Ordering Cleaved Fragments: Study with Video Lessons, Practice Problems & Examples

To determine the sequence of a protein, it is essential to cleave it into smaller peptide fragments using techniques like Edman degradation. However, using a single cleavage method may not reveal the correct order of fragments. Employing at least two different cleavage techniques allows for overlapping peptide fragments, which helps reconstruct the original protein sequence. This process highlights the importance of understanding peptide bonds and the role of specific reagents, such as cyanogen bromide and chymotrypsin, in generating distinct fragments for analysis.

Ordering Cleaved Fragments

In this video, we're going to talk about ordering cleaved fragments and how that even relates to Edman degradation sequencing. So from our previous lesson videos, we know that Edman degradation sequencing is limited to sequencing small peptides with less than 50 amino acid residues, which means that most proteins in nature, which are much larger than 50 amino acid residues, are going to need to be cleaved down into smaller peptide fragments before we can sequence them with Edman degradation. And so after fragmenting a large protein down into smaller peptide fragments, we know that we need to separate those peptide fragments and then sequence each of them separately via Edman degradation. And so you can imagine if we have a large protein, we're going to need to cleave it down into smaller peptide fragments, separate and then sequence each of those peptide fragments separately via Edman degradation. But then there's a question that arises and that is, how do we determine the order of these peptide fragments in the original protein? And so that's exactly what this question here is asking. It's asking how do we determine the order of the fragments in the original protein sequence. And so down below in this image, we're going to help across to the right of the diagram, and then we start with step 2 down below and move our way to the right again. And so, with this original protein here, notice what it has are question marks inside of the amino acid residues, which means that it has an unknown sequence. And just for, limitation on the amount of space I have on this page, I only have 7 amino acid residues in this original protein. But we know that most proteins in nature are much larger and have several hundreds to several thousands of amino acid residues. So I want you guys to imagine that this original protein here has many, many more amino acids, several 100 or even 1000. And so if we want to determine the sequence of this large original protein here, we know that we're going to need to first fragment it down into smaller peptide fragments so that we can sequence it with Edman degradation. And that's exactly what this first step is. It's to cleave our protein into fragments. And we've talked about many different protein cleavage techniques including chemical cleavage and proteases or peptidases. And so here we're using chemical cleavage with cyanogen bromide. And we know that once we take our original protein and fragment it down, that it's going to generate a bunch of protein or peptide fragments here. And so we have 2 dipeptide fragments and one tripeptide fragment. Notice that they still have these question marks. So after fragmenting our protein into fragments, we're going to need to separate those protein fragments and then sequence each of them via Edman degradation. And that's exactly what step number 2 is down below. So notice that we're going to separate each of these fragments over here, and then we are going to sequence each of the fragments separately via Edman degradation. And notice that we've changed all of these question marks into actual one-letter amino acid codes because we've revealed the sequence of the fragments. So now, this is exactly where this question comes into play. How do we determine the order of these fragments in the original protein sequence up above? So maybe this fragment here showed up at the very beginning, but maybe it showed up at the very end, or maybe it showed up over here. How do we determine exactly the order that these fragments came in? And so that's exactly what step number 3 is. It's what the order of the fragments is. It's what is the order of the fragments in the original protein in terms of being first, second, or third from the N terminal to the C terminal end. And so at this point, what we're going to do is a little bit of experimentation. So what that means is we're going to fill in these blanks here, but then later on, we might change them. So keep that in mind as we move along. And so, maybe the order of these fragments is pretty easy. Maybe it's literally in this exact order, where this is the first, fragment, and this FL here is the second fragment, and maybe this third fragment here is the 3rd fragment. I don't know. Let's check. So let's say that that is one possibility, and that seems to be what maybe this is trying to tell us over here, where here we have our original peptide, our original sequence, and we're trying to determine the possibilities for our original sequence. So maybe this RM fragment came first, just like what we said. And maybe this FL fragment came second. So, let's go ahead and put FL in for the second one. And then maybe this GYM fragment came 3rd. So, let's put that in a different color over here, GYM. So, maybe this is a possibility, but we have to remember that we cleaved our peptide, our protein, our original protein with cyanogen bromide, and cyanogen bromide cleaves next to methionine residues on the carboxyl side. And so that means that this methionine residue here, this peptide bond on the carboxyl side of it is going to be cleaved. And so if this were the original sequence, essentially this peptide bond would be cleaved. And this methionine residue doesn't have a peptide bond on its carboxyl side since it's literally the last residue of the peptide. And so that means that if this were the sequence, we would generate 1 dipeptide and then 1 pentapeptide upon cleavage with cyanogen bromide. But that's not the results that we actually got up above. We got 3 different peptide fragments, 2 dipeptides and one tripeptide. And so, it turns out that this here is not the correct sequence. So we can go ahead and get rid of that, and this is not the correct order over here. And so it turns out that the only way that we are able to generate 3 different fragments in this order is if the FL fragment is actually at the very end. And so if you don't understand that, it's okay. It's I don't really expect you guys to fully understand this process yet, but it turns out that this FL needs to go at the end. If it goes anywhere else, we won't be able to generate 3 fragments just because of how cyanogen bromide cleaves. And so we know that the FL fragment is going to be the 3rd, the last fragment at the very end, for sure. Otherwise, we wouldn't get 3 fragments. So now the question is, does the RM fragment come first, or does the RM fragment come second? And does the GYM fragment come first, or does it come second? And so notice over here with this first possibility that we have the RM fragment coming first. And so, if the RM fragment is coming first, that means that the GYM fragment must be coming second. And so, what we can do is put in the RM fragment over here. And so it turns out that both of these are valid possibilities because if we cleave with cyanogen bromide, notice that they are going to generate 3 different fragments. So it will cleave after the methionine residue here, and then also after the methionine dipeptides, and one tripeptide, just like what we saw up above. So possibility number 1 is a valid possibility. And then, looking at possibility number 2, it can cleave after this methionine residue here, and it will cleave after this methionine residue right here. And so that will generate 2 dipeptides and one tripeptide, just like what we have up here. So this sequence is different than the sequence possibility from number 1, and they are both valid sequences that seem to generate the same exact peptide fragments. So the question is asking, can you guys tell which sequence is correct? And the answer is actually no. You cannot tell which sequence is correct. So, what this means is, which of these sequences is correct with the provided amount of information. And so that actually presents an issue, that presents a problem that we have. And the problem is that by cleaving a protein with only one cleavage method, like we did up above here with cyanogen bromide, it's possible that the proper ordering of the fragments may not be possible. So it's possible that we may not be able to determine what the correct sequence is if we only cleave with 1 cleavage method. And so what that means is, typically in most scenarios, we're going to need to take our original protein and cleave it in multiple different ways. And we'll be able to understand that idea better when we get to our next video where we'll talk about how 2 or more different cleavage techniques are required to order the fragments properly and determine which of these possibilities is actually the correct possibility. And so, this here concludes our introduction to ordering cleave fragments, and how it relates to Edman degradation sequencing. And again, we'll be able to get some more practice as we move along in our next video. So I'll see you guys in those videos.

Ordering Cleaved Fragments

So in our last lesson video, we presented the problem that if we treat our protein with only one cleavage technique, then it may not be possible to order the fragments and to determine the sequence of our protein. And that's why typically a minimum of at least 2 different cleavage techniques are required in order to properly order the fragments. And so the reason it works like this is because when we treat the same protein separately with different cleavage techniques or different reagents, it will generate different peptide fragments. And so those different peptide fragments, we can actually align all of these different peptide fragments so that the overlapping peptide fragments will actually reveal the original order of the fragments and the actual sequence of the original protein.

Let's take a look at our example down below so that we can better understand this overlapping idea and the ordering of the cleaved fragments. Notice in our diagram what we have in the first box up here is one particular cleavage technique on our original protein, and then down below, in the second box here, we have the same exact original protein as up above, except we're treating it with a different cleavage technique, cleavage technique number 2. Notice that it generates 2 different sets of peptide fragments. Then, in step 3 over here, we are essentially ordering all and overlapping the cleaved fragments from both of these cleavage techniques in order to determine the original protein sequence down below.

Notice in this first box up here, this original protein, we are treating it with our first cleavage technique, cleavage technique number 1, which is with cyanogen bromide, and it generates these particular fragments that are shown: these three fragments, 2 dipeptides and one tripeptide. And you'll notice that these are the same exact fragments that are generated from our previous lesson video in the example. And now, down below here, we're treating the same exact original protein with a second cleavage technique, cleavage technique number 2, using a different reagent. This time, we're using chymotrypsin. And it generates all of these fragments here: a dipeptide, a free leucine residue, a free leucine amino acid, and then a tetrapeptide shown here.

In step number 3, we're seeing how we can overlap and order the fragments in order to reveal the original protein sequence. Now in our next lesson video, we're going to talk about an actual strategy for how to overlap and order the fragments. But for now, just to give you guys a quick little glimpse and insight into the strategy that we're going to use, typically we're going to start with the longest fragment. So the longest fragment is this tetrapeptide here amongst all of the fragments. So this is the tetrapeptide, and all we need to do is recognize that this tetrapeptide here is the same one as this one over here. We can fill that in. So notice it's MRMF, so we can put in MRMF. Then what we can do is look for overlapping fragments. Notice that if we check this other fragment over here, the RM, that it overlaps perfectly with the RM here. This RM fragment can fill in for the RM up here. And what you'll notice is that the F from this fragment right here will overlap with the F from this fragment over here. We can put in FL down below right here because of the overlap. And what you'll see is that we have an M here that also needs to overlap, and it overlaps with the M from the GIGYM fragment. We can put in GYM. And, of course, what you'll see is that we've got a GY fragment over here with the yellow that we can fill in. So we can put in the GY here. And then, of course, the lone leucine over here will overlap with the leucine down below. Essentially what we mean by these overlaps are these vertical overlaps. Notice we have a glycine here confirming the N-terminal amino acid residue down below in our original protein sequence as being glycine. So we can put glycine right here, and that is our first residue. Now moving on to the next set of overlap, we have Tyrosine which is confirming the second residue. So the overlapping fragments confirm a tyrosine here. And then applying the same strategy, what we have is overlapping methionine, confirming the 3rd residue as methionine, so we can put in methionine here. And then, what we have next is arginine, and then we have methionine following that, so we can put in arginine and methionine. And then of course, what we have remaining are phenylalanine and leucine. So we can put in phenylalanine and leucine.

Recall that when we use this cleavage technique all alone, we actually had 2 valid possibilities that we were not able to determine the order of these fragments, order the overlapping fragments from the order the overlapping fragments from these, fragments generated from that cleavage technique, with these fragments generated from a different cleavage technique, that we're actually able to determine the original protein sequence, which we've confirmed down below here. Essentially what we're saying again is that 2 or more cleavage techniques, cleavage methods, are needed to order the fragments and reveal the sequence of the protein. Again, we're going to talk about an actual strategy for how to overlap and order the fragments in our next lesson video. But before we get there, let's get a little bit of practice. I'll see you guys in that practice video.

Overlap, align & order the following peptide fragments to reveal the sequence of the original protein.

Fragments from cleavage method #1: 

Fragments from cleavage method #2: 

So this practice problem wants us to overlap, align, and order the following peptide fragments to reveal the sequence of the original protein. And what you'll notice is we have the fragments from the first cleavage technique shown on this line here, and then down below, we have the fragments from the second cleavage technique shown here. And so, in our next lesson video, we're going to talk about more detailed fragments. But for now, we can pretty much eyeball it and just look for these overlaps. And so, we're going to start here with this largest fragment here, and what you'll notice is that this glycine isoleucine arginine portion matches perfectly with the glycine isoleucine arginine from up above. And so essentially what we can do is we can take this fragment right here that's circled in red from fragment 2 and overlap it with the fragment from number 1. And so all we need to do is rewrite this down below so that we can see this overlap a little more clearly. So let's rewrite this red fragment right here. So we've got Leucine, Tryptophan, Glycine, Isoleucine, Arginine and that is it for this red fragment. And then for the blue fragment up above, notice that it overlaps with the glycine, isoleucine, and arginine portion here. And so essentially, when we have an overlap that's confirmed, And so now we're looking for a leucine tryptophan to overlap with. And what you'll notice is that the leucine tryptophan on this portion right here actually overlaps with the leucine tryptophan from the fragment up above. And so essentially, all we need to do is overlap these two fragments. So we can take this blue here and just overlap. So we can have our leucine tryptophan here, and it's attached to a lysine. And so essentially what we have is we've got the leucine tryptophan overlapped, and so now this entire red fragment is done, but now we have this overhanging lysine that needs an overlap, that would be this lysine here. And so what you'll see is that there's a lysine down here that this lysine can overlap with. And so all we can do, what we can do is we can take this red fragment from down here and overlap it with this lysine. So, we can essentially write this in backwards. So we've got the lysine from here and then, we've got, let's see, the phenylalanine is coming next. So essentially just writing it backwards like this. Then we have methionine coming next and glycine coming next. And so now that we have an overlap for this lysine here, we can get rid of the lysine up here and we can get rid of the lysine down here. And so the only part that's left now is glycine, methionine, phenylalanine, which matches the glycine phenylalanine fragment from up above. So we've got the red fragment already drawn in, so all we need to do is overlap this blue fragment that's left. So we've got glycine, methionine, and phenylalanine. And so now we have all of the fragment pieces overlapped. So we finished this piece and we've also finished this piece here. And so this is really the sequence of the original peptide. These overlaps vertically are telling us the sequence. So the sequence is going to be in this order, glycine, methionine, phenylalanine, lysine, leucine, tryptophan, glycine, isoleucine, arginine. And so the answer to this practice problem is this highlighted green portion here. And so that concludes this practice problem, and in our next video, we're going to be able to get an actual more detailed strategy for how to overlap these fragments. So I'll see you guys in that video.

Here’s what students ask on this topic:

What is Edman degradation and how is it used in protein sequencing?

Edman degradation is a method used to sequence amino acids in a peptide. It involves selectively removing the N-terminal amino acid of the peptide, which is then identified through chromatography or electrophoresis. This process is repeated to determine the sequence of amino acids one at a time. However, Edman degradation is limited to peptides with fewer than 50 amino acids, necessitating the cleavage of larger proteins into smaller fragments before sequencing.

Why are multiple cleavage techniques necessary for determining the sequence of a protein?

Using multiple cleavage techniques is essential because a single cleavage method may not provide enough information to accurately order the peptide fragments. Different cleavage reagents generate distinct sets of fragments, which can be overlapped to reveal the original protein sequence. This overlapping helps to confirm the order of fragments and ensures a more accurate reconstruction of the protein's primary structure.

How does cyanogen bromide function in protein cleavage?

Cyanogen bromide (CNBr) is a chemical reagent used to cleave proteins at methionine residues. It specifically targets the carboxyl side of methionine, breaking the peptide bond and generating smaller peptide fragments. This specificity makes CNBr a valuable tool in protein sequencing, as it allows for predictable and reproducible cleavage patterns.

What role does chymotrypsin play in protein sequencing?

Chymotrypsin is a protease enzyme that cleaves peptide bonds on the carboxyl side of aromatic amino acids such as phenylalanine, tyrosine, and tryptophan. In protein sequencing, chymotrypsin is used as a secondary cleavage technique to generate a different set of peptide fragments. These fragments can be overlapped with those generated by other cleavage methods, such as cyanogen bromide, to accurately determine the protein's sequence.

What is the importance of overlapping peptide fragments in protein sequencing?

Overlapping peptide fragments are crucial in protein sequencing because they help to accurately reconstruct the original protein sequence. By aligning fragments generated from different cleavage techniques, researchers can identify overlapping regions, which confirm the order of amino acids. This method ensures that the sequence is determined correctly, even when individual cleavage methods alone are insufficient.