid revision for some, um, and maybe new for, uh, for others. And then in the second lecture, in lecture 14, we'll discuss the analytical aspects and how we go about understanding phosphorylation and signal transduction on a really, firstly, on a small scale and then on a very, very large scale and you get a tiny little snippet of that right at the end of of this of this lecture. So by means of kind of learning objectives today, we're gonna talk about what signalling is. As I said, it's a little bit of a refresher for those people who did the proteins in cells Unit 2002 or 2902. last year, Uh, we'll talk about how really kind of this paradigm of how we sense changes in our environment And when we've sensed those changes in our environment how that signal is trans through cells, uh, tying in a little bit to what we learnt with that idea of the regulo and stimuli. Because ultimately what's gonna happen here is we're gonna end up changing the activity, at least of trans transcription factors. So really kind of everything from sensing a change in the environment through how that signal is transmitted within the cell to ultimately result in a change in gene and protein expression. Uh, at the at the the kind of, uh, DNA or gene regulation level, there's really three mechanisms I wanna touch on. As I said, I did three lectures on this last year, and we're gonna just take little snippets of those because you don't need the detail that we talked about in second year. We're gonna talk about firstly, bacterial two component systems. We've kind of touched on these already when we looked at the regul. So these kind of bacterial like transcription factors or response regulators because really, the signal of the way the signal is trans even in single celled organisms is actually shares quite a lot in common with the way signals are transducer in eukaryotic organisms as well. So then we'll go on. We'll talk about the two major classes of of receptor and signal transduction mechanism in humans. Those are G protein coupled receptors or G, PC, RS and receptor tyrosine, kinas or RT KS. Talk about how those signals are transmitted via protein kinas and phosphatases. Using a couple of examples, uh, which will be firstly a an example of G protein coupled signalling which will be adrenergic type signalling or adrenaline and then insulin signalling, which is, of course, the most famous example of the role of receptor tyrosine kinas. The one in here is quite important. It's kind of the the The journey that we're really on today is understanding how signals are sensed by cells. How those signals are transmitted by protein, kinas and phosphatases via the addition and subtraction of phosphorylation to proteins and then ultimately leading to a change in gene expression. Uh, that enables the cell to ultimately adapt to or respond and then adapt to a change in the environment. So it's pretty simple. Biochemically Or at least, uh uh, the the at least the way the phosphorylation occurs is pretty simple. This is an a TP dependent process, so adenosine triphosphate protein kinas use a phosphate from a TP, so this is energy dependent protein. Kinas. Use a phosphate from a TP, converting it to adenosine diphosphate to add a phosphate to a target protein. And you see here that it's written as PO 42 minus. In this case, it's showing the activation of a target protein. So it's showing this as kind of that molecular switch that we touched on in lecture, uh, in lecture 12, where we talked about the different ways in which modifications could enable interactions or enable, uh uh, different functions of target proteins. But here what's happened is that the protein kinase targets the protein it phosphors and the protein becomes active, does its job somewhere in the cell or transmits the signal to another protein. And then once that signal is gone, or once the cell has responded adequately to that signal, a protein phosphatase which is like a control mechanism, will come along and it will remove the phosphate from the protein. And it removes this as, uh uh. It removes this as HPO 42 minus the protein, then becomes inactive. And the target site then has a hydroxyl group which is left alone like this. So good way of thinking of this, uh, and really one of the take home messages from the post Translational modification series in this unit is to say, under normal circumstances, kind of homeostasis within cells, you have a pool of a protein that's sitting there inactive. When that activity is needed within about 1 to 10 seconds, that protein can get phosphorated and become active. And so this really hugely cuts out the middle man of trying to, you know, express enough protein to get this protein to be in an a sufficiently active form. So you have pools of protein sitting within cells. Not all proteins are like that, of course, but some proteins are, particularly those that are involved in signalling. They can be phosphorated to become active, and phosphatases can then uh, uh, remove that phosphorylation to send them back to that pool of protein sent back to its inactive form. I should point out at this stage that it's also fair to say that there are just as many cases where the phosphor related form of the protein is inactive. So the protein might be sitting in the cell in an active form that you don't need that activity for under a certain signal and that the protein gets phosphorated to become inactive. I think that's a really important distinction to make. We're gonna see the classic. Example of that is the glycogen synthese and glycogen phosphorylase. So one leading to glycogen storage one leading to glycogen being, um, converted into glucose for the need of activity both dependent on phosphorylation. One is an inactive and one is inactive. OK, to start very simply, I think we'll just talk about bacterial two component systems. Uh, and again, as I said, this follows on a little bit from what we talked about in lecture 11 about the the regs and stimulants. The reason that I like to kind of discuss this here is because there are really lots and lots of similarities in bacterial signalling versus eukaryotic signalling. But it's much, much simpler. OK, much, much simpler than what we see in New Karic systems. So just like we see in New Karys as we're going to see, bacteria have these proteins called sensor histidine kinas, and this is the first component of the two component system. It acts very much like a G protein coupled receptor or a receptor thyra in Kinas. It has a domain that sits outside the cytoplasm that senses a stimulus. That stimulus gets transducer and it gets trans duced by auto phosphorylation, which means that the protein phosphors itself OK, the only difference to what we would see with a receptor tyrosine kinase, which also phosphors itself as you will see, is that the is that this occurs on histadine and the name of the protein tells you histadine sensor kinase. So it auto phosphors itself on histadine when the stimulus is present, that phosphate group then gets phosphor transferred to a response regulator that is then active as a transcription factor. There's no multiple steps in the middle here. As we're gonna see with eukaryotic signal pathway. It's stimulus auto phosphorylation one phosphor transfer step to the transcription factor or the response regulator that is then active and combined to DNA to modify, uh, gene expression. So, yeah, I think it's quite important to understand that all of these proteins, you know, we think of them such a you know, we often think of them as just names, and we don't do a lot of kind of structural biology in this, um, in this unit. But I think it is important to understand that just because there are classes of proteins that are called receptor thyra zine or histadine sensor kinas doesn't mean they all look exactly the same. And if you think about it, it makes a hell of a lot of sense. And I think it helps to understand what these proteins actually do again, We've talked a little bit about the architecture of the histadine kinase. We see it back here. It's shown in sort of cartoon form here, but here we start to look at it structurally, and we've got this part of the protein that sits outside the cell. That's the sensor domain that senses the signal. That, of course, has to be variable, right? It has to be specific for a given signal. So when you look at these by, uh, when you look at these by kind of crystallography and structural biology approaches and you look at the three dimensions of these, uh, of these sensor domains, they all look somewhat different. And of course, they do because they sense they're specific for sensing different, uh, signals from the environment. The transmembrane helices are conserved transmembrane helices are pretty conserved across all transmembrane proteins. And then the part of the protein that actually phosphors itself so kind of helping in transmission of the signal is also pretty conserved across these types of proteins. It's actually a bit of it's actually a dimer. You've got two a TP binding domains. You've got this DH P domain, which stands for dimer and his phosphor transfer. And that is the site at which a TP is used to phosphor this conserved histadine. So these parts of the protein, which are kind of seen over here they all look pretty much the same. They all have these quite long helices, uh, and conserved residues in them. And same with the A TP binding domain that binds the A TP that is needed for phosphorylation. Because, as I said, this is an energy dependent process that is also conserved. There's a little tiny domain in here. Don't worry so much about what Hamp and pass and Gaff stand for. These are really the the S what I referred to as the signal transducer. So the thing that takes the signal that has been sensed by this very different looking sensor domain passing through the transmembrane helices into one of these domains. And again, these are specific for the, uh for the signal that has been sensed by the sensor domain. So these again look quite different a little bit more conserved, uh, than the sensor domains, Uh, in that they can be actually grouped into these different types of domain, which we won't go to in any in any great detail. But they're specific. I think again, um, for the li and and for any kinds of second messages and other kinds of activities that that protein might have and we were not gonna go into that in any great detail. But again, just to be aware that when we talk about these classes of protein, they do look different or at least parts of them look different. And that's because you have to have different Uh, you have to have different signal, uh, proteins, uh, for different type of environmental signal. So really, really easy kind again. I really kind of get you thinking about signal transduction is Really Is it really kind of a two step process or a really a really short process in, in at least these simple single celled organisms, you've got the histadine kinase that sensors the signal it auto phosphors itself. So that's one signal transduction event. As the signal binding to the sensor domain gets trans via that, uh, signal transduction domain to the phosphorylation domain. It gets phosphorated, and then it passes that phosphorylation over to an aspartic acid. So this is different to what we look in eukaryotes, histadine and aspartic acid. Once this response regulated, this bacterial transcription factor is phosphorated. It becomes active and it can bind to DNA. And that's what's shown here with this structural. Um uh, crystallography of one of these response. Regulators. You've got this kind of, um, you've got the the domain up here. That's the phosphorylation domain. The end terminal domain here. And you've got this variable, uh, transcription factor type domain that binds to DNA specific to to specific pieces or stretches of DNA. And does that the reason it does that, like all transcription factors, that's to promote or repress uh, given, uh, given genes the genes that are needed under certain circumstances. The classic example of this, the first one that was ever discovered. The first two component system that was discovered was PPFQ that was discovered in salmonella. FQ is the histadine kinase. It senses in this case, low magnesium faux P is the response regulator and the conditions where there's low magnesium in the environment. This signal is transducer to the response regulator. It gets phosphorated. It binds to the promoter region of this gene called MGT a that gets it gets transcribed and translated, and MGT a encodes a magnesium transporter. If you don't have enough magnesium, the best way to get it is to put more specific transporter on the surface to try and acquire as much of that minimal magnesium that is present in the environment as possible. Second thing it does here. I mean, it does lots of other things as well, but it's just saying that two component systems can regulate other regulators. So in this case, the activated phosphorated response regulator, acting as a transcription factor, also promotes the expression of a second transcription factor called in this case RST. A. But again that the the the specifics of this are not important. It's just really how the mechanism of phosphorylation signal in the environment transducer by phosphorylation ends up, resulting in a change in transcription factor out the whole lungs. SPEAKER 1 They got the whole and to this is a test SPEAKER 0 only be really great if they did that between lectures as opposed to in the middle of people's lectures. But we're gonna get that in a second. But I have to keep moving on. OK, so we need to think now we've kind of talked about bacteria. We need to think about eukaryotic cell type signalling and really, the best definition here is that signalling is a biochemical mechanism that mediate sensing of the environment to facilitate a cognate response by the genome and I. I think it's an important phrase here. I've kind of said already that, you know, there's sort of two things happening here, and I don't want you to get confused. We've talked about phosphorylation activating and deactivating proteins without respect to the genome, right? That's of course, true. But part of doing that in signalling is to ultimately activate or deactivate a transcription factor such that there is an adaptation process occurring within the genome by the expression or repression of certain genes. SPEAKER 1 Test only no need for action test only. SPEAKER 0 Sorry about that. It really does kill your train of thought. Um, so, yeah. So, I, I kind of I really want you to get that distinction between you. You know what we've been saying about? Yeah, you can activate and deactivate certain protein functions, but in a signal pathway while you're doing that as part of the signal transduction event. Uh, ultimately, the end product of this is to is to induce a change of the genomic level, induce a change in gene expression, and that's fundamentally what's shown on this slide here. I mean, there's a number of different mechanisms. Some signals are, uh, are capable of diffusing into the cell. They combine to a cyto zoic receptor, which can then modify a, uh or can directly become a transcription factor or activate a transcription factor to pass into the nucleus to modify gene expression. But the kind of signalling that we we generally tend to think of the classical type of signalling is more what's shown over here, where you have a surface receptor exactly kind of like a histadine sensor kinase that sits inactive on the cell surface. When its signal is present, that signal binds to the cell surface receptor. It activates it and induces a signal cascade that can involve lots of protein phosphorylation and, in some cases, the presence of second messengers. And we'll talk more about them in a minute. And that ultimately will result in a transcription factor either passing into the nucleus or more likely, uh, an effect of protein, modifying a transcription factor within the nucleus in in higher organisms. Those transcription factors generally tend to be nuclear located, as we talked about when we talked about uh, Subcellular proteomics and looking at the nuclear proteome and then looking at the DNA binding proteome to find those transcription factors um um without the other abundant proteins present. OK, so really, what are we looking at here? Biochemically with these kind of major features of cell signalling and, firstly, cells respond. Cells have to respond to these extracellular signals or stimuli, Um, and that these activate membrane associated or sometimes cyto zoic receptors as we saw in this previous slide. But I won't talk more about that kind of signalling throughout this lecture. We'll really be concentrating on this type of signalling. That's, uh, on the right hand side of this slide. The major ones in eukaryotes they are G protein coupled receptors or G, PC, RS and receptor tyrosine kinas. I'm gonna talk about both those groups, uh, of receptor, uh, specifically as we move along. As I said in the case of cyto zoic receptors, some of these can act as direct transcription factors or or indeed regulate downstream pathways. And we saw that really on this side. Oops. On this side here, where we've got, uh, signal transduction events in the presence of second messengers. That signal transduction that's kind of shown in cartoon form here is really all mediated by the phosphorylation and dephosphorylation of proteins. and often involves the activation of a protein kinase, which can then activate other proteins by phosphorylation. And this actually enables the signal to be amplified significantly. Because if the signal, uh, if the signal activates one protein kinase that protein kinase may then be able to activate several different targets on different proteins. So one kinase can often be very, very promiscuous in the sense that it can, uh, activate or deactivate a range of other targets. Importantly, to make the distinction here between what we talked about with histadine and aspartic acid, we are primarily from here on in both this lecture and in the lecture on Wednesday, we are going to talk about phosphorylation events that occur on the hydroxyl groups of sine thine and thaine residues. The overwhelming prevailing view that has been around for probably 20 years or more is that, at least in the human proteome, serine is by far and away the most common amino acid to be phosphorated. It accounts for probably 85 to 90% of all uh, phosphorylation events. Three amines, The SEC second most common one, about 5 to 15% and tyrosine is the least common used to be thought that that was as low as 0.1%. But now probably somewhere between one and 5% of all phosphorylation events. And there are hundreds and hundreds of thousands of phosphorylation sites now known in the human proteome. It may even be close to a million phosphorylation sites that are known in the human proteome. And the reason for that is, of course, the large-scale proteomics kind of approaches that we'll talk about in the in the lecture on Wednesday. But very important to understand. I'm gonna talk a little bit more about why thyra zine is less common than the others. Um, but I won't talk about why serine and thine are are are are are differential. Uh uh uh thought to be differential, uh, in their in their ability to be targeted. But I will talk about thyra zine as we move on. OK, so we want to think about how these signals interact. Firstly, um, and how the signalling event is initiated to begin with. As I said, we're gonna concentrate really on two forms here. We're gonna concentrate on the G protein coupled receptors. We're going to concentrate on the receptor. Uh, tyrosine kinas. We won't talk too much about gated iron channels. The reason here is that gated iron channels effectively just the binding of the li just enables the cell to take up more of that li be it calcium or sodium or potassium or something like that Iron. Uh, these gated iron channels just open in the presence of a needed, um, element. So not much to it won't talk too much about the diffus type, or it won't talk more about that. We mentioned that a little briefly with the cytosol receptor, the idea of a Liga that can just diffuse into the cell and interact potentially either via a single protein within the cytoplasm or direct to the nucleus to interact with the transcription factor. So we'll really be sitting over here because these ones are the major ones and they involve kinase cascades, the G protein coupled receptor. Again, as we saw in the previous slide, both of these types of receptors exactly the same as a histadine kinase. They have a signal sensing domain that sits outside of the cell. In this case, this is the plasma membrane. Here's the Liga that binds. You've got a protein coupled receptor that is inactive. The Lagan binds to it and it becomes active. This is pretty much exactly the same as what happens with a receptor thyra zine kinase. It sits outside the cell. In this case, it's Dymer sits outside the cell, the Liam binds and signalling is initiated. Where they're different is in how they transmit the signal. The G protein and it's called a G protein coupled receptor because it binds to a G protein and a G protein has that name because it's a GTP binding protein guanine triphosphate, binding protein. What happens is that this is actually a protein complex. You've got the receptor sitting outside. You've got the receptor, part of which sits inside the cell, part of which the li and binding domain or sensing domain sits outside the cell and the G protein that sits inside the cell. Actually, it has a little transmembrane region that allows it to move along the membrane. When the liga binds, GTP binds to this G protein, activating it. It dissociates from the receptor and goes and finds a membrane bound enzyme, and that enzyme produces a second messenger, which is here referred to as X. And it's that second messenger in that case that initiates Kane's activation and ultimately a signal cascade that results in the activation of a transcription factor, which is here, designated as tea that then either initiates or, uh, represses gene expression with the receptor thiazine kinase. What happens is that the Lagan binds the dimer. The individual monomers of the dimer come together. When the Lagan binds, they phosphorylation each other that initiates, signalling a kinase cascade or a signal transduction cascade and again. Ultimately, that leads to the activation of a transcription factor, which can then switch on or switch off gene expression. OK, so let's look at those, uh, G protein coupled receptors first, um, so G protein coupled receptors and G protein coupled receptor signalling these are are doesn't really matter what kind of proteins they are. I think it's important to understand that they are transmembrane proteins. Clearly, they have a cytoplasmic part of the protein, and they have an extracellular domain. But they are alpha heal integral membrane proteins, integral membrane proteins, meaning they are transmembrane proteins. They bind the G protein, which is shown down here, which we discussed on the previous slide to give just a little bit more specificity to this. The G protein itself is a trim. It's a hetero tr that's made up of three different, Um uh, subunits the alpha beta and gamma subunits. They are membrane associated through this little part of the protein or through one of the sub units here. This is the, um uh, the, uh, gamma subunit. I think we see it on the next slide. Um, that are bind guana zine nucleotides. When this gets activated, when the liam binds to the G protein coupled receptor, the G protein gets activated through the binding of guana zine triphosphate and that dissociates from the receptor and binds to an enzyme that's the third component in this pathway. That's an effect enzyme. It's regulated. It only is active when it's bound by, uh, activated G protein. It's an enzyme, and then it produces a second messenger, and the one we'll talk about will be cyclic a MP. OK, so the best example of this, um uh, actually, before I go too far, I should have said, because I wanna contrast this before I should have said that. G protein. Um, G protein coupled receptors are actually very interesting from a a proteomics perspective and from the perspective of the human proteome. We talked a lot right at the beginning of the lecture series about how you know 50% of the proteins in the human proteome. We don't really know what they do, and there is a project. And we come back to these big scale projects towards the end of semester when we talk about the human protein atlas. But there is another project called the Human Proteon Project. And what part of that project is doing is looking at finding evidence of for the expression of all these genes as proteins. And as of last count, there are somewhere between about 3070 proteins that are not Yet there is no evidence yet for their existence. They are nearly all G protein R, nearly all predicted to be G protein coupled receptors. And this is a big class of protein, and it's about 800 proteins predicted in the human proteome to be G protein coupled receptors. So out of 20,800 is a sizable, uh, I think, a a reasonably sizeable proportion. It's sort of 4 to 5% of the total. Predicted Proteome is predicted to be G protein coupled receptors, UH, 800 proteins. Lots of these are still functionally unknown, and the reason for the difficulty in finding them and the reason for the difficulty in understanding their functions is because they're very specific for very well thought to be very specific for very specific signals. So lots of them are are are thought to be auditory. Lots of them are thought to be olfactory. So for specific smells and tastes and things like that, Um, and so they're really, really hard to find because they're only found or thought to only be found in very specialised cells involved in those processes. So I think that is important to kind of say so. This is AAA class of protein that's really being, you know, significantly investigated. Um, for those reasons, are very, very specific to very specific cell types. And then, of course, there's others that are really well characterised, like the G protein coupled receptor that's responsible for beta adrenergic, um, type signalling. So I think a lot of you I think everybody knows what the fight or flight response is. The fight or flight response Obvious, obviously, is mediated by, uh, a hormone called adrenaline. The sort of more, um uh, scientific name for adrenaline is epinephrine, and that's a hormone that's made in the ad adrenal glands. And it mediates the stress response. I think everybody knows what the fight or flight response is. It mobilises energy generating machinery. So if you're gonna run away, you're gonna need glucose to drive the production of a TP in your muscles. So the release of adrenaline is, uh, the is the initiation, uh, event and that adrenaline or epinephrine binds to a G protein coupled receptors, and these are very well characterised. They're called beta adrenergic receptors, and they're found on, you know, many, many different types of cell in muscle cells, heart muscles, um, skeletal muscle. They're found in liver cells and so on. And really, the the kind of key element here is to get glucose, get as much energy as possible to get away as quickly as possible, or to give you the energy to fight right in adipocyte. This will increase the hydrolysis of lipids so again, trying to generate, in this case, energy from lipids increasing the heart rate in myocyte. And so on the G protein in this, uh, process to just kind of dig a little bit deeper onto the G protein side of things. It's an activator. And remember, I said that it was a hetero trr, an alpha, a beta and a gamma subunit. And here we're gonna talk to one of we're gonna talk about one of those sub units as being the activator enzyme, or the stimulatory G protein, or GS, and in this case, in beta adrenergic signalling. The effector enzyme is called Adonis Cycle, and the second messenger is cyclic a MP. So this kind of schematic diagram shows the structure of the G protein in a lot more detail than we previously saw. You can see the three subunits here. I was wrong. It's the alpha subunit. That's the catalytic subunit. So the stimulatory G protein is a three is atria that consists of an alpha subunit, a beta subunit and a gamma subunit. That's down here, and it's the gamma subunit that kind of partly sits in the membrane. And the alpha subunit also has this little membrane hook. As as well, So what happens under the when the When there's, um, adrenaline present in the blood or epinephrine, it binds to its specific receptor. It's held down in here. That signal is then transmitted to the G protein that removes GDP and replaces it with GTP guana Zine diphosphate versus guana zine triphosphate, the G protein, or the the alpha subunit, at least of the G protein. Dissociates from both the receptor and the other two subunits. The beta and gamma subunit passes along the membrane, using this little membrane hook as kind of like a slide rule along the, um the the the lipid bilayer here binds to the enzyme Adonis cycle, as you see, is sometimes referred to as Adonis with one YL. And Adonis is with two YLS. Uh, I'm just gonna call it Adonis Cycle, just cos it's easier to say, and, uh, that becomes activated when the G protein binds to it. It then uses a TP. Excuse me Right then it's I'm really losing my voice. Oh, wow. Sorry. Fit their users, um, a TP don't know if that's dust from out there. It uses a TP to create cyclic a MP the cyclic a MP then activates protein kinase a and you get a stimulation of the, uh of the signal transduction pathway there is We're not gonna talk about the recycling of this so much. There is an enzyme called cyclic nucleotide phosphoria esterase. Now, when the signal is removed, when there's no more signal present, when the the fight or flight response is over, this enzyme will breakdown any remaining cyclic a MP to five prime a MP, Um, and that therefore, um, protein kinase a will not be activated. So really, from here we're gonna talk about this part of the pathway where you have the second messenger that increases the activation of a signal transduction domain through protein kinase a So protein kinase a is a Cine three andine type phosphor. Uh uh uh uh protein kinase It, uh um is very rapid. It can, um, phosphorylation in less than a second. It's quite co ordinated with the presence of, uh, phosphatases. It's very easily reversible. So right back on slide three, where we just showed the protein kinase using a TP, the protein phosphatase removing it. That's ac absolutely. The way that protein kinase a works. I discussed a little bit about the amplification of the signal. The idea that a kinase, um or the activation of a kinase can lead to the phosphorylation of many proteins, and it can also act as a molecular switch. So by phosphorylation uh, um, proteins that can activate or deactivate as we're going to see in specifically in beta adrenergic, signalling that production of the second messenger cyclic a MP by Adonis cycle activates this protein kinase. Its full name is cyclic a MP dependent protein kinase. So it tells you that cyclic a MP is the activator activates protein kinase a which in turn, phosphorylation lots of target proteins, including, uh, glycogen phosphorylase, which is an activating event that converts glycogen stores to glucose. How do these protein kinas work? Well, they phosphorylase they recognise a particular sequence motif. So you remember back in lecture four, we talked about, uh, sequence motifs, uh, protein kinase. In fact, I think we referred to an example of one that was a tiras kinase motif. Here, it's, uh, protein kinase a motif. It will recognise that S that, uh, particular piece of sequence and that particular fold in that piece of sequence and it will phosphor at that site Of course, proteins don't have to be phosphorated only by a single kinase they they can be, uh they phosphorated by a number of different kinas. Some kinase are specific for one protein and others can phosphor many proteins such as protein kinase A. And each of these phosphorylation sites can have a different impact on the on the sort of structure function relationship, and rev and result in a different function occurring. Definitely have to keep an eye on the time here because it's starting to run out just to show that that adrenergic binding event has lots and lots of downstream events through the actions of protein kinase A. I'm not gonna ask you, you know, I don't want you to go and memorise all the all of these kind of, um, you know, proteins. It's just really the kind of take home message here, and it gives you the protein kinase. A consensus motive. Here is where you've got the stimulation of glycogen or or the inhibition in fact, of glycogen synthesis, the stimulation of glycogen breakdown, stimulation of glycolysis and pyro, acetyl coa and lots of other things that are going on as well via the phosphorylation of different proteins. So even things like synthesis of dopamine. And even there's the, uh, there's a feedback mechanism here via tyrosine hydroxylase to reduce the synthesis of epinephrine, for example, because you don't want to keep, you know, people who are constantly there is a disease that's called, um, it's like excess adrenal tumours and things like that where people are constantly making too much adrenaline. Um, and, uh, this is obviously very bad, um, for people to be constantly under that kind of stress, but lots and lots of things. So this is really the take home message from this slide? Is the idea of a single kinase being able to target lots of different proteins to get lots of different functional outcomes. Um uh, through signal transduction. So probably there's two, I think, quite complex slides in the lecture, and this is one of them. And it's just an example of how a G protein coupled receptor signalling, uh, actually occurs. And again, this is, uh, part of the, uh, adrenaline pathway or the response to adrenaline. You've got the binding of your Liga to your G protein coupled receptor. We've talked about the G protein activating the enz, the the the enzyme in this case, the Aden cycle and the production of cyclic a MP. So what cyclic a MP does to the cyclic a MP dependent protein kinase or protein kinase A Is it actually binds to these, um uh, to the, uh, uh the the the receptor domains or the regulatory domains of the PK A. And by doing that, it actually allows the catalytic domains to dissociate and become active. So that's quite important here. The phosphorylation event, uh, or the cyclic a MP binding is associated with the, um, dissociation of the catalytic sub units from the regulatory subunits. Those catalytic subunits then pass into the nucleus where again, using a TP, they phosphor a protein. In this example, this is just one example and we saw on the previous slide There are many examples by different mechanisms by which this occur. Phosphors, A protein called creb CREB stands for cyclic a MP response element binding protein much easier to remember it as creb. It gets phosphorated phosphorylation enables to associate with a second protein called CBP or P. 300 CBP stands for CREB binding protein. So it's good nomenclature CREB and CREB binding protein This protein complex phosphorated dimer of CRE of CREB associated with CBP can interact bind to DNA, so the CRE is the cyclic, a MP response element in the promoter regions of target genes. The interaction the double phosphorylation biop protein kinase enables the interaction with CBP that enables an interaction with RN a polymerase too, which can then transcribe these genes. So protein phosphatases remove phosphorylation in response to the stim to a stimulus or the removal of the competing stimulus. Not as many phosphatases there are kinas, I think I mentioned before. There's about 650 protein kinas in the human genome protein phosphatases. There's much fewer. They have really kind of. The whole nomenclature of kinase is really awkward. You know you have map kinase that's phosphorated by map kinase kinase and map kinase kinase. That's phosphorylation by map kinase kinase kinase. Um so it gets quite confusing, but the protein phosphatase is a much smaller group. You've got phosphatase that dephosphorylation sines and three amines and you've got phosphatases that dephosphorylation primarily thyra zines. This pathway, I think, really shows that really, really effectively. Um, how this occurs so again. This is still adrenergic signalling. We've got lots of cyclic a MP that is present the cyclic a MP activates protein kinase a and that does a number of things that are important in that in this process. The first thing is is that the phosphorylation of protein kinase a is an activator. Uh, sorry, uh, is, uh, activates those catalytic subunits And this enables the phosph correlation firstly of glycogen synthese and that phosphorylation is an inactive event. So glycogen synthese, the protein that makes glycogen from glucose is inactivated by phosphorylation. We don't want glycogen being made from glucose when we're trying to run away from something. At the same time, it phosphors a protein called glycogen phosphorylase kinase. That is an activating event that enables this kinase to phosphorylase, a second protein called glycogen phosphorylase. And that enzyme stimulates the production of glucose one phosphate from glycogen, so breaking down glycogen stores to make glucose available at the same time. Over here we have the inhibition of phosphoprotein phosphatase. We've got an inhibitor that's called the inhibitor of phosphoprotein phosphatase phosphorylation by protein kinase a enables it to bind to the protein phosphatase and inactivate it it can't bind to the protein phosphatase in its inactive form. That's not phosphorated. Of course. When we remove the adrenaline signal, this phosphorylation event doesn't happen anymore. This doesn't bind to the protein phosphatase. The protein phosphatase becomes active. It then removes phosphate from glycogen synthese, enabling it to be phosphorated and enabling glucose to be stored as glycogen. Because this is glycogen synthese same time it removes the phosphorylation from this one glycogen phosphorylase kinase sends it in into an inactive form, removes from this one glycogen phosphorylase and sends that into an inactive form. No more breakdown of glycogen. OK, so I wanna end today by talking about the receptor thaine kinas as I might not fully get through all of the slides, but I might Let's see how we go. Remember G protein coupled receptors. Big family, 800 proteins. Receptor thyra zine kinase, small family. There's only about 60 of these known and they, unlike G protein coupled receptors. But like histadine sensor kinas, they transduce an extra cellular signal via auto phosphorylation they phosphorylation themselves. Why is tyrosine phosphorylation much less represented in the human phosphor proteome? Because there are so few comparatively tyras kinas There's only about 60. Which means there's, you know, 10 fold less than there are sine three andine type kinas. As we'll see they have again. They all share an extracellular, uh, ligand binding domain, uh, single transmembrane span and the enzymatic active kinase domain, which is found in the cytoplasm. And that's a tyrosine kinase. And they act as specific receptors for growth factors. And for insulin is the example that we will look at so pre pretty simple again. Like all we've seen with the others, they have these extracellular Lagan binding region and they occur in the membrane in an inactive form as monomers. They're poorly catalytic as monomers. When the light began binds in this case something like insulin. The two monomers come together and they phosphorylation in the, uh, in the, uh, cytoplasmic activation domain. They actually phosphorylation each other again, using a TP when the dyer is phosphorated. What you actually get is an activated kinase that enables phosphorylation of additional residues within that cytoplasmic domain. And each of these provides a different docking site for an interaction partner. OK, so these phosphorylation events are mainly to do with protein protein interactions. They enable other substrates to come along to bind and to be phosphorated. The example that I want to look at is uh, uh, insulin. Um, insulin interacts, uh, with the cell. And this is the classic example of receptor tyrosine kinase signalling. I think most of you will know quite a bit about insulin. It's a peptide. Uh, it's produced as quite a large protein that he's then cleaved into a quite small polypeptide of around about six Kools to be active. But it's produced by the be the beta cells, and the islets of Langerhans in the pancreas. Obviously passes through the bloodstream goes to a lot of dis different destinations. Cells within those destinations have an insulin receptor, which is a receptor tyrosine kinase, and the binding of insulin initiates signal transduction pathways or insulin signalling that leads to increased glucose uptake and increased glucose metabolism. And I think you're all aware that obviously, if you can't sense or make insulin, that is generally, um, uh referred to as diabetes. The famous example. The most famous example of this. Some of you would know that we, uh, that the university is very lucky. That has Professor David James here and Professor David James discovered Glut four. and so one of the things that insulin does is when it binds the insulin receptor by a signal cascade that we'll look at on a subsequent slide. It actually, and it actually enables the translocation of Glut four to the cell surface to take up glucose and Glut four is an interesting protein. It's a membrane protein. It normally sits inside the cells stored within these membrane vesicles. But when there's glucose present and insulin binds to the insulin receptor, those intracellular um vesicles will endo zom move to the plasma membrane. They fuse with the plasma membrane and they bed glut four on the surface, and then glut four takes up glucose. When there's no more glucose present, they get stored again. You get this little budding event that happens down here by endocytosis and the, uh, glut four receptors get stored again within the endos within the cell. This is probably the second most complex slide within the lecture. And it just shows one, signal event that is occurring in this type of, uh um signalling this receptor thyra in kinase signalling again. You've got insulin binding to the insulin receptor. You're getting multiple phosphorylation sites on the insulin receptor that become docking sites for other proteins. In this case, what we see here is the insulin receptor substrate. One binds to one or more of these phosphorylation sites and becomes phosphorylation itself. This then enables the IRS one or the insulin receptor substrate one protein to interact with a protein complex that includes a number of different proteins, including GRP two S OS, Rasin, RAF. When that binding event occurs, a protein kinase called mec mitogen activating protein kinase a Mac kinase. It is Mac K. Uh, MEC will bind to this and phos and become phosphorated. So the phosphate is transferred through the complex via the IRS one from the insulin receptor through to this protein kind called mec. Very famous kinase mec phosphorylation is another one called the extracellular signal kinase. Or when it's phosphorylation, it can pass into the nucleus. It can combine with a number of transcription factors to again enable the initiation of signalling. The example it's shown here is SRF and elk One, uh, I think SRF stands for serum response factor. Uh, elk. One is I don't know what elk. One specifically stands for um In this case, it's not particularly important. But what the phosphorylation enables is the interaction of these two proteins together to become an activated transcription factor and the, um, initiation of cell signalling just to show that there's lots and lots of phosphorylation that occurs here. It's not just map kinas that are, uh, activated. As we saw on the previous slide, there are a whole other ones as well. The lipid kinase P I three K can become, uh, activated by the insulin receptor substrate one. Um, but the most famous one is probably a KT. The thing to take the message to take home from here is again that idea that these Kaisers can have multiple targets and the multiple targets here include FOXO, which is involved in glucose production. Uh, TSC two, which is involved in lipid and protein synthesis. GSK three B is the one I'm gonna talk about in T BC one D four, which is involved in glucose update. You see this little line here? What that little line means is that when a KT phosphors these things, it's actually an inhibitory event. So inhibiting GSK three BGSK three B, glycogen synthese kinase inhibiting glycogen synthos kinase inhibits phosphorylation of glycogen synthese by glycogen synthese kinase and keeps glycogen synthese in its active form. So you can see that both G protein coupled receptor signalling and, um uh, receptor thaine kinase signalling can actually interact with each other. I think I have one slide left. Um, yeah, I'm gonna really try and do this last slide leading into Wednesday's lecture. Insulin signalling We've known about known about insulin insulin for 100 years. But even as recently as 2015 and I can say there's been studies since being able to use large scale phosphor proteomics provides both a temporal and a global view of insulin signalling that has provided much more depth to what we've known from the previous 100 years. And this is just showing you a kind of schematic to give you an in an an idea of the time frame and the and the and the the the magnitude of the change that is induced by this simple, binding event of insulin to the insulin receptor. What we see down here is phosphorylation of the insulin receptor. Within seconds within five seconds, you have a 17 fold increase in the phosphorylation of the, uh, auto phosphorylation domain within the cytoplasm caused by insulin. It then passes that signal on to IRS one a five fold increase within five seconds. Uh, a AAA four fold increase of a second site on that a KT kinase phosphorylation at different sites. SPEAKER 0 few are getting into distinction. Zip it. Oh, we got lots to get to. So I've been talking. OK, record to the cloud. Apologies. Uh, hide that one. Hide my unpleasant image. OK, let's move on. Um, we're definitely seeing a a drop off in lecture attendance. I'm not surprised by that, because, um, there's a lot of you that do need to catch up quite a lot. We'll be talking more about that, not next week, but the week after, when we do a tutorial, I doubt it'll be the people that turn up to the tutorial. That'll be the ones that are behind. But we record it, and hopefully people will get the message. And I'll probably send an announcement about that too. Yeah, we will do a tutorial in a couple of weeks time. The last Wednesday before the, uh, mid mid sem break. Um, yeah. The in sem break, I think is probably the better thing to say. Ok, lots to get through today as we talk about phosphor proteomics. Um, so you remember last time. Obviously, we talked quite a lot about the kind of biochemistry of phosphorylation and signal transduction and the way these lectures. Run, uh, this series on PT MS. We're gonna talk about the biology and the biochemistry first, and then we're gonna talk about the analytical aspects. Second, I'm just gonna make sure that that is recording properly. Ooh, look at that. It's quite low. That's better. OK, so by the analytical aspects, I'm really sorry to everyone that's here and back. Um, listening. But there is a fire alarm going off in the law building, and it is terribly, um, distracting. Um, so apologies about that. It's definitely at DEFCON three now, um, which is the bit where everyone really has to evacuate, So there must be an actual issue in there. Um, must be a lawyer getting underpaid. Um, anyway, so today we're gonna talk about I know there's people doing science law here. I shouldn't say that. Um, there's, um and, uh, you know, the idea today is that we're gonna talk about the analytical aspects. We're gonna talk about proteins first. Phosphor proteins talk about peptides. You know, hopefully, you know, the the practical and lecture series a little bit disconnected this year. And this now catches us up a little bit. With what we've been doing in the last couple of weeks in the PRAC series, so we'll start off. You know me. I love the two D gel side of things because they're very visual. So they're gonna show you how we might do this at the protein level and show you the kind of principles behind this, including some phospho specific stain. We'll talk a little bit about affinity chromatography the idea of purifying or enriching for, uh, the phosphate group on proteins or peptides. And then we'll talk about mass spectrometry and large scale phosphor proteomics, including the enrichment approaches that you used in the laboratory class. The most difficult thing about this lecture is we're going to introduce this concept MS to the third. So you've already heard of MS one. So looking at the mass of a peptide, that's a peptide mass fingerprint. You've heard of MS two, which is tandem mass spectrometry where we sequence a peptide, and here we're gonna look at M S3 or EMS to the third, and we'll look at some. Hopefully if we've got time, which, knowing me, we probably won't. We'll do a look at a a kind of case study at the end of this. OK, so let's start simply and we'll talk about phosphor proteins. Uh, first. So I think we're all pretty familiar with the idea of two D electrophoresis now that the separation is based on charge in the first dimension, and the separation is based on mass in the second dimension. And so when we add a phosphate group, or when a phosphate group gets added to a protein, it changes the charge of the biomolecule. It changes the charge of the protein. And so, depending on the the the mass of the protein, uh, the addition of a phosphor group can result in anything up to around about a 0.4 of a PH unit shift, um, to the acidic side, and you'll see this as isoforms or variants across the gel. The term that I prefer to use is proteoform. We'll talk about the concept of proteoform in a LA in a much later lecture. Um, but we often refer to these as isoelectric point variants or isoforms. But but technically, the the correct term is proteoform. But of course, you know, just seeing a spot across a two D gel is you know, not enough to confirm that a protein is phosphorylation so and lots of other PT MS modify charge as well. So So we need to do more. We're gonna look here at phospho specific stains. They work quite well, fluorescent stains that are specific for the phosphate group, and it's a really neat little technique that we can do in conjunction with two D gels. The third thing that we can do is actually blot so we can take the proteins from the gel and use anti phospho antibodies. So antibodies that are specific for phosphor related amino acids to look for the distribution of those phosphor proteins on a gel. And then finally, prior to a gel, we might do some kind of enrichment like iMac chromatography that we talked about in the lab class. We can purify proteins using a mobilised metal affinity chromatography and then run those bound proteins on a two D gel. Very difficult to do that with titanium dioxide. Ah, because the proteins are are are kind of too big for the what's called multi dentate binding. But I'll talk a little bit more about that in this lecture, So the easiest one to understand is this really neat technology. We're, uh, one of the first groups to have this, um, many, many. About 20 years ago, this this, uh, pro Q diamond fluorescent stain came out and it's a phospho specific fluorescent stain. And the really big benefit of what you can do with this stain is that you can run a two D gel exactly the same way you did in the lab class. You stain it. Firstly, with the phospho specific stain, you image it, you then wash it, put it into Kasi blue or into another stain. And you have two images of the same gel, one that's specific for the phosphor proteins and one that's specific for the total proteon. So what you're seeing here is firstly, in the upper panel, you see all the the intensely staining phosphor proteins. So this one, which is HSP 90 a couple of other ones Here, you look in here and you got a little bit of background, a little bit of non-specific staining. Perhaps you got these two quite strong binders or quote two strike quite strong, um, staining images. You take that same gel, you image it with Kasi blue or with another total protein stain like a cyber ruby. If you want to use a fluorescent stain and you get the total proteome and then you can overlap these two images because it's the same gel just imaged with two different stains. And you can see that this one is this one here. And this one down here, which is quite heavily phosphorated, is actually this little one down here. And these two here and here are these two here and here. And this works really, really effectively. And this is a little power dime that I'm going to use throughout this, uh, throughout this lecture, and I actually use it in a couple of lectures. It's a study that that my own group did, and it just shows a couple of things that are really important to teach about phosphor proteins. So what has been done here is this is just a little panel on two little panels on a two D gel in the upper panel. Is this time the total protein stain? The middle panel is the phospho stain. And then what the third panel is is showing. These two images overlapped, so the total protein stain gets a red channel and the phospho stain gets a green channel. And when you overlap those two images, you can actually see which proteins are phosphorated. So we're gonna talk more about these ABC 12 and three. This is a protein called Alpha beta crystalline or alpha B crystalline. And this one over here, HSP 123 and four are heat shock protein 27. And these are four variants of the same protein translated. But obviously they have different migration on the two D gel, so therefore they have different post translational modifications on them. So when we look at the phospho stain, the second thing, that's really important. And probably the only thing that I'm gonna comment about this side of the gel this side here, over on the right hand side is that what you can see sometimes are very intensely staining phosphor proteins that must be of relatively low abundance because they stain very heavily with the phospho specific stain. You can see this big green spot here, but it doesn't have a corresponding red spot. Maybe this tiny little thing is kind of almost like a smudge in the background. This really big one here doesn't overlap with anything here either. So these are really intensely, heavily phosphor rated proteins that are of low abundance because we see a lot from the phospho stain. But we don't see a lot from the total abundance stain from the Kasi blue or the cyber ruby that's shown up here. You see, other examples here are very abundant proteins, very abundant spots, but they're not phosphorated at all. I mean, this is just background here, So when you overlay these two images, you actually get quite a lot of information. You can see these ones. This one is, uh, phosphorated. It's quite it's quite strongly phosphorated. This one is phosphorated here. There's actually another one here that you can make out here that you don't see a lot of here, but it's really this big spot here. And the big spot, I think, uh, next it these two are not really seen here at all. They're actually sitting, uh, here and here. And so sometimes, you know, we talked a lot when we did our peptide mass fingerprints about, you know, we cut out a spot and we identified a protein. You know, I think sometimes There might be some, um, proteins sitting in the background that don't stain, but the mass beck is sensitive enough to pick those up. So, um, sometimes you might think you're identifying this one. But really, you're identifying this one here again, but to take message heavily phosphorated but not very abundant. You'll see that with the phospho stain abundant and not phosphorated, you'll see that with the, um, total protein stain. And then sometimes you have proteins that are abundant and phosphorated. But again, this one this 12, and three these three copies, if you like of the alpha B crystalline we're going to talk about in quite a bit of detail. As we move along through the lecture, you can see that one of them is phosphorated. And I'll come back to that in a minute so we can use uh, ant phospho antibodies. I think I mentioned very briefly in the in the pre practical um tutorial or the intro talk that you can buy antibodies that are specific or supposedly specific for, uh, for phosphor related amino acids. So ant phosphoserine ant phospho ant phosphatase. Some of these work better than others. The sine and thine one. It's not a big chemical handle for an antibody. A phosphate group on an amino acid with a hydroxyl group, right? It's not a big, uh, it's not something that's easily recognisable, so you tend to get a lot of false positives with poorly specific antibodies. The tyrosine one is a little bit better, so you tend to get quite good specificity with ant phospho thaine antibodies. But the principle is very similar to what we've seen previously. You might have your healthy cells. You might have your disease cells. You get your proteins from these, you use an antibody against, um, against one of these phosphorated amino acids. Let's say between healthy and diseased cells and you can compare, uh, what doesn't bind the rest of the proteon between those cells? Michelle, you have a question. So if the phosphate is quite small and it's not SPEAKER 1 a very unique chemical, yeah, you stick a bunch of SPEAKER 0 phosphor in them. Yeah, yeah, yeah, yeah. Sometimes they got a little bit more to them like they have a little bit of an adjuvant to them as well. But yeah, that's pretty much so. If it's buried, I mean, it's buried. It probably doesn't get phosphorated. But it it Yeah, yeah, it They tend to be a little bit non-specific but the thyra zine because it has. Yeah, yeah, but the the thyra is better because it's a bigger amino acid with the ring structure as well. Ok, then, the next thing we could do is obviously we might have these proteins on a gel. We might go. Well, you know, we've we've cut this spot out. We know it's phosphorated. We don't know where it's phosphorated. We haven't confirmed that. It's phosphorated. So we need to start thinking about how are we going to confirm the phosphorylation side? How are we going to confirm that this is a phosphor protein? So first thing that you've done, of course there is that you've isolated those spots. You've cut them out, you've subjected them to peptide mass fingerprinting or tandem mass spec. Um, and you've identified them. The next thing, we're gonna do exactly what you've just been doing in the lab class. You're gonna do a selective enrichment. That could be by affinity chromatography that we, um uh that we didn't do with antibodies. It could be with a mobilised metal affinity chromatography. Or it could be with titanium dioxide, ch chromatography and titanium dioxide is kind of the gold standard for doing all of these experiments. Pretty much everything that's done, Um, in the world of phosphor proteomics these days is done with titanium dioxide. Fun fact. Titanium dioxide is the active ingredient in sunscreen. So when you put your sunscreen on, you can purify your skin phosphor peptides at the same time. Yeah, yeah, they're very similar. And you know, that's a great question. We're gonna talk more about them. There's absolutely no doubt that the principle behind them are very similar, but not 100% the same. I'm gonna touch on how they're different, but I don't want to get into it too much. But you can think of it, really. The principle is the same charge based, binding, negatively charged phosphates, positively charged resin, titanium two plus and, uh, immobilised metals. It can be gallium copper. Iron can be a whole heap of them, but always positive. That's the easiest way Titanium dioxide. As you'll as we'll see, it's actually called multi dentate binding. There are other elements that are involved as well. Um, but I'm not really gonna go into them. Primarily that the major, uh, way that, uh, phosphor peptides bind to titanium dioxide is again that charge based interaction gonna tell, teach you a neat little trick, an on site phosphatase treatment, and then we're gonna do some troubleshooting and talk about MS to the three. So if we're going to characterise these phosphor proteins and phosphor peptides in a perfect world, the best thing that could happen would be we digest our protein, we get our peaks. We've all talked about it. We've talked about it kind of ad nauseam. In our brief reports, you get that peak list, you put the peak list in mascot. Some peaks match some don't, OK. And everyone says, Well, some of those could be post translational modifications, right? They they're not. But some of you people say, because they could be right so we could get lucky. We could then say, Well, why don't we open the search space up and say, Tell me if there's any phosphors in here, as well as methionine sulfoxide and and acrylic and, um um propion amide for cystic. And I'm gonna show you the one example in my entire career where that worked. OK, so that's comparative mass spectrometry. And I'm gonna talk a little bit more about comparative mass spectrometry. So you've got those we saw with that ABC. 123. We had three spots across the gel. We cut them all out. We look at the Spectra, and instead of comparing gel images, we compare the spectra which peaks appear and disappear. This is pretty much what she did with the fuin. Example in the lab class. Then there's a neat little trick that I can tell you about the on target phosphatase treatment, a bit of affinity, enrichment using those approaches we just talked about. And then finally, we're gonna talk about that large scale tandem MS. So, as I said, this is kind of summarising what? I've literally just said that we do this standard peptide mass fingerprint using MT MS. We get that list of matching peptides and you always get this list of non matching peptides and for someone you know, experienced like me, who's done a million of those things, I can immediately look at the peak list and go well. 1470 five's keratin and 19 forties keratin and 10 Fif. Whatever. 1066 is matrix iron, all that kind of thing. But even I'll see a bunch of peaks. No idea. It could be two, Miss Cleavages. Three Miss Cleavages. You just don't know. It could be other peaks within a different form of keratin. Lots of people have different types of keratin. There's lots of mutations in keratin. Everyone has kind of their own specific parts of keratin, especially curly hair versus straight hair. It's all how Keratin forms disulfide bonds. Um, very, um, you know, interest, actually, quite quite an interesting area. Could be, Uh uh, So But it could be these post translation modified peptides. But this is super fraught with danger, as you saw in the lab class, you know, Did you see any of those phosphor peptides in a background of non modified peptides? You didn't really see them in the raw peptide mix until you enrich for them. And then you get these huge signals because there's no other ions there to suppress them. And this is the what we mean by suppression. Is this concept of that? Remember that? That what you're looking at at the Y axis in a peptide mass fingerprint is not only the abundance, but it's the relative intensity caused by how well that PE that that biomolecule ionises So the ones that are modified, they ionise really poorly. And there's lots of reasons for that, and we'll talk more about that in a second. It's mainly because the modifications are much more, are very labile, and they can kind of fall off in the source, in the in the mouldy source or in the electrospray ionisation source. So we have to be really careful to try and keep them on. And and but But they so they don't ionise well in the presence of the other ones that do so, we have to enrich for them. And that's and that's really normally what we see and why a standard PM F, where you've got your non matching peaks modifications particularly phosphorylation quite rare in there. More likely you're seeing maybe methylations um, you might be seeing acetyl stations, but phosphors much rarer. So let's talk a little bit about how this goes. So this is my best case scenario. The the one time that I did this in, and, uh, it came up with something that was absolutely true. So this is this idea of comparative mass spectrum? True, We've got two spots on a gel. We cut them out, we digest them with Trypsin, and they both come back with an answer that says they are thiol peroxidase. OK, it doesn't matter what they what it is, but they come back and tells you they're the product of the same gene. It's the same protein, but it's in two different spots, so we know it must be chemically distinct. They're chemically distinct species. So we look at the spectrum from the one on the right and we look at the spectrum from the one on the left and we can see immediately that this one on the left has this big peak here at 994. And the one over here doesn't have a peak at 994. The rest of it looks exactly the same. The two of them look exactly the same. We then go down and we say to the database very similar to like what you did with the four proteins. The phosphor proteins we say to to the database show me any of these masses that I haven't matched whether any of them are posttranslational modified and it comes up. And it says to me that this 994.49 which is what I see here is potentially phosphorated and you can see here there's only one site that can be phosphorated here, and that's the searing at position 125. Now, I can tell you that when I saw that, that's the only time I've ever seen a phosphor peptide actually ionise efficiently like that in a multi top mass spectrometer in the presence of all the other ones. It's the only time I've ever seen it. This was not enriched. Now I'm gonna show you that in in a second. Yeah, So this is just a straight peptide mass fingerprint, and we got this, and obviously I had to prove it, right. I mean, 994. How do I know it could be the phospho? But that's just a database search. I have to go and prove it. So the second way of doing this is, uh, uh is to enrich for it. So, with compa, I'm gonna talk just a little bit more about the com. The idea of this idea of comparative mol EMS and I'm gonna go back to that three P I variants of ABC alpha B crystalline. So here again, like I did in the previous example, you can compare the spectra from multiple P I variants. Not just the two. As we saw in the previous example, um, and we can look for both missing peaks and new peaks. So a phosphor peptide would be seen as, uh if it's a single phosphorylation. It's an 80 mass unit Shit. 79.96 or something like that. It's 80 mass units. The quantity here and the abundance is not quite so important. But what you're more likely to see is what we kind of see over here is that, uh, normally, what you would see is that a peak would be missing. So the peak is kind of missing. And so let's look at that example Those three examples now a again, this is ABC 12 and three. The reviewers of the paper didn't pick this up, but in this figure, it's called a alpha BC. 123 ABC and alpha BC 12 and three. They are exactly the same thing. So again, just to remind you that you've got three spots, you've got three variants of this particular protein. Alpha B crystalline. You've got the one over here. ABC three, you've got the one in the middle that's much less abundant. ABC two and you've got the one over to the left. That's ABC one again. Cut them all out of the gel. Run your peptide mass fingerprints. And here, instead of looking at the peak lists, we're kind of looking at the Spectra. But the principle of looking at the peak list is exactly the same as you did in the lab class. So you look at these and you come across and you say, Well, first thing, the first thing that I notice here is that there's a peak here at 14 30.72. That's only present in this one, and it's not present in these two. And then there's also a peak here at 14. 78.75 that's present in these two, this one, and this one, but not present in this one, which we know is the phosphor is the phosphor related form of this protein, so that's interesting to us. Because if we look at what the sequence matches for this peak, we see that it has a serine in it. It has a three anine in it. It has another serine in it and so on. So it's residues 57 to 69 so that immediately starts to tell us. Well, this is a good candidate peptide to account for this phosphorylation If we go out, what's 80 mass units on top of 14. 78.75 15, 58.75. Ah, quick maths. Um 15. 58.75. If we look for that here, we don't see it, OK, and the reason we don't see it is cos all those other non phosphor related peptide peaks are suppressing it. We think so. We have to go and do a little bit more. So what we're seeing here is that that 14 78.57 M over Z peak, it disappears in ABC one most likely because it is phosphorated in this proteoform. But we don't see the corresponding peak at 14. 78.57 plus the phosphate, which is 80 why I've just said that the other peaks suppress it. It's, uh, the other ones ionise much more efficiently than the phosphor pide. Now kind of jumping a little bit here. So one thing that you can do that's really neat and we used to do this in the lab class. It's actually really quite cool. To do this in the lab class is that you can exploit what happens in nature. What happens in cells. There's kinas and phosphatases. You can buy and tonnes of biotechnological phosphatases, and you can do something that's actually really, really cool. You can take your mouldy plate. You know, that big metal target plate where you put your matrix on and you can say, Well, I think this is phosphorated and you can come along and you literally put down a drop of alkaline phosphatase onto the top of that spot where it's all there you put the matrix, you put the spot, you incubate it with a little bit of, uh, you know, a wet, um, tissue in incubate it for about half an hour. At 37 degrees, you come back out, put a bit of matrix back on the top, stick it in the mass spectrometer and you see something. If it's phosphorated, you see something that looks like this. This is probably the oldest spectrum in this entire unit. In fact, it's definitely the oldest spectrum in this entire unit. There's no isotopic resolution here. This is a theoretical peptide. This is a synthetic peptide. It's not a triptych peptide, but it has a phosphoserine on it here at this position for put it on the target. Add the alkaline phosphatase. After half an hour, you see the loss of 80 mass units because the phosphates fallen off. And that was the technique that I used to prove. This one all those years ago just took the plate out, put some alkaline phosphatase down. Found out 949, 94 disappeared. And I got a big peek at 9. 14. Confirms it because there's only one searing in that in that uh, um, sequence. So that's a neat little trick. You could do this. Here's a classic example. This is the beta caseine. This is the beta caseine that you used in the in the lab class. As I said to a number of you beta caseine ha is the archetypal phosphoprotein It has so many phosphorylation sites. It has a quadruple phosphorated peptide in it. That surprisingly enough we did see the 2966, peptide. If you treat without phosphatase here, just 20 femto moles of of, uh, of phosphatase treatment. You actually see that this tiny little quadruple charged phosphor pide loses its phosphates and becomes a quite nice, intense high mass peak and the same down here? You've got a sly phosphorated one here barely can see it above the intensity of all the non phosphorated peaks. And again, it loses 80 mass units and you see it quite clearly here. Nice little trick. OK, so let's talk a little bit more about affinity enrichment. So the specific analysis here of phosphor peptides alone and we wanna be as selective as possible, we're gonna talk a lot about enrichment techniques in in In the PTM block of lectures, the affinity techniques for phosphor peptides are by far and away the best they are. If you don't get a 95% enrichment and identify 20,000 phosphor peptides from a human cell line, you've done something wrong. OK, 95% enrichment pre that means of all the peptides you identify with these enrichments, 95% of them should be confirmed phosphor, phosphor related peptides. So it's very selective. And that's because of how good titanium dioxide works. So those chrome So really, this is a chromatography step prior to the mass spectrometry, and we look again at bound and unbound peptides. We might use those antibodies. I kinda dis them a little bit. But as I said, the ant phosphatase ones are quite good. The two approaches that we talked about in the lab class, the immobilised metal affinity and the titanium dark side. I do want to talk a little bit about what can happen with false positives and negatives, and then we're gonna get on to the idea of large scale analysis by tandem aspect. OK, lots of writing on this slide a little bit. We covered in the lab class, but this is a mobilised metal affinity chromatography, or iMac. The way that the binding occurs is exactly what I talked about. It's charge based interaction, negatively charged Phospho and a positively charged metal iron. Those metal ions in the immobilised metal affinity chromatography they can be copper can be gallium it can be iron. It can be others as well. I say there that they do have different properties. There is literally a plethora of literature looking at the relative binding capabilities of different metal ions in a mobilised metal affinity chromatography for, um uh for phosphor peptides. And to be honest, I, I cannot tell you which one is better or worse, because lots of people in their hands have one being better than another. We typically would use iron, but, uh, look any of those and understanding the principle. It's charge based interaction. Bond is broken at alkaline PH. This is exactly the same as what you did in the lab class for titanium dioxide, the really big, important one here is this idea of negatively charged amino acid stretches so glutamic acid and aspartic acid that both have negative charges they can bind as false positives. This is also true for titanium dioxide, but it's much more true for for iMac. The other thing that makes iMac not quite as preferable as, um, titanium dioxide is that the interaction the the charge based interaction is not so strong that sometimes if you have a very big singly phosphor related peptide. So a long one, it just doesn't bind or it binds very weakly. It gets washed off. Shorter ones are OK because the negative charge is more, uh, is is is a bigger component of the biomolecule than on a really long, uh, phosphor peptide. So sometimes those singly charged one, singly modified ones don't bind very well. And actually, if we have time and we talk about a large scale approach, you know, the kind of thing that a lot of groups do is you get all the multiple phosphorated ones with iMac, and then you get the singly phosphorated ones with titanium dioxide. There's a really good reason for that, too. OK, so those are the two kind of, you know, things you gotta be careful of with iMac, you've got the carboxylic acid moed negatively charged amino acids that can bind as false positives. And if you get big peptides with only a single phosphate on them, you can get false negatives because they don't bind very strongly. And the approach beyond that is very much if you've ever done a purification of a his tag protein, it's very much the same kind of thing. So this shows what I just talked about on the previous slide in kind of a schematic form again, this is with proteins. So if you have your control cells and your treated cells and you wanna compare the phosphor proteome, uh, of these two, conditions you do your iMac chromatography, the phosphor peptides bind, and you can compare the the the phosphor proteome by two DG, for example. You then identify any differences that are shown here. There's a third spot here that you cut out and you identify it. And you just know at the phosphor protein level what the differences are. If you want to find the sites of modification or you wanna do large scale shotgun type phosphor proteomics, then you do your protease digestion. You do some desalting and you do the capture. Uh, exactly this same principle negatively charged phospho peptides. In this case, as opposed to negatively charged phosphor proteins in this case will bind, and you can label them with your TMT or your eye track or your IC AD or your CAC beforehand. All of those can be used, um, and you can compare your control and test conditions just for the phosphor proteon. And the really neat thing as well as is shown here is everything that doesn't bind is representative of the total proteome. So you can actually look at both the total proteome and the phosphor proteome by both of these, uh, methodologies now with titanium dioxide, the Y. You know, as I said before, you can really think of it as very, very similar in principle to what we just discussed with iMac. Simple Samp. Simpler sample handling because you don't have to put the, uh, metal ions. You don't have to make the column with the with the metal ions. So you just use the resin itself. You get fewer false negatives and I'll talk a little bit more about that and you get fewer false positives. So I don't have it explicitly written here. The fewer false negatives, the binding is stronger between titanium dioxide and iMac. And I know that sounds a little bit kind of nebulous. It's stronger, but I don't really wanna go into it, or we will be doing three chemistry lectures on titanium dioxide. OK, so the binding is stronger. So what that means is that you bind those singly phosphorated peptides, but there's a negative side to that as well. There's a downside to that really multiply charged phosphor peptides bind so strongly that even at very high PH now you used ammonia solution PH 13 to get these off the column. But at very high PH. Sometimes those multiply charged phosphor peptides stay stuck on the titanium dioxide in the lab class. We actually got the quadruple charged beta caseine phosphor peptide at 2966. So we did actually get it off. That doesn't always happen. And that's taken quite a long time to find the right PH to help get the those strong, uh, very multiple phosphorylation peptides off the column. False positives I mentioned before the exact same false positives as we would see with iMac, negatively charged glutamic acid aspartic acid. Here, though, it's much more specific. You just have one or two. They don't bind. In my experience, if you have, you have to have three in a row. DDDDEDEED. If you have three in a row and a peptide, it will bind to titanium dioxide OK, and it will be eluded as a false positive. So this is definitely the current method of choice. This is used for all the phosphor proteomics in every lab around the world. That's doing phosphor proteomics. Um, uh, but, you know, two years ago in the masc facility, you know, 24 masks, I would say 50% of the work we did was phosphor proteomics. How does it work again? I I'm not going into it in too much detail. You've got the titanium dioxide is basically produced as these little microparticles You make a column or you make a you can actually make a slurry. And you can just incubate your peptides with that, uh, titanium dioxide slurry. Um, they attach you loot with a high PH ammonia solution, and you get your phosphor peptides. This is pretty clear down here. This is showing the binding interaction, the negatively charged, uh, hydroxyl groups and the positively charged titanium. As I said before, this is multi dentate binding, which means there's lots of things going on in the binding, but charge based interactions are the major principle here. OK, so we can do this. We can, you know, separate our gel separated proteins. We digest them with trypsin. We've got a mix containing normal unmodified peptides and phosphor peptides. We do exactly what you did in the lab class. You pass your, uh, peptide mix through a titanium oxide column. It goes orange as you saw in the lab class. Anything that goes through you can spot as the unbound. Anything that binds can be eluded and and checked as well. So gotta come back to this example. We gotta solve our You gotta do. We gotta solve our biochemical problem. We gotta solve our problem with the mass spectrometer. Remember, we've got our three spots. ABC three, ABC two and ABC one. Remember? We saw in ABC one. This peak disappeared, but we couldn't see a peak 80 mass units higher. What we're then gonna do is we're gonna take all of these and we're gonna put them through a titanium dioxide column exactly like you did in the lab class. What we see ABC three no phosphorylation at all. That's just all noise. See, the baseline goes up to 100% so always you get 100% and the baselines there. No peaks there at all. ABC two, the one in the middle. No peaks. There no phosphorylation as there shouldn't be because we don't see them Stain with the phospho spec specific stain. But when we come down to ABC one peptide mix bound to titanium dioxide is this very nice peak at 15. 58.7 80 mass units higher. Those of you who are very keen eyed would say Hang on. How do you know that that residue is the phosphor rated one? Not that three. And in there or not, that sine there. Of course, you should ask that question. We didn't know at this stage, but you take that mix and you put it on to tandem mass spectrometry and you sequence it and that's what we're gonna do right now. OK, so we need the tandem mass spectrometry for the site verification. So prior techniques We've got to the point where we've managed to purify a phosphor peptide. But let's look at the sequences like in the last example. We had three possible phosphorylation sites. We had a serine at position. What's that? 57 58 59. We have the three anine here and we have the serine here at position 69 68 67 66 but we don't know. Sometimes you get lucky. You have something like this like we saw with that thiol peroxidase, you purify a phosphor peptide. It's only got one possible phosphorylation site, which is the three. And in here. You just look for the plus 80. We saw it before. What it looks like on a spectrum has to be that here. However, when we've got a Y here or an S here, both of them could be phosphorated and we don't know which one it is. Could both of them be phosphorated? Well, it depends on the mass change, of course cos it'd be two times 80 if both of them are phosphorated. But we need MS MS to identify the exact site. And here's where things go a bit problematic. Phosphor peptides, typically not always can cause a lot of difficulties in tandem mass spectrometry. Remember, the principle of tandem mass spectrometry is that we break the peptide bonds between the amino acids. But there's also a bond that forms between the hydroxyl group and the phosphate. Right. When we add fragmentation energy, we have to make it and and we have to give it enough energy to break the peptide bonds. The problem is, is the bond between the amino acid and the phosphate is way weaker than the peptide bonds. So what you sometimes see is something that looks exactly like this. You put a phosphor peptide in the tan in the tandem mass spectrometry. You add the energy required to break the peptide bonds, and all you see is the loss of phosphate. And here it's seen as a loss of H three PO four. Because, remember, it's an intensity scale. It's not, you know, it's it's absolutely based on the most intense peak will go to 100%. So the loss of phosphate here is 100%. The the the precursor ion is 2322 and the fragment ion is 2224 The loss of H three PO 498 mass units. We don't see any real other fragment ions down here. We don't see a Y one. We got a couple of little peaks here, So the efficiency at that energy of the loss of phosphate is basically a a hundredfold more efficient, more labi than the peptide bond. So this creates us a really big problem and when this occurs, we don't get any sequence data. In other cases, that doesn't happen all the time. But of course, in other cases when we do tandem mass spectrometry of phosphor peptides, we might get a much richer MS MS spectrum. But it's much more complicated because of course, each of the fragments that contain a phosphate will also lose that phosphate under that same energy. So here what you do you look for fragment irons that have lost the H three PO four. They lose 98 so phosphate added is 80. But when it's lost, it's lost as H three PO four. So this is a little bit complicated because I'm showing a BIN series here and each of these B ions if you can see a loss of 98 you know that that be iron has a phosphate on it. So again, remember, with tandem mass spectrometry, we're looking at Y ions, which are those that are the C terminal fragments. And we're looking at corresponding B ions that are the N terminal fragments. So as we move along here and I'm gonna show this, uh, in a simplified form in a minute. Any fragment ion, be it Y or B that contains still contains the phospho. We'll lose 98. So that's actually really helpful to us in identifying the phosphorylation site. Because here we've got a sequence that has two sines and a thyra zine. We don't know which of those three amino acids the phosphate might be on. So let's look at it. Here's our peptide we've got uh uh we we know here. I'm telling you that the phosphate is on, uh, this sine at position five. I'm showing you where the fragments occur. They also occur obviously here as well. And let's start to sequence this the first iron we create here or the the the the first one we're gonna look at At least remember they're all happening at the same time. Is the Y one, which is arginine doesn't have a phosphate on it. Cos Arginine is not phosphorylation. We don't see loss of 98 the corresponding B iron. We do see the loss of 98 And if you go back B eight, we see loss of 98. If we keep moving through here we go to Y two YR No loss of phosphate. So we can immediately say while we see the corresponding B seven iron losing phosphate. Uh, losing the the 98 that corresponding B iron does have phosphate. So the tyrosine cannot be the site of phosphorylation in this example and we keep going VYRY three B six has the phosphate on it. B five PV YR for Y four doesn't have the phosphate on it. We've got to our two s's. And then suddenly we see a switch over the Y five SPV YR has the phosphate on it. The corresponding BINB four does not have the phosphate on it. We can then say that the position of the phosphate attachment is this Seine at residue five. And you can kind of maybe if we go back, we can maybe see some of those. You can see B four here. So B four is E LP S. You can't see a loss of 98 from this. Ok, I'm not, um, magnifying that to show you it, um uh, to show you it, but you know, trust me, it's not there. Ok, now here's the Here's the final complex thing from from this lecture. Let's see how we go. So as I've said, we see lots of examples where all we see is that loss of 98. We don't see all the other Y and B irons. We just see that loss of 98. And so there's something really clever that we can do when we're in that kind of example where we've just got two big peaks, the precursor and the loss of 98. Again remembering that the bond between the phosphate group and the modified amino acid is much more labile than the peptide bonds. And in tandem mass spectrometry, the fragment ions that result from the loss of phosphate can be much more intense. So this can make the Y and Bion peptide series quite difficult. And you get much lower peptide identification. So MS to the third takes advantage of this loss of 98. It says if we go all the way back here, we set it up and we tell the instrument if you see this loss of 98 stop, accumulate this peak. This iron that has lost 98 Accumulate it. Hold it in the iron trap. Hold it in the Orbi trap. Hold it in the, uh, in quadruple two. Let everything else leave. I just want you to fragment and hold this. 2224 iron. So you've done MS one. You've measured the peak. 2322 You've done MS two and generated 2224 M S3 says, let's sequence 2224.2. OK, so MS to the third is literally just taking advantage of that precursor minus 98. Dalton Peak generated in MS MS. Sorry, generated in the MS two. So you get the sequence identification. The problem is, what have you lost? You lost the 98 haven't you? Cos the 98 already fell off and you're holding that cos you want to know what the sequence of the phosphor peptide is. But then you got that problem. You can't localise the phosphorylation site if there are more than, uh, one CN 39 or tyrosine in the sequence that is generated. So, what does it look like? It looks a little bit like this in reality. So we do MS one MS two M S3. And we see something. You know, we might see something like this. Oh, this is what we might normally see. MS we get a peak, we might get a nice MS two, spectrum and be able to sequence it sometimes with phosphor peptides. We get something like this. So panel B is what I've just been talking about. We get that same peak when we go to perform tandem mass spectrometry. All we really see is the loss of 98. And so that loss of 98 we can then isolate that and we can sequence it. OK, so we get the sequence. What we can then do is kind of overlap these two, These two features this feature here and this feature here, and we can then join those two spectra together, the MS two and the M S3 Spectra. We can overlay them and get more rich sequence information. And this is called multistage activation. You don't really need to know what it's called. It's just this idea that sometimes, of course, you get a very nice um MS, uh MS two. Sometimes you only see the loss of 98 and very little sequence information beyond the loss of the phosphate group. If you go to M S3, you isolate the non phosphor related form. The peak that has lost 98. You get a nice rich sequence. Um um fra, uh, fragment iron pattern for that in M S3. Combine those two images get as much sequence and positional information as you can because you're getting the A little bit of positional information from very low abundance fragment ions in here, but getting richer sequence information from the M S3. OK, have I done I? I have. I've got through and I'm I'm probably gonna be ok here. So the last thing just to talk about is the idea of doing this at large scale. We talk very much here about purifying proteins on gels, but really, these days, everything is going through a shotgun proteomics, um, type approach, like we talked about in lecture eight. When we talked about, um, shotgun proteomics and lecture nine. When we talked about quantitative, uh, shotgun proteomics. So here we're doing it at scale. We're gonna digest the whole cell, we're gonna digest it all into peptides, and we're gonna look at the phosphor proteome by, uh, shotgun proteomics. So here we're generally using titanium dioxide chromatography. I'm not gonna talk about heli. At this point in time, I'm gonna talk more about that when we talk about glyco. Uh, glycosylation analysis. If we combine with a temporal dimension, you know, I talk touched on this when I talked about insulin signalling at the end of the last lecture. This is the approach that was used to to to elucidate that insulin signalling. If we combine it with label based approaches, be it Silla or tandem mast es or IRA, then we can get a great idea of what's happening in signal pathways during disease. So this is the C. This is actually the workflow that my lab uses. And that's why the slide is slightly different. Um, in colour, because this is from actually one of Melanie's, um uh, talks. Um, don't need to worry about the sample you've got. Your proteins get extracted, they all get digested with drips and yada is bery tags. The really take home message here and I touched on it earlier. Is this idea of combining iMac and titanium dioxide? Why do we do that? IMac. You can look at the multiply charged, uh, the multiply phosphorated peptides. Titanium dioxide. Those ones stick too hard you can't get them off very well. And titanium dioxide. You look at the singly phosphorated peptides because they don't bind very well to iMac. Don't worry about what the different illusions are here. This approach is actually called C iMac simultaneous. Uh, enrichment from iMac is what that stands for. And we do, uh, mass spectrometry on on these all these different fractions and you get something like this. This is actually a little older study, but you've got 13,000 nearly 14,000 quantified phosphor peptides from about 4.5 1000 proteins. That's about mm. That's about two thirds of the number of proteins that are in the heart, and you can quantify them over a time course. In this case, they're quantified over a time course of myocardial ischemia, which is heart attack. And so, after one minute, look at how much things are changing after one minute of a heart attack. That's what you're seeing in the graph. Here is the big changes that are occurring in the phosphor proteome. As early as one minute of ischemia, you can cluster these. So for us, we're interested in what happens early in a heart attack so these ones that go up really early are interesting to us, but some of them don't go up until necrosis starts to happen. So there's all signalling that happens when tissue starts to die, and that's called necrosis. But there's all other kinds of salvage pathways that happen early on to try to salvage the tissue validation you can Western block. There are There are antibodies available for specific, uh, antibodies available for specific sites in specific proteins. Just wanna contrast this with antiphospholipid phospho thine. These are antsy. 367 in stat five. So they're generated against a quite more much more substantial piece of sequence that's got a phospho on it. And therefore those antibodies work really well, this is a kind of different slide. This is actually erythropoietin. But I just wanted you to get used to this idea of looking at changes and how they can be. The phosphorylation antibodies can be compared to the total anti, uh, for the antibody that shows the total abundance of that protein. So there's a pool of stat five here, for example. That doesn't change at all across this, uh, time course or or actually ex exposure course, um, to erythropoietin, in this case. But the phosphorylation of the protein goes up, um, in a dose dependent manner with the amount of erythropoietin. The reason I wanna show that is because I wanna show that this is how this work gets used is you've got a kinase here that is heavily, um, associated with the early stage of a heart attack. It goes up quite a lot. This is the proteomics data shows that it goes up at 12 minutes starts to go back under baseline by 60 minutes. That's absolutely what you see. Compared to the, um, uh, the non ischemic control goes up very early on. You see the effects a little bit better with the mass spectrometer. It starts to go down around about five minutes, which is pretty much what you see here as well. But then that makes you out of 50 thou, uh, 15,000 phosphorites. You've now got a hypothesis about one. Is that event helpful to the heart, or is that event detrimental to the heart? Is that actually activating pathways that are gonna in instigate necrosis or is that kind? Is that, um, phosphorylation event going to try to salvage the heart. So you come along with an inhibitor for it and you add the inhibitor. And these are all animal models. So you put the inhibitor in of that particular phosphorylation event that happens there. And you see, when you add that phosphor, uh, when you add that inhibitor that the heart does not recover very well. So this is just showing you heart functionality that RPP is called rate pressure product. It's a function of how hard and how quickly the heart contracts. So this event, there's a single phosphorylation event that we captured by phosphor proteomics here, one of the just one of these 4000 things that change. If you go and make a hypothesis about it and find an inhibitor, you can see that actually kind of keeping this phosphorylation active would actually help the heart recover better or not. Be as, uh as um, susceptible to, um, an ischemic event, because by inhibiting that event that phosphorylation event, you see much worse recovery. And that's what's shown in the red part of the panel. Obviously, none of that's examinal. It's just kind of the or the idea of it is what would be examined more. The idea of you know you're doing a big scale phosphor proteomics study. You know, what's the what's the end goal? Why? I mean, are we stamp collecting? No. We wanna make some hypotheses about biology, and that's really what that's trying to show you. OK, so I'll finish there. Sorry, I've got a couple of minutes over. This is I think it's the biggest lecture in the entire unit. It's got 36 slides, so there's a vast number of modifications and we talked about those contaminants and that idea of what the peak list has in it that didn't match the protein and what they mean. Phosphor peptides. They rarely fly very well. This is in, uh, mouldy or in electrospray ionisation. And that's because of iron suppression effects by non modified peptides. So you need this enrichment when you get them. You need to use independent methods of verification, be that by a Western blot or by other types of mass spectrometry approaches that we look at in the final week of the semester. And then, of course, what does the SPEAKER 0 OK, we'll make a start. Um, yeah. Apologies about that confusion there. For those of you listening at home, we thought we didn't have a room and we had a lecture