 Welcome to MOOC course on Introduction to Proteogenomics. Interpretation of mass spectrometry data is very challenging. There are many softwares which are commercially available or open source softwares which make our job relatively very easy. However, the basic knowledge of mass spectrometry data and its interpretation becomes very crucial. In the last lecture, you were introduced to the basic concepts of mass spectrometry and sample preparation. Today, we have a hands-on session which aims to provide some basic knowledge required for the manual interpretation of mass spectrometry data. Let us welcome Dr. Karl Klauser for today's hands-on session. Now, other than to say that we are going to try to assign fragment ion types and these are the things you should keep in mind for the various fragment ion types. One of the things I did not mention so far is that there are ammonium ions which give you composition information, but they do not give you stoichiometry. So, if you see a 120 ion in your spectrum, that is a good indication that you have phenyl alanine, but if you have a really tall 120, it does not mean you have two or three of them. So, you do not have the stoichiometry information. In order to form an ammonium ion, the fragmentation has to occur in two places along the peptide backbone and it includes the side chain that is unique to the amino acid. By the techniques that we are going to be using in the spectra that are being shown, the ammonium ions that are likely to be present are shown in green. The ones that are shown in black are essentially not, you can only form those if you have a high energy collision type of instrument, which is not the same as saying at HCD. So, actually from a terminology standpoint that is a distinction between the manufacturers. So, if you have a QTOF instrument that comes say from Psi X or from Agilent or Brooker, they might call that fragmentation technique CID. That fragmentation is exactly the same thing as what thermal calls HCD and the reason is that before thermo created the current instruments, they had ion trap fragmentation that was resonance excitation and they called that CID, ok, all right. So, they had to have another name for when they did it the same way as other people, ok, all right, next, ok. These are the amino acids. If you knew these by hand by heart, it would be easier for you to look at the mass differences if you had the opportunity to print them, that would be helpful. So, these are some things that is particularly helpful for you to know that we will make use of in interpreting the spectrum, ok. So, an A ion is 28 Daltons less than a B ion and if you can't decide whether an ion is an A or a B or a Y and if it has something that is 28 Daltons less, then you can probably call it a B, that is why it is useful, ok. All right, if you know your amino acid masses and you just add them all up and add one, that will give you the mass of the B ion. If you add up all your amino acids and add 19, that will give you the mass of the Y ion, ok. And then if you fragment between two amino acids, B ion is going to go one way, Y ion is going to go to the other. If you add them back together, you would have the whole peptide, right, ok. But when you fragment, so I am always going to talk about the prerent masses being singly charged, ok. The B ion is singly charged, the Y ion is singly charged. So, if you add the two together, you get the M H plus plus 1, ok. And those are complementary ions as shown up here. So, B ion plus Y ion is the same thing as M H plus plus 1, ok. Some software packages, they will strip out the charge, I think that is silly, ok. Because we measure ions, we do not measure masses, right. And furthermore if you, when you are dealing with high accuracy masses, you have to trust that they have built the, to subtract the right amount, because it is not 1.0, it is 1.0078 minus the mass of the electron, ok. And these things matter, right. So, I think it is easier just to think in terms of ions, ok. All right. So, I keep the charge, all right. Those are the useful things to know. What kind of spectrum would you call this? This would be on the quiz, come on, what, if you were to give it a cute little name, what kind of, you want to call it a white picket fence. I was going to call it a royal infield, ok, right. So, this is, this is a relatively beautiful spectrum. It is going to have, you can see it has got ions that span the mass range, they look nicely spaced, ok, all right. So, before you start doing some, some math, ok, so the M H plus is 1,000, ok. This right here is at 500, ok. So, if you look, you will see some nice symmetry. Lycine has a Y1 ion that is 147, ok. Arginine has a Y1 ion that is 175. You should now be able to tell me what the C-terminal amino acid of this peptide is, if you are looking at the spectrum, all right. So, if this is the, where the precursor mass is, and I, you can see that there is some symmetry, right. So, these, these two peaks are about equidistant from the center, and that, so that means they are probably a BY pair, ok. So, I am just going to do this kind of thing here, then I bet you if you look, where the red, where the red carrot is, ok. All right, and yet, so the other thing you can do to check, if you can't really tell the symmetry, you are going to add up the masses, 414 plus 587 should be equal to 1001, all right. So, similarly, we can go like this, ok, those are going to be symmetric, I already told you that if, if it is a tryptic peptide, Y1 is 175, which would be consistent with that being an arginine at the C-terminus, right, ok. And if you, that Y1 would be symmetric with 826, that means this should be a B-ion, ok. And then, there is probably a couple others in here, so 230 going all the way to 771, ok. So, you can see there is lots of symmetry present, all right. Now, let us do some, let us do some arithmetic. My advice, whenever you are going to start doing arithmetic is, and you have to choose where to start, make one of two choices. Start with a big peaks or start at an end, ok, I am going to go with big peaks, but and when you start at an end, it is helpful to, it is nice to have one that you already have an ion type for, one of the things I try to resist doing is starting at the low end, ok. Because there is more often going to be multiple things happening at below the precursor mass, you are going to have multiply charge things, there is going to be internal ion fragmentation, usually have a cleaner spectrum up at high mass, all right. So, now, let us just do some arithmetic, ok. So, this mass gap here is 101, no, 58, 13, 71, ok. This mass gap here is 113, ok, then 587 to 513 is 74, that is not an amino acid mass, 488 to 587 is 99, that is an amino acid mass, ok. 488 to 414 is 74, that is not an amino acid mass, ok. Let us go 826 to 727, that is 99, ok, 727 to 612 is 115, ok, 612 to 513 is 99, ok, 513 to 414 is 99, ok, all right, can we go 414 to 353, that is 71, right, 343 down to 230, 113, so now then I would go 175 to 274 is 99, ok, 115, all right, then can I go up here, 99, ok. If this is a Y ion, this should be a Y ion, this should be a Y ion, if this is a B, that should be B, B, B, B, ok, valine, oh, let us do one other thing, this is mass 100, 1000, sorry, can you read that, that is not a fake peak, we know that that is reasonable because we measured the precursor mass before we did the MSM spectrum, the precursor mass is 1000, ok, all right. So, and then if it is 1000 we can go from here with 129, precursor mass is like Y ion, it is like the last Y ion, ok. So, now we could write down the sequence and if we go with Y ions, we would write down the sequence backwards, ok, arginine already, 99 is valine, isolucine is 113, oh no, wait a minute, ok, what, lucine is 113, but yes, so when you do de novo sequencing, usually if we cannot tell, we just choose lucine and I think the simplest reason is that there are 6 codons for lucine and 3 codons for isolucine, so if you cannot tell, might as well go with a chance that it has got more codons, right, but at the end of the day, no, I cannot tell that it is lucine rather than isolucine, ok. 115 is aspartic acid, ok, 99 is valine, ok, where are we here, 71 is alanine, ok, 99, so there is a bunch of valines here, right, alanine, 115 is aspartic acid, that is valine and this is glutamic acid, 129 is E, ok. Alright, there is a whole lot of junk on the spectrum on the page now, but if I follow it carefully, this 99 would be valine and it would be aspartic acid and it would be another valine and then another valine and alanine, lucine and glutamic acid and now if we try to read off the BINs, let us see if we get the same thing, so we got lucine, no, lucine, alanine, valine, another valine, aspartic acid D and then valine and then we would go all the way to here in the arginine, so it looks like there is something that is not quite right, ok, so what do we do wrong, so I said E, L, A, V and oh is it going to be, I got it partly backwards huh, no V, D, V, D, V, V, A, L, E, ok, so the E, the E is not right, should be N, D and why is that, alright, the E, how did I have the, oh that is, so I cannot do my, bad math, ok, so this is, this is 229, alright, it is not 129, ok, so we need and that is right, that matches our, this B1 ion here, ok, so we have some combination of amino acids that adds up to 229, ok, ok and there is a couple of ways you can do that, you can do 115 plus 114 and that is D and N, ok, so if we now take a look at the way that this spectrum is labeled, we do not have fragmentation between N and D, so this would start at 230 and then this last gap here is 129, ok, so the sequences is complete with exception, we do not know the order of N and D, ok, so from a pure de novo standpoint, we do not know whether it is N, D or D, N, ok, but if we then add the requirement that this peptide has to come from the human proteome, turns out one of the, only one of those two choices is present, ok, right and so it is the N, D part, alright, so let us go to the next one, so this one is going to be easy because there is not very much to work with, alright, so you take a look at the spectrum, there is not a lot of symmetry, but there is, if you look at this, this is doubly charged, ok, 2 times 480 would give you 960, right, there is two charges on this, there is only one charge on this, alright, what else have we got, ok, this is 1220, is there any symmetry here, 262 to 959, that should be a BY pair I think, right, 221, ok and then the mass difference between these two is 26, is 28, alright, what is the ion type of the peak at 262, ok, this was the one of the things I gave you of useful information, the difference between a B ion and an A ion is 28, so we could assign that as A and that as a B ion, if this one is a B then that has to be Y, ok, what else can we do here, so then that would mean that this is Y doubly charged, ok, then if we take a look at this mass gap that is 97 and that mass difference is 25 is 87, ok, that is searing, proline, alright, alright, there is not a whole lot else here, so we do not have down to low mass, ok, but if we, so at some point there is some sequence here that is PS and then the distance here is something that adds up to 261 and the distance out to here would be 775, this is, this one comes from a triptychpeptide experiment and it, so there should be either an arginine or a lysine here, ok, that is about all the information there is in that spectrum, ok, there is the PS part, that is about all the information there was, that information all together is enough to produce that one answer as a peptide from the human proteome. In today's hands-on session you are introduced to the fine points of mass spectrometry based data interpretation, the spectra they are having all the information, but how to get the most relevant information, think about if you are locating even the post-translational modification such as phosphorylation or even glycosylation, the data interpretation becomes very crucial and very challenging. However, in today's hands-on session Dr. Klauser tried to explain you how to get the best interpretation from these complex spectra and even to glid into the PTM based analysis. As it was discussed in today's lecture, the B ions differ from the A ions by a mass of 28 Dertens. This observation helps in better understanding and differentiating various fragments. In the next session you will be provided with comprehensive information on interpretation of further more complex spectra. Thank you.