Mass Spectrum - Video Tutorials & Practice Problems
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Mass Spectrum
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So now that we've reviewed the basics of mass spectrometry, in this video, we're gonna talk about a mass spectrum. So a mass spectrum is literally just a plot of the mass spectrometry data or the mass spec data, and really just plots the relative abundance on the y axis of the plot and the mzratio on the x axis of the plot. And so if we take a look at our example down below on the left hand side, what we have is a mass spectrum being shown, where indeed, on the y axis, we have the relative abundance, and on the x axis we have the mz ratio. And throughout our mass spectrum, we have a bunch of these different peaks being shown. And we know from our previous lesson video that most of these peaks correspond with smaller peptide fragments that result from cleavage of our original peptide peptide at only one peptide bond. Now, later in our course, we're going to talk more details about how a peptide actually fragments during mass spectrometry. But before we get there, I want you guys to know that a mass spectrum can actually be analyzed to reveal the primary protein structure. And recall that the primary protein structure is the amino acid composition, or the number and types of amino acids, as well as the amino acid sequence or the order of amino acids from the n terminal end of a protein to the c terminal end of a protein. And so the way that amino acids are identified in a mass spectrum is by looking at the mzratio differences between the appropriate sets of peaks. And so let's take a look at our example down below to clear up this idea of how a mass spectrum can potentially be analyzed to reveal the primary protein structure. And so notice that above our mass spectrum, what we have is a pentapeptide being shown, or a peptide with 5 amino acid residues represented by these 5 different squares. And we know that in general, proteins are considered from their n terminal ends to their c terminal ends. And we also know that most of the peptide molecules in, mass spectrometry are going to be fragmented at only one peptide bond. But it is possible for some of the molecules of our peptide to get away with not fragmenting at all. And so if our peptide does not fragment, that means that all of the peptide bonds are gonna remain intact, and all of the amino acid residues are gonna remain intact as a single unit. And this single unit is gonna have its largest possible mass, and the largest possible mass has the largest m z ratios, and the largest m z ratios show up furthest to the right of our spectrum. And so this peak here in light blue actually represents our unfragmented pentapeptide. And so the reason that this peak is blue is because the leading end terminal residue is also blue. Now even though some of the peptide molecules can go unfragmented, we know again again that most of the peptide molecules in mass spectrometry are going to be fragmented at only one peptide bond. So let's imagine that this peptide bond here is being fragmented. Well, essentially what's gonna happen is this residue here on the end is going to be cleaved off, and this fragment here is going to be detected by the mass spectrum. And so this fragment here is gonna have the next largest MZ ratio, and it's gonna correspond with this highlighted peak shown there. And so really, the only difference between this yellow peak here and the original blue peak of the unfragmented peptide is the, mass of this one residue here. And so if we take the mass difference, the delta mass between these two peaks right here, which comes out to 99.08, that will give us the mass of this one residue that was cleared off. And so looking at our chart over here, we have amino acids, listed on the left, and then the molecular weight of the residue in gram per mole listed on the right. And notice that, the difference in mass between these two peaks came out to 99.08. And analyzing all of these molecular weights, notice that it matches the molecular weight of unique molecular weights. And so a molecular weight of 99.08 corresponds with a valine amino acid residue. And so that means that this blue, residue up here is a valine residue. And we were able to obtain that just by imagining the cleavage of this one peptide bond. Now let's imagine that a not this peptide bond, but perhaps a different peptide bond is cleaved. So let's imagine that this peptide bond is cleaved this time. Well, essentially, what's gonna happen is this entire fragment here is gonna be cleaved off and the only part that's gonna be remaining here is this fragment right here which corresponds with the next largest, Mz ratio peak, which would be this green peak right here. And so essentially, the only difference between, this green peak and this yellow peak is the loss of just this one, this one residue here. The only difference between, these two peaks is the loss of just this one pink peak here. And so essentially, what we can see is that the difference in mass is 137.18. And looking at our chart, notice that it matches with the molecular mass of asparagine, 137.18. And so we can put asparagine's 3 letter code here. And asparagine would be this residue. And so you can see here how we're literally most of the amino acids have unique most of the amino acids have unique molecular weights. And so you can kinda see how the pattern is working for this. So if we were to continue this, essentially what we would do is cleave this peptide bond, we would cleave off this fragment, and we would get this fragment here showing up as this peak right here. We would take the difference between these two peaks, and the difference between these two peaks comes out to 71. Point 8, which matches with alanine 71.08. And so, essentially, what we can do is put in alanine here and up here. So we're going to continue to analyze all of these residues. And, of course, if we were to cleave this last peptide bond here, we would fragment we would release this fragment. This one would be detected right here. And so we can take the differences between these two peaks to reveal this amino acid, and that comes out to 114 0.08, which is a histidine. So we can put histidine here and histidine up here. And you guys are getting the pattern of this, so essentially what you can see is that this last residue here is going to be all by itself as a single peak. And so its mass is literally going to be determined by the difference between the 0 mark on the x axis and the peak itself. And so that comes out to a difference of mass of 57.08, which matches with the mass of a glycine residue here. So we can put glycine here, and glycine as our last residue. So now, what you might may or may not have noticed is that literally, our mass spectrum spectrum from the right to the left in order to reveal our sequence from the N terminal end to the C terminal end. And so that's actually one of the major points that I want you guys to take from this video. And so, what I want you guys to know is that mass spectra are typically analyzed from the right to the left of the, the spectrum to reveal the peptide sequence. And so again, we're starting here, it starts at valine to reveal the n terminal sequence, and then we continue to read it from the right to the left to reveal our sequence from n terminal to c terminal. So the reason for why we need to do that, we're actually gonna talk about in another lesson video a little bit later in our course. And again, for now, I just want you guys to know that we typically analyze spectrums from the right to the left. Now, one of the actual limitations of mass spectrometry is that it actually struggles to differentiate leucine from isoleucine. And the reason for that is because leucine and isoleucine are isomers of one another, and that means that they have the same exact chemical atoms, just arranged in a different format. And so because they have the same chemical atoms, they have the same exact mass. And so, when we have a mass difference, it could correspond to either isoleucine or leucine, and mass spectrometry cannot differentiate between them. And so that's actual limitation of mass spectrometry. So this here concludes our initial introduction of a mass spectrum, and again, in our next lesson video, we'll be able to talk more about how a peptide actually fragments during mass spectrometry. But before we get there, let's get a little bit of practice. So I'll see you guys in our practice video.
2
Problem
Problem
Use the mass spectrum below to determine the sequence of the peptide.
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concept
Mass Spectrum
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Video transcript
So now that we understand mass spectra a little bit better and we know that we can use them to reveal a protein's primary structure, in this video, we're gonna talk about how how a protein actually fragments during mass spectrometry. And we're going to define b and y ions and how they contribute to the mass spectrometry spectra. So we already know that mass spectrometry ionization fragments most of our protein molecules only one time at a single peptide bond. And so it turns out that when a peptide bond is actually fragmented during mass spectrometry ionization, it actually leads to the generation of 2 prominent sets of ions, and those 2 prominent sets of ions are the b ions and the y ions. Now the b ions always contain the n terminal amino acid residue, and those b ions are read from the left of our spectrum to the right of our spectrum, so from left to the right. Now the y ions on the other hand, they always contain the c terminal amino acid residue and those peaks are always read from the right of our spectrum to the left of our spectrum, so in this direction here. And so remember that that's the same direction that we tend to analyze our spectrum to reveal the sequence of our protein. And so what that tells us is that the y ions here must be key for revealing the sequence of our protein. Now another way that helps me distinguish between b and y ions is that because b is the second letter of the alphabet, it's towards the beginning of the alphabet. And so b ions always contain the beginning amino acid residue of our protein or the end terminal amino acid residue of our protein. And they're always read from the beginning of our spectrum to the end of our spectrum, so from left to right. Now for the y ions, because y is the second to last letter of the alphabet towards the end of the alphabet. That helps me remember that y ions always contain the amino acid residue on the end of our protein, or the c terminal, end of our protein. And those y ion peaks are read from the end of our spectrum to the beginning of our spectrum, and so they're read from right to left. And so if you can remember that, that'll help you distinguish between the b and the y ions. So let's take a look at our example down below to clear up this idea of b and y ions. And so, what you'll notice is that we have the same exact pentapeptide from our previous lesson video. And we know that during mass spectrometry ionization, most of our protein molecules are going to be fragmented. And so you can see that we have fragmentation as our first step here. And we know that when that fragmentation occurs, most of the molecules are only going to be fragmented one time at a single peptide bond. And when that single peptide bond is fragmented, it's going to generate 2 prominent sets of ions. And so let's take a look at our example and imagine that this peptide bond here is the one that's being cleaved. Well, essentially, that's gonna generate 2 prominent sets of ions, b ion and a y ion. And so the b ion, remember, always contains the beginning amino acid residue or the end terminal amino acid residue. And so that fragment is going to be this entire fragment here, and that's the b ion that's generated from this single peptide bond that's broken. Now the y ion always contains the amino acid residue at the end or the c terminal residue. And so that's gonna be this residue right here. And so down below, you can see that we have the same exact, fragmentation here. You can see that we have the Y ion here, and we have the B ion over here. Now, through the color coding here, we can see that this, y ion that's generated from this peptide bond fragmentation is just gonna be glycine, so we can put in gl y here. And because this y ion only has one amino acid residue, it's called the y one fragment. And because the b ion has 4 amino acid residues in it, it's called the b 4 fragment. And so you can essentially see that by fragmenting this one peptide bond, we generated these 2, ions, b ion and y ion. And that was just from cleaving this one peptide bond that's being shown, here. And so, what you can see is that if we were to cleave different peptide bond, say it wasn't this peptide bond, instead it was this peptide bond that was cleaved. Well, essentially, we're still gonna get 2 prominent sets of ions, the b ion with the n terminal amino acid residue, and the y ion with the c terminal amino amino acid residue. And that's exactly what we see down below, the y ion and the b ion over here. So we can fill in the residues that are missing. So the blue corresponds with valines, the pink with histidines, and the green with alanines. So, you can see the pattern here, and you can see that we need to fill in the blanks for the rest of these fragmentations. So if we were to fragment our next peptide bond over here, we would get valine and histidine right here. And then, for the y ions, we would get alanine, asparagine, and glycine. So we can fill those in, alanine, asparagine, and glycine. And then, of course, for our last peptide bond that gets fragmented, we're going to get, the, b ion of just a valine shown here, and then we're going to get the y ion of this entire fragment here, so we can fill that in as well. So we have histidine, alanine, asparagine, and glycine. All right. So now we've generated all of the B and Y ion fragments that could result from breakage of any of these peptide bonds. And so because b ions and y ions are both ions, they both have a charge on them, and they're both gonna be deflected in an electric field, and they're gonna be detected by the detector and show up on a mass spectrum. And so it's important to note that, intermixed, so down below here, intermixed b and y ions can actually show up on a mass spectrum. But the y ions are actually more stable than the b ions, and they're structurally more stable because the b ions are very short lived and they tend to fragment further into other ions. But the y ions, because they're more stable, they, tend to have higher intensity and abundance, and they're often going to be the most prominent peaks in the spectrum. And so essentially, because they are the most prominent peaks in the spectrum, we tend to have a preference to analyze the y ions in a spectrum. And so because we have that preference, the b ions ions over here, essentially, we can cross off since we're not really gonna be analyzing their analyzing them very much moving forward. But the y ions, again, because they're the more prominent, peaks in the spectrum, these are the ones that we're typically gonna focus on in a mass spectrum. And so you can see that in our mass spectrum up here, you can see that we only have y ions being shown, and so you can see the y one ion, is being shown here. We have the y two ion being shown here, the y three ion showing here, and the y four ion showing right here. And then, of course, the y five ion is going to be the unfragmented ion that is the original peptide. And so that's something important to keep in mind that, these y ions are gonna be the most prominent peaks. Now, again, this, spectrum is only showing the y ions, but in a real spectrum of a protein, there is going to be intermixed b and y ions showing. So there would be some b ions that would be smaller, less prominent peaks if this were an actual spectrum. So that's another important factor to keep in mind. And so because we focus on the y ions and the y ions are read from right to left, from the right to the left of our spectrum, what this means is that in most cases, it is safe to assume that the mass spectrum is analyzing the y ions and is read from right to left, especially moving forward in our course. And so, essentially what you can see is that, that is why, we read our spectrum from the right to the left to reveal our amino acid sequence. So this here concludes our lesson on the difference between B and Y ions, ions, and we'll be able to get some practice in our practice video. So I'll see you guys there.
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Problem
Problem
Upon fragmentation of a peptide bond during mass spectrometry, what ions can be detected on the spectrum?
A
b ions.
B
y ions.
C
b & y ions.
D
Ions are deflected but not detected.
5
Problem
Problem
Use the mass spectrum below & the indicated y-ion peaks (red) to reveal the sequence of the peptide.
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6m
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Problem
Problem
In your mass-spectrometry of a pure protein with an m/z of 1,582, you found peaks of y ions with the following m/z ratios of 1,582, 1396 and 1283. The mass in Daltons for the possible relevant amino acids are provided: Y (163), N (114), W (186), D (115), G (57), L (113) and M (131). From this data, it is obvious that the C-terminal amino acid residue of the 1,582 fragment is:
A
G.
B
L.
C
N.
D
W.
E
Can't be determined.
7
Problem
Problem
Use the mass spectrum below & the provided chart with amino acid masses to determine the sequence of a hexapeptide (6 amino acid residues). In the mass spectrum, y ion peaks are indicated with 'y' while b ion peaks are indicated with 'b.' The N-terminal residue is given as Leu and the C-terminal residue is given as Lys. Determine the remaining amino acid sequence using either the y ions or the b ions.