 Hello, so my name is Laneth Martens and I'm going to walk you through a tutorial about some mass spectrometry basics. For those of you who care, I'm located at VIB in Gent University in Belgium and my group is the Compomics group. We make a lot of software tools in the field and so we try to teach people how to use the tools, but it's very important to know how mass spectrometry works as well and that is the objective of this lecture. Now, before we start with the actual lecture first I'll do a small advertorial because I so happen to have written two books on the topic text books with some other people from Norway. One of them is computational methods for mass spectrometry based proteomics and the other one is computational and statistical methods for the specific application domain of quantitative proteomics. And so these two books obviously are a little bit biased. I make a very small amount of money for each of these books but they might be useful to you. They're real text books and they read like text books and they could be informative if you want to know more. Now enough of that, the concept of these tutorials is that we actually go through several sections and each section will be a separate tutorial film. So the first one will be about amino acids and proteins because we have to understand the things we're analyzing in order to go ahead with that. The second part will then be about ion sources of the mass spectrometers. The third one about the analyzers in mass spectrometers. The fourth one about the detectors. Now, there's two types of mass spectrometers that are quite different from these which are free transform ion cycles from resonance machines and orbitrap machines. They actually combine the analyzer and the detector, which is why I keep them for a later point after we've seen all the individual parts. Now, when we've summarized up all of these and these four lectures we will have gone through all the concepts and the components of a typical mass spectrometer. At that point, we will know about amino acids. We'll know about proteins. We know how they're analyzed in a mass spectrometer and all of the parts that function together. Then we'll talk about a more advanced topic, but it's crucial to proteomics and also metabolomics these days, which is tandem mass spectrometry. Also dubbed mass spectrometry, mass spectrometry, or mass spectrometry squared because essentially we're going to use two stages of mass spectrometry in sequence to analyze fragments of what was the original analyte. That sounds a bit complicated. That's why we dedicate one lecture to that. The final lecture will then be about what happens when we fragment these peptide ions and how we're going to expect the resulting spectra to look like. Once we get there, we've kind of covered everything you need to know for the basics of mass spectrometry and you would then be ready to continue looking at the tutorials about our tools. So as I said, one tutorial topic per film or movie, we'll start now with amino acids and proteins. So without further ado, let's have a look at these beasts. In life, amino acids are crucial. We all know about nucleic acids, how they make DNA and RNA, but ultimately the majority of the biochemical reactions in the cell are done by proteins and proteins are built out of amino acids. Now you can see immediately that amino acids are very different from nucleic acids because amino acids are chemically very distinct from each other. That is shown here in this chart of the 20 common amino acids that we share with all of life, from E. coli's over Archeobacteria, through yeast, through eukaryotic organisms of multicellular kind, to vertebrates, to humans. All life has these 20 amino acids. Here and there you find a few other strange ones, but these 20 are very common. They're practically everywhere and practically the whole of life is built only on these 20 building blocks. They're organized, as I was trying to tell you, in different groups based on their chemical properties. Now we're going to go into a lot more detail because this is quite important. And the main thing to carry home right now is that the different groups are really very chemically different. I'll give you a simple example. These positively charged amino acids here, like lysine and arginine, and to some extent histidine, are positively charged. Whereas we have the exact opposite as well, aspartate and glutamate are very strongly negatively charged. So we can already see they're very different. These guys are positive, the others are negative. This difference is because we want to have as broad a range as possible in chemical function and chemical nature, because that's, coupling all of that together, allows us to do different functions. And that's where you see these are effector molecules, that when you put them together in a certain way you can do all kinds of crazy chemistry. With amino acids, with nucleotides, this is a bit harder. Nucleotides tend to be all very similar in chemistry. Having said that, there are RNA molecules that do chemical reactions as well. Now let's have a little closer look at these chemical differences that are so important to sustain life as we know it. Let's start by having a look at the chemistry. Now the first thing you will see when you look at any amino acid is that they're all at the top have this carboxyl group, COO minus group. You see that? Every single one of these amino acids has this group. And this group is a very simple carboxylic acid and it gives half of the name of amino acid. Obviously it's the acid part of the amino acid. Now where does the amine come from? It comes from this group here, the NH3 plus group, that again every single amino acid has. And this is the amine amine group. And so we have amino acid. We have the two groups together and they constitute what makes an amino acid. We call this the backbone of the amino acid, especially when we combine it with this carbon atom here that joins the two together. So the backbone of every amino acid is the same. Now you may have noticed that this carboxyl group is always negative and this amine group is always positive and that's because they are very basic and acidic. And that they either lose a proton, so for the acidic one or gain a proton for the basic one at physiological pH. Now one plus and one minus makes a net of zero and that's important. We call this in chemistry a zwitterion and a zwitterion means that it's negative and positive charges even out. So the net result for the backbone is that it's non-charged. Okay plus one minus one makes zero. Okay so this is what they all have in common and why they are all defined as amino acids. What makes them special each individually is what is shown in pink which are all the different side chains and the side chains are really what distinguishes the different amino acids. So let's have a look at them. There's a very small one here glycine which only has a hydrogen as a side chain. It's the smallest amino acid and it's really really tiny. It's essentially a backbone. One step up is alanine which is a little bit bigger and it contains a methyl group and then you get what they call the non-polar sorry aliphatic amino acids in this group. The remainder are valine, leucine, methionine and isoleucine and they contain only non-charged apolar groups which means that they are they don't really like water. So they're hydrophobic and they're afraid of water and they like hydrophobic substances like oily substances to be in. So one good example of a biological system which very oily is a membrane. So a biological membrane is a natural habitat for these guys and you can see that they're very apolar because they have all these sorry very non-polar CH3 groups everywhere or CH2 groups which means that they really don't have any charges or any ways to bond with water easily. One other thing you might notice is methionine especially it has a sulfur atom which is one of the few amino acids. There are actually only two. Now as opposed to the non-polar aliphatic ones we have the polar uncharged ones. So they're polar which means that there actually is a little bit of a charge distribution within the molecules. The molecules are a little bit more positive towards one side or negative towards the other but they are not charged. So their net charge remains zero but there is some kind of polarization going on and it's very easy to see how this works because we have what are known as the alcohol-containing amino acids, serine with the alcohol here, threonine with the alcohol here and then cysteine which doesn't have an alcohol but a thiol which is the sulfur equivalent. So now we have our second sulfur-containing amino acid, we have methionine for the sulfur and cysteine for the sulfur. Cysteines you might know from cysteine cross links. So one cysteine can bond another cysteine and create a strong cross link in the structure of a given protein and so they are quite rare in biology because they have this very special function of creating cross links. Another thing that they do is do reductive chemistry like in glutathione for those of you who know about that. And then other members of this polar but uncharged group are glutamine again very clear why it's polar asparagine, you have the amine and you have the aldehyde here and then proline which is a very special amino acid because it folds back upon itself. So the the amine of the backbone is actually connected to the last atom in the side chain and this creates a five ring which is quite stable and proline is a bit weird because it has a strange bond angle. If you bind another amino acid as we will see later it actually will create a little nick in the in the flow of the of the backbone chain and that will actually change the structural properties. So if you're in a helix it will actually break the helix, it creates a special bond and it has a cis trans conformation so it can actually do this hinge function as well which is also used in some structural properties. Now these guys don't have a charge, there's one other group that doesn't have a charge and they're the aromats. The aromats all contain a benzene ring as you can see here for phenylalanine the same benzene ring here for tyrosine with a hydroxyl on it with an alcoholic side chain and then here tryptophan which is a really big one that has the aromats and then a five ring. Now these guys obviously they all have a benzene ring so they're very hydrophobic but it also gives them a few special properties which are particularly relevant to some kinds of mass spectrometry I will talk about that later so keep that in mind. These are also quite large in volume so they take up a lot of space and tryptophan 2 is quite rare because it's so voluminous and because it's water-hating it doesn't tend to be on the outside it has to be on the inside and so within the protein there has to be sufficient space to accommodate this big boy and so if that space is not there it's very difficult to mutate one in because it will start pushing the structure apart. Interestingly there's only one codon in the standard genetic code that codes for tryptophan so you can see again it's rare it needs only one codon it's not that important to have redundancy there. Now that sums up here all the uncharged amino acids the charged ones are also very interesting and these are either positively or negatively charged as I showed you before and the negatively charged ones are aspartate and glutamate and quite simply put they have a carboxyl group at the end of their side chain which makes them negatively charged. The positively charged ones they come in three varieties two seriously positively charged ones and one with the possibility of getting positively charged so lysine is quite simple it has a long aliphatic chain just like these guys of methyl groups and then finally it has a amine a free amine at the end so that amine is very similar to that amine on the backbone and this particular amine is of course charged then. Arginine ups the ante it has a shorter aliphatic chain and then it has what is known as a huanidinium group which has three amine groups and these two amine groups can actually share the double bond and the positive charge so you can imagine that there's a resonance structure where this double bond resonates between these two which actually spreads out the positive charge which makes it more happy more easily accommodating when it comes to receiving this positive charge and we'll come back to that. Histidine finally is also positive because this nitrogen here can take on an additional proton and as it does that it becomes positively charged so it can become positively charged but it's quite a bit less basic than these two in real life this is used a lot in enzymes where it fulfills in the active center the role of a proton acceptor so it's a temporary place to put a proton while you're doing a reaction and then later on get the proton back and put it in a new location so this is much more convenient for enzymatic reactions because it's so easy to give it a proton and then take it back taking one back from arginine is very hard taking it one back from lysine is also pretty hard right so that sums up our overview of the amino acids let's see how this chemistry translates into a bunch of different numbers that we can work with in chemistry so that's this table here where you can see all of the names of the amino acids then you have the three letter code which summarizes their name in a slightly more convenient format and then finally and this is used a lot so you should learn how to read this the single letter code it's a little bit tricky because for instance alanine is a as you would expect but arginine is r which maybe you don't expect and asparagine weirdly is n to make it difficult aspartate or aspartic acid is then d so it's a it's a bit tricky to learn this nomenclature but it's very worthwhile because most people in the field use only exclusively the single letter notation for brevity now the first thing that we see here is that they all have different weights obviously tryptophan is going to be much heavier than glycine however there is one notable exception here which are isoleucine and lucine because they have exactly the same mass it's 131.18 dolpons now how can this be it's quite simple let's have a look at their structures lucine has one two three four carbons in the side chain and so does isoleucine one two three four carbons and then it has two three six nine hydrogens in the side chain and this one has isoleucine has three six nine so they have exactly the same chemical composition they are not exactly the same however because this methyl group is at a different location than there so essentially this methyl group when you swap it down one you get lucine they're different in structure they're different in volume they behave differently in the protein but they have exactly the same mass so a mass spectrometer cannot easily tell them apart it's the only notable exception all the other amino acids have a clearly distinguishable mass in principle we have therefore a molecular formula which is just a number of carbons and hydrogens oxygens and sometimes sulfurs that are there we have a residue formula we'll come back to the residue later and the residue weight again we'll see where that comes from later and then we have four very interesting variables that we really need to understand in order to understand how we do proteomics today there's the p k a p k b p k x and m p i now these three the p k's they all belong together they're all about the same thing and p i is something slightly different so let's start with the three p k's you may remember from chemistry that the p stands for minus log so it's a 10 base logarithm transformation of some value or other and what value is being transformed is the k which is an equilibrium constant now the equilibrium constant is defined by the subscript lowercase letter that follows it in this case it's a lowercase a and it stands for acid so here we have an acid equilibrium constant and here we have a b so if a is for acid then b will be for base now let's have a look what could the acid be and what could the base be well when we look at our structures we have an acid for every amino acid and we have a base for every amino acid as well so the actual p k a refers to the acidity of this carboxyl group and the lower this is the easier it is for for this particular group to be deprotonated at a neutral pH and we see that most of these numbers are between one and a half and two and a half roughly which means that they're actually quite acidic so if you put them at a pH of three which is already quite acidic most of these will still be very strongly deprotonated so this carboxyl group in physiological pH which is a bit more than seven will tend to be completely dissociated in COO minus and NH plus and that's why we write COO minus okay now another thing that you should notice is that there is a difference between the different amino acids because on the face of it each of these groups is the same right but they're not exactly the same because they do have a different side chain attached to them and that difference in side chain really can make a difference on the chemistry of this particular group and you see the effect where for instance tryptophan here has a very low acidity constant whereas the other ones let's take one that has a very low one here we have for instance aspartic acid and aspartic acids where are we here that carboxyl group actually deprotonates much more readily than the one for typtophan so there is an effect of the side chain on the acidity of the backbone carboxyl group we see exactly the same thing for the basicity so where would that come from well obviously we have only one clear candidate which is the amine group on the backbone and here we see that this is usually between nine-ish this one is 880 but nine-ish and 10 although here we have one at 1060 and so essentially at a pH of seven again there will be fully protonated and fully protonated means that of course will be NH3 plus which means that there will be positively charged now let's look at the exceptions we have 880 which is asparagine so asparagine is particularly low in basicity so it's very hard to accept the proton there and which ones are very high in basicity particularly proline so let's have a look at proline again proline here remember is this circular amino acid that folded back on itself and we here have this NH2 that is very happy to accept an additional proton that is because at NH2 it's already over protonated and this will have repercussions on the way that peptides fragment later on so keep that in mind proline is a bit special its end terminus is very positive very easily made positive you may have also noticed if you've been paying careful attention to this table that this minus here for pyroglutamic acid is actually telling you that there is no acidity or basicity what is pyroglutamic acid it's also a circular amino acid and it's what happens when you take glutamine and you fold it back onto its own backbone so it becomes similar to proline but in this particular case there's not going to be any basicity or acidity left or it has not been measured at least this is relevant because pyroglutamic acid is encountered a lot in mass spectrometry it's a standard artifact that happens when this amino acid is at the end terminus of a peptide when it's at the head it can fold back on itself now that's a chemical modification of a standard amino acid so it's not really a standard amino acid the final value that we have is pkx so pka was this carboxyl group the pkb was this amino group so now we are left with the side chain and as you can see a lot of side chains don't actually have a pkx that means that they don't act as bases or as acids so if we look here for instance in non-polar aliphatic groups a molecule like isoleucine from this pink highlighted part is not going to lose an amino a proton anytime soon it's also not going to gain one they they are not acting as bases or as acids on the other hand arginine and lysine should pick up a proton very easily and aspartate and glutamate should give it away very easily so let's have a look at the table again when we look up aspartate there we go aspartic acids you will see that the pkx is 3.6 so it does actually dissociate into a proton and the remainder of the molecule the negatively charged carboxyl side chain group at a very low pH already it is less acidic though than the backbone so on the on the structure this carboxyl group is not as acidic as this one okay there's a slight difference when we look at the positively charged amino acids that's lysine and arginine predominantly let's have a look lysine has a pkx of 10.5 which means that it's quite basic it's quite ready to accept that proton mind you 10.5 is very close to what we saw here remember 10.6 which was for proline so the proline and a amino group here is very similar in elasticity to the lysine side chain that will become important later on as well when we look then at asparagine finally you will see that asparagine has 12.5 pkb which means that it's much more basic than lysine how much more basic is it well you remember that the p stands for minus log and a 10 base logarithm if you go two units on a 10 base logarithm you're a hundred fold more basic so arginine is a hundred fold more basic more ready to accept the proton than lysine again this will become somewhat important later on so we've got all the pk's covered now so the pkab and x which are essentially basisity or acidity equilibrium constants proton donor proton acceptor for these different amino acids backbone carboxylic group backbone amine and side chain whatever amine or carboxylic group the final column then is the pi the isoelectric point so what is the isoelectric point it is defined as the point at which a particular amino acid obtains this particular pH I'm sorry it's a particular pH with a particular amino acid or peptides or protein achieves a net charge of zero isoelectric means charged zero and the point is defined as a pH unit this is very important because we use pH to separate proteins or peptides the way it works is very simple you can create a small plastic strip and on the small plastic strip you put a polymer and the polymer has an embedded pH gradient so it starts at a low pH and it ends at a high pH and as you go through this polymer the pH changes it's a polymer because that locks the pH in place the next thing you do is you apply a voltage onto this and of course any charged molecule like a peptide that you now put in this in this polymer in solution it's going to feel the electrical field as all ions do and it's going to be attracted by the opposite pole so if you put all the peptides in the solution where it's negative at the basic or at the acidic side so at the acidic side it tends to be negative and then you put the positive pole on the other side all these negative molecules are going to migrate towards the positive pole what is going to happen is as they migrate they're going to move through this pH gradient and at some point the peptide or protein is going to find the pH at which it's net charge will be zero as soon as the net charge is zero the molecule precipitate it will stop feeling the electrical field it will become blind and impervious to the electrical field so this is very nice since a lot of peptides and proteins will have different pH points at which they reach a zero charge depending on the pis of the amino acids they're composed of they will migrate different distances through this gradient it's a great way to separate peptides or proteins through isoelectric focusing and that depends primarily on the isoelectric point we won't come across the isoelectric point inside the mass spectrometer anymore we will come across the pkab and x and especially the pkx is very important as well as the pkb the pka not so much so keep in mind that we have this difference so we've essentially seen three key differences that help us in proteomics we have mass differences we have differences in acidity or basicity and we have the differences in isoelectric point for the separation prior to mass spectrometry okay so now we know a lot about amino acids let's see how we take these amino acids and join them together into a peptide or protein this is done via a bond that is known as an amide bond or a peptide bond because of course peptides are built from this and what happens is you have a single amino acid in this case it's uncharged just for clarity reasons but of course in physiological pH you would have NH3 plus and COO minus here I've put NH2 and COOH that has the amine group the carboxyl group the backbone carbon and then the side shape so for glycine this would be this would be H for alanine this would be this would be CH3 and so forth and so forth okay then we have another amino acid whatever that may be with its own amine central carbon and carboxyl group backbone what is going to happen is this amine group and that carboxyl group are going to make an amide bond splitting off water so you can essentially visualize it as if you would split this off and then bind these two together what you get is you get an amide bond as shown here in the blue highlight where you have a carbon group that comes from the carboxyl of the first amino acid bond to the amine group of the second amino acid and this bond between the carbon and the nitrogen of the two different amino acids is what's known as a peptide bond or amide bond this bond is actually quite weak you know this bond because it's actually present in a lot of light drinks so if you have a soft drink that is sweetened with the sweetener aspartame aspartame is none other than aspartate coupled to phenyl alanine now when you look at this particular type of drink it will say that you cannot store this in sunlight and you should keep it away from warmth or heat the reason for that is very simple in sunlight which contains UV radiation this bond can easily be broken by the energy in the light so what is going to happen then you're going to break the aspartate away from the phenyl alanine and the result is not sweet anymore in fact it's going to taste horrible so you're going to completely ruin the taste of your drink it's one of the great reasons to keep light drinks out of the sunshine and heat has the same effect because this bond is very weak remember now the other thing you will read on these labels is that drinks with aspartame are not suited for people with phenyl ketonyria which is a very annoying metabolic disorder that prohibits the people who suffer from it from metabolizing correctly phenyl alanine so the phenyl alanine leads to to illness and in fact is going to attack your nervous system at some point so in order to make sure that they don't get an overdose of phenyl alanine which for these people is very very quickly they cannot eat anything with aspartame the reason for this being your stomach is acidic and acid is going to dissolve this bond again it's very weak so acid will attack it your intestine is basic and again basis will also dissolve this bond so it's very certain that aspartame will split into aspartate and phenyl alanine and then the phenyl alanine is going to make these people ill so they cannot at all drink or eat anything that contains aspartame so the take-home message here is that is a very weak bond thank god it's a weak bond because we can that way split peptides or proteins into smaller chunks by cleaving them easily after we've done using after we're done using them so you can break proteins apart you can make them very easily it's a simple bond to click things together amino acids together and remove them again so if we do that in a sufficient number what we get is something like this we get a polypeptide backbone and the polypeptide backbone starts with the first amino acid remember this was our first amino acid which still has the original free amine because there's nothing before it so this is still the free amine of the original first amino acid and then you have the carboxyl of that amino acid bond to the amine of the second amino acid which gives us the amide or peptide bond now then we get the side chain of the second amino acid we get this carboxyl the amine of the third one amide bond again and so forth and so forth so we keep doing this until we come to the end or c-terminus so this is convention by the way the end terminus is the start or the head of the peptide or protein whereas the carboxyl group is the tail or end of it and that still has the original carboxyl group of the original last amino acid as you can see that leaves for any amino acids that's somewhere in the middle of this chain it leaves only the N the H the carbon and the oxygen so it has lost an NH bond so one hydrogen is gone and here we see that it loses an OH so one OH is gone and one hydrogen is gone together of course they make H2O or water and that is what you see on the previous table where we talk about residue formula you will see that they have subtracted H2O from the formula and that the residue weight is the weight of the original amino acid minus the weight of two hydrogens and one oxygen H2O so this residue weight applies to every amino acid in this polypeptide chain even the first and the last although we have to keep in mind that we then need to add one hydrogen here if it's uncharged two hydrogens if it's charged and we have to add an OH here because they still remain there so this is residue plus OH residue plus one of two hydrogens and this is how you write out a protein sequence how it's built out of the individual components of amino acids so with that we're done with the introduction we now know about amino acids we know about polypeptides or proteins how they are made from these amino acids and the different properties of the different amino acids that contribute to this diversity in proteins and that we can then measure in a mass spectrum in an experiment so the next lectures will be about mass spectrometers how they are built which components they have and we'll kick it off by talking about iron sources that will be the next lecture this sums up this lecture thank you very much for watching