 Hello, Nanak Magtis from Gantt University, PIB. And we're again looking at mass spectrometry basics, and now we're in the sixth installment of our tutorial series. So in the first one, we talked about amino acids and their properties and how they form to create proteins and how we can then use these properties to analyze these proteins or parts of them peptides in a mass spectrometer. Second lecture talked about mass spectrometry and its overall concepts and about the two ion sources that are prevalent in the study of proteomics, which is Maldi and EZ. In the third lecture, we talked about the mass analyzers that actually find out what the inertial masses of a particular ion, and in fact, it usually the analyzers give you mass over charge. Then the third lecture was about detectors that finally detects the movement of the ions in the instruments and they're an important part in making the mass spectrometry work, obviously, and they can also give us signals about the quantity of a particular ion that is there with some gradients. And then in the fifth lecture, we talked about free transform ion cyclotron resonance and orbitrap instruments, which are a special breed of instruments because they essentially combine an analyzer and a detector and they use some fancy mathematics to weed out the inertial masses or mass over charges, but they do so with very good resolution and that makes them very interesting instruments, especially the orbitrap because it's easy to use. Now with that, we actually summed up everything we needed to know about the actual instruments themselves. Now we're going to look at some interesting new ways of doing mass spectrometry, and the most important one of those is tandem mass spectrometry, or MS, MS, or MS squared. Now, tandem mass spectrometry is meant to break an analyte and then analyze the fragments and the reason why it's called tandem mass spectrometry or in this case, shorthand MS followed by MS or MS squared is very obvious and we'll look at it right now. What happens in tandem mass spectrometry is that we literally use two stages of mass spectrometry in sequence. So again, we have a source, can be electrospray or Maldi, and that source is going to create ions. These ions are then going to go into a first stage of mass spectrometry and remember that I used to color green throughout these lectures to indicate a mass analyzer of some sort. So here we use a mass analyzer, not so much in order to measure the masses of the ions that come in, but to make sure we only have one mass that makes it through this particular ion selector or mass analyzer. That's why we call it the ion selector. Now, you may remember when we talked about analyzers that there was one analyzer who was particularly well suited for this and then it's the quadrupole. So very often this ion selector is in fact a quadrupole. If you will recall, the quadrupole throws every mass over charge out that does not fall within a very limited range that is definable and only this mass over charge will have a stable trajectory and will make it through the quadrupole. Another mass spectrometry that can do this ion selection is an ion trap. Because in an ion trap, what we can do is we trap all the ions and then eject them from smaller ions to bigger ions or rather from lower mass ions to higher mass ions. And then we can actually discard all of the ions that come out of the trap, save for a particular patch of ions that we want to use or rather we could even try and keep these ions in the trap and not eject them at all. And whereas we eject all the other ones. Even a time of flight can be used by putting a gate at the end and only opening the gate at a given flight time. So at a given time after the extraction of the ions. Now, very often it's a quadrupole or an ion trap. After this ion selector, we now have a very small amount of ions left. The small amount of ions corresponds to only those ions with the required mass over charge. Now, the reason why we take only these ions is because we can then fragment them. We can break them apart into bits. This is a very important step and I'll try to explain you how this works with a rather morbid analogy in a second. And these fragments that we obtain, so the bits and pieces that an analyte breaks into are then analyzed by a second mass spectrometer, a second mass analyzer. And this mass analyzer can be anything, can be a time of flight, an ion trap. You could in principle use a quadrupole, but it's not that often used. Could be an orbit trap, could be a free transform instrument. And the idea here is that all of these fragments are then analyzed, just like as if you were in the normal stage of mass spectrometry as we've seen before. And the detector, of course, detects the fragments at the end. Now you see why they call this MSMS or tandem MS because you have one stage followed by another stage of mass spectrometry and they're in series. And this is why we call this tandem mass spectrometry MSMS, MS squared. Now why would we do this? The reason why we do this is because our objective is to identify the analyte that actually came into the mass spectrometry in the first place. Now we could try and do that by its intact mass. So suppose we have a protein, we have used strips into digested into smaller bits which are called peptides. And these peptides fly into the mass spectrometer. Now we can determine the mass in a single stage of mass spectrometry as we've seen in the previous lectures. We can determine the masses or the mass of a charge rather as you will know of these individual peptides. But you can liken it to a room full of people, say a room full of students and you measure the weight of each of these students. The question is, will we have sufficient information? Just knowing the weight plus minus a certain error of our set of scales that we use, will it be enough to uniquely identify every student and say a 60 student classroom? And that is actually going to be very difficult. So one way that we could fix that problem, of course it's difficult because many people could have the same body weight and in fact, this is likely to happen. So the solution might be to not look at the intact body weight alone. Imagine that we would weigh somebody and then, and here's the morbid bit, we would then have to chop them into little pieces. We could weigh the arms, the head, the torso and the legs separately and then we could use the combination of the intact body mass together with the masses of the individual body parts we get after cutting them into fragments. And that combination is likely to be unique because two people who have the same body weight may not have the same weight of arms, legs, torso or head. What you can also imagine is if we're going to go through with this very gruesome experiment and we would have all the students come in at once and we would chop them all up at once, we will have a giant mess to take care of. It would be very difficult to find out which parts belong to whom. It would be possible, but it would be tricky. So what we do is we put a bouncer at the door and we only let one student in at the time. And that way we know that the fragments that we obtain come from a given individual. So of course, don't try this at home. It's a really gruesome example. But hopefully it will help you understand what is going on here because we do exactly the same. Instead of letting all the peptides into the fragmentation chamber at once, we use a bouncer like a quadrupole or an iron trap to keep everybody else out, only let one type of iron in, that type of iron will then be fragmented into its constituent parts and then we measure all the fragments. And since we know which kind of mass we limited to, we know what the intact mass is with a certain amount of error because that's the mass that we let through and we know of all the fragments. And so the combination of the fragments and the intact mass should be enough to uniquely identify a particular analyte. This is the reason why we do all of this. Now, mind you, modern approach is called data-independent acquisition methods of which SWATH is probably the most prominent example, but also what this is, MS2, MS2, the ESORI method does this, actually gets rid of the iron selector by and large or sets a really wide iron selection window and then does fragment multiple ions at the same time. Obviously the corollary there is that the fragments you get are no longer attributable easily to a single precursor analyte. And so what has to happen there is some kind of trick in the software, either in the interpretation or in the pre-processing of the data to try and disentangle this mass and say these fragments are likely to come from this precursor or to interpret the very messy spectrum that you get of all the fragments jumbled together to try and get the right identifications out. These approaches are very new, they're very interesting and they're very actively being developed right now. But by and large, MS2 still is done with a selection and then fragmentation and fragment analysis. I've already told you that you can use the same kind of analyzers here as we use here. And so in fact, there's nothing new to learn there. The only thing is conceptually, we use these, this first analyzer as a selector. Now the key point in all of this is that we try to break peptides into fragments. And the thing is we have to understand how the peptides break because otherwise the fragments would be meaningless. So we have to have some kind of structural insight. So let's have a look at what happens. I've put here a peptide, a very small one. It has one, two, three, four amino acids. That's actually too small to identify typically, but for the sake of argument and for the clarity of the presentation, it will suffice. I've whimsically put two CH2 groups here. That's just to change the picture a bit, but ultimately this can be anything. It could be a glycine as well. Remember, we have the amino terminus of the peptide and here we have the carboxyl terminus of the peptide. And then in between we have the peptide bond. You will remember the peptide bond. This is the carboxyl group of the first residue that has paired with the amino group of the second residue and creating an amide bond, which is known as the peptide bond because all peptides consist of a series of amide bonds. I've also told you that this is a pretty weak bond. That's important because it will now come become clear why that is important. So we can in fact break here. And if we break between the central carbon atom of an amino acid and its carboxyl group, the bit that still contains the amino terminus of the original peptide is called an A ion. Now we can also break here between the carboxyl group of the first residue and the amine group of the second residue. And this is the amide bond or peptide bond. And if that breaks the fragment that contains the amino terminus is called B. Now you can almost, I hope, guess what the name will be of the bond of the ion that we get when we break this bond between the central carbon of the second residue and the amine nitrogen of that residue. That bond, if it breaks, will generate a C ion. So A, B and C for this ion, this ion and this ion. If you know your Roman alphabet, you know exactly where the nomenclature comes from, it's quite simple. Now, these ions are generated with the maturen terminus. However, we can do exactly the same kind of fragmentation but then counting from the C terminus onwards. So we can break between the central carbon of the fourth residue and its own amine group. This ion is similar to this ion or this breakage is similar to this one. This ion that now contains the carboxyl group of the original peptide, so it is the tail of the original peptide, is called a Z ion. The next one down is called Y ion and that's the result of the breakage of the peptide bond or the amide bond. So this is the carboxyl group of residue three and the amine group of residue four. And then finally, we can also break between the carboxyl group of the third residue and its central carbon atom and that is called X. It's very simple, you've got A, B, C for the ones that contain the N-termini, X, Y, Z for the ones that contain the carboxyl group and of course it's repetitive because when we add other ions, you will see that we can break here, here or here and then we repeat. So we get another A ion, it's the central carbon and the carboxyl is always A. The peptide bond is always B and the central carbon and the amine group is always C and then A, B, C again and of course this is the first one, so A1. This will be the second one, so it's A2. This will be the third one, A3. Okay, it's a simple counting. Now something else that you may have noticed is that a Z ion creates this, of course this tail Z ion, but it also creates on the other hand this really big C ion. When you break a certain bond, you always get two resulting fragments. One will be C terminal and the other necessarily will be N terminal. So we call these complementary ions. If you were to take these two fragments and you would glue them back together again, you would again get the original peptide. Together they should thus constitute the peptide mass again. It's quite important in some approaches that you can figure out which ones of these are, which ones of the ions in the fragmentation spectrum are complementary. Now the question is which of these ions are we most likely to see when we fragment a peptide? Now it depends a little bit on the fragmentation method and we'll get into that in some more detail later, but you should remember again that this is the weakest bond, okay? It kind of makes sense because we can use a reasonably gruesome analogy again. The gruesome analogies I apologize for, but they're quite useful in making you remember things. Imagine that this is a person, each amino acid is a person and the people make a peptide by holding hands. So they make a chain of humans and they all hold hands. The peptide bond is comparable to two people holding hands. Whereas the bond between the central carbon atom and the amino group can be seen as the right shoulder and the bond between the central carbon and the carboxyl group can be seen as a left shoulder. So what is most likely to break when we start pushing people or moving people very strongly, we're holding hands. Obviously, we will probably not lose our arms very quickly because this attachment is much stronger than the attachment of the hand holding. And this is exactly what we see here. This is a weak bond that is easily split. This is a strong bond that's much harder to split. This is a good thing, otherwise our amino acids would just fall apart and we would have a very hard time building peptides. So keep that in mind about the energy. The last note is the nomenclature. This particular nomenclature with ABC XYZ is called Beeman nomenclature, but it was originally published by Rupestar for full month. So we should actually call it Rupestar's nomenclature, but fair enough, history has its way of changing these things, I guess. Now, how do we actually get our fragments? So we talked about fragmentation being necessary because it allows us to uniquely identify peptide analytes. We talked about the structural effects that such fragmentation could have on the peptide backbone. So the different kinds of ions, ABC, XYZ. Now we're going to have a look at how we're going to go about breaking peptides. And what we do is we make a little chamber of horrors for peptides in two different ways. The first thing is the blind alley driving. So what is going to happen is we have a collision cell. This is a chamber that has a small electrical field over it. So a voltage differential. And you know why we have a voltage differential? It will accelerate the ions. It will move them faster through this cell. And of course, you can guess what's going to happen by the name, it's a collision cell. We fill it with a certain gas, a collision gas, which is either a noble gas atom, like xenon or argon, or it is molecular nitrogen. So N2, the reason for this is we want something that is inert. It should not do any chemistry to our analytes. We don't want the analytes to be modified in any way. And both the noble gases and molecular nitrogen are essentially inert chemically and so they will not react with our sample. They will just be there in this room and form obstacles. Now, here is our selected peptide. Remember we selected it, right? There's a mass analyzer here that only lets a particular mass over charge ratio through. So the sample is simple. This peptide is now going to enter this chamber. It's going to feel the voltage differential and it's going to start moving faster and faster to the opposite side. As it does so, it's going to smash into multiple of our gas molecules. And every time it smashes into a molecule, it essentially gains energy, okay? And this energy is going to be translated into vibrations. And the vibrations between the atoms are going to spread out over the backbone of the peptides and they're going to amplify each other. So each time there is a collision, you get more energy into the system and more energy and more energy. And this energy floats freely, you can imagine it like that, floats freely, vibrates freely throughout the molecular structure until the energy is sufficient to break the weakest bonded encounters. And this is important. The weakest bonds, as we have seen before, are where the amino acids are holding hands. This is the peptide bond or the amide bond. So very often, these ions will ultimately, after a few collisions, break apart according to the peptide bond. And this yields B and Y ions. So in this kind of collision-induced dissociation, CID, you can see where the name comes from, the collisions induce the dissociation. We do expect B and Y ions predominantly. A spectrum of that looks like this. It's essentially mass over charge and intensity. And then there's peaks everywhere. And some of these peaks are noise, but a lot of these peaks will be derived from the individual masses or mass over charges of fragment ions that were obtained after fragmenting a peptide. Now, that's not the only way we do this. The second style, it's very similar. We also have a fragmentation cell, but it's no longer called a collision cell. This is sniper alley. So what happens here is we have, instead of neutral atoms or molecules, we now have electrons. And electrons are the basic element of chemical reactions. Chemical reactions revolve solely around electrons. And this is what we're going to use to our advantage. Our sample ion is going to come in. It's selected, of course, for a certain mass over charge as before. And it's going to enter this fragmentation cell and now there are going to be electrons everywhere. And these electrons are going to be attracted to our positively charged analyte ions. As soon as the electron hits the ion, it's like a bullet, a high-velocity bullet, is going to hit it, and that spot is going to break apart. There is now one electron too many, and this creates an immediate breakage of the bond. This is very different from the collision-induced association. The collision-induced association is a build-up process over several iterations of bumps into our collision gas, and slowly the energy spreads and finds the weakest point. With a technical term, this is called an ergodic process. So the energy has time to spread out and find the weakest point. This is not. Here, the electron hits, and the point of impact is the point of breakage. So there is no time for the electron to wander around the structure. It's going to break the point it hits. It's a non-ergodic process. So while the actual spectrum looks very much the same, in fact, here I'm being facetious, I used exactly the same spectrum, it's indistinguishable. At first sight, it has a very different type of fragmentation because it just so happens that this kind of fragmentation, which is called ETD electron transfer dissociation or electron capture dissociation, there are slightly different methods to accomplish the same thing. It depends on the instrument you use. These methods, this method will yield C and Z ions. So this is a different type of ion that we get. So these peaks, they're usually not B or Y ions anymore, they're C or Z ions. So you have to interpret the spectrum in a very different way. You have to calculate the theoretical ions in a different way. Now the unfortunate bit about this is that it works best for high charged peptides. And you remember that we talked about charges being very important because if an ion does not have a charge and becomes a normal uncharged molecule, the mass spectrometer cannot analyze it anymore. If your peptide would have a single positive charge, but it gets hit by one electron, now the single positive charge is negated by the electron negative charge and the net charge becomes zero. So if you have a singly charged ion, it will simply disappear off the radar as soon as it's hit by the electron. That's why this is typically better for highly charged ions and also they are more efficient in attracting the electrons. There is something very, very special about this method though and that is the non ergodic aspect. And now we have to take a little sidestep. Remember the analogy of holding hands, the amino acids hold hands. Some of these amino acids can be modified with post-translational modifications. The most common one is phosphorylation. Now imagine that phosphorylation is like somebody wearing a hat in this chain of people. If you're going to rock this chain of people, the first thing that is going to fall off is the hat. It's going to fall off before they even let go of the hands. And so the phosphorylation and collision-induced dissociation is very often lost almost immediately. With ETD or ECD, this non ergodic process, if the bullet strikes anywhere, the hat doesn't have time to come off and the hat will stay on. And so this is a method that is used for post-translational modifications a lot because it maintains the post-translational modification on the residue in the peptide intact even during fragmentation. This is where this method shines. The other thing where it's quite useful is for intact proteins because they have a high charge and they're very amenable to this type of fragmentation. A final thing I'll say about it is that the fragmentation procedure is slightly less efficient and it is harder to get a good coverage of all the possible fragment ions along the backbone. As a result, what most people do is they actually enhance the fragmentation and they use a kind of mixture. They use ETD or ECD to fragment as the primary mechanism but at the same time, and this is often done in trap instruments, in the trap they increase the energy by switching the voltages to high, so by setting the voltages higher and the switch will then create more violent back and forth motion and this more violent back and forth motion leads to collisions between the peptides and generates Y ions and B ions to some extent as well. So in those cases, you get a mixture here of C, Z, B and Y ions and that gives you better coverage but it makes for a slightly more difficult to interpret spectra. Nevertheless, this is quite popular. With these two methods, we have seen the two ways in which you can break a peptide into bits. We'll not talk about the interpretation of these spectra because that is a completely different topic and it's about proteomics informatics but it's quite important to realize how this happens and what goes on. In the next lecture then, which will be the last one, we'll talk about some concepts behind how CID works. We'll primarily focus on CID and I forgot to mention this but there's also it's very closely related sibling HCD. The HCD stands for Higher Energy Collision Induced Dissociation. It is a very similar mechanism but a higher energy is used there. It has certain consequences, mostly that the B ions are less prevalent. So it's more Y ion centric, although usually you will find the B2 possibly the B3 ion as well. But we'll talk more about how these things actually fragment in our CID slash HCD fragmentation primer, which will conclude our basic mass procurement lecture. See you next time.