Ordering Cleaved Fragments

in this video, we're gonna talk about ordering cleaved fragments and how that even relates to Edmund Degradation sequencing. So, from our previous lesson videos, we know that Edmund 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, much larger than 50 amino acid residues, are gonna need to be cleaved down into smaller peptide fragments before we can sequence them with Edmund 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 Edmund degradation. And so you can imagine if we have a large protein, we're gonna need to cleave it down into smaller peptide fragments, separate and then sequence each of those peptide fragments separately via Edmund 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 its asking. How do we determine the order of the fragments in the original protein sequence and so down below in this image, we're gonna help clarify exactly what we're even talking about. And so notice in this diagram, we start with this original protein up here, we make our way across to the right of the diagram, and then we start with step two 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 seven amino acid residues in this original protein. But we know that most proteins in nature are much, much larger and have several 100 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 hundreds or even thousands. And so if we want to determine the sequence of this large original protein here, we know that we're gonna need to first fragment it down into smaller peptide fragments so that we can sequence it with Edmund degradation. And that's exactly what this first step is it's to cleave are protein into fragments. And we've talked about many different protein cleavage techniques, including chemical cleavage and Proteus is or peptide aces. 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 to die Peptides, fragments and one try peptide fragment and notice that they still have these question marks. So after fragmenting our protein into fragments, we're gonna need to separate those protein fragments and then sequence each of them via Edmund degradation. And that's exactly what step Number two is down below. So notice that we're gonna separate each of these fragments over here, and then we're going to sequence each of the fragments separately via Edmund degradation and notice that we've changed all of these question marks into actual one letter amino acid coz 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 three is. It's what is the order of the fragments in the original protein in terms of being 1st, 2nd or third from the end terminal to the C terminal end. And so at this point, what we're gonna do is a little bit of experimentation. So what that means is we're gonna 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 third 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 Pepto, um, 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 FLN for the second one. And then maybe this G y m fragment came third. So let's put that in a different color over here. G Y m. So maybe this is a possibility. But we have to remember that we cleaved our peptide are protein, our original protein with cyanogen bromide and cyanogen bromide cleaves next to meth I ning residues on the car boxes side. And so that means that this Matthias residue here, this peptide bond on the car boxes 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 myth, I Any residue doesn't have a peptide bond on its car boxes side, since it's literally the last residue off the peptide. And so that means that if this were the sequence, we would generate one die peptide and then one Penta peptide upon cleavage with cyanogen bromide. But that's not the results that we actually got up above. We got three different peptide fragments to die peptides and one shrike peptide. 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 Onley way that we're able to generate three 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. Um, it's I don't really expect you guys toe 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 three fragments just because of House I engine, bro. Mike leaves. And so we know that the FL fragment is going to be the third, the last fragment at the very end, for sure. Otherwise we wouldn't get three fragments. So now the question is, does the RM fragment come first or does the RM fragment come second and does the G m y the G Y M 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 G Y M fragment must be coming second. So let's put in the G Y m fragment over here and then in the second possibility, we have the G Y M fragment coming first and that must mean that the RM fragment over here 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 clean with cyanogen bromide noticed that they're gonna generate three different fragments. So it will cleave after the thinning residue here and then also after the meth I inning residue here and that will generate to die peptides and one try peptide just like what we saw up above. So, possibility number one is a valid possibility. And then looking at Possibility number two, it can cleave after this myth einen residue over here. And it will cleave after this myth einen residue right here and so that will generate to die peptides and one try peptide just like what we have appear. So this sequence is different than the sequence possibility from number one, and they're both valid sequences that seem to generate the same exact peptide fragments. So the question is asking, Can you guys tell which sequences correct? And the answer is Actually, no. You cannot tell which sequences correct. So what this means is that possibility Number one is equally is valid as possibility number two. And there's really no way for us to be able to determine this sequence. Which of these sequences is correct with the provided amount of information? And so that actually presents a new issue that prevents that presents a problem that we have. And the problem is that by cleaving a protein with Onley one cleavage, uh, method one cleavage methods such like we did up above here with cyanogen bromide that it's possible that or it's ah, 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 Cleve with one, uh, cleavage method, and so what? That means is typically in most scenarios. We're gonna 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 to orm or different cleavage techniques are required. Thio. Order the fragments properly and determine which of these possibilities is actually the correct possibility. And so this here concludes our introduction to ordering cleaved fragments and, um, how it relates to Edmund Degradation sequencing. And again, we'll be able to get some MAWR practice as we move along and our next video so I'll see you guys in those videos.

So in our last lesson video, we presented the problem that if we treat our protein with Onley one cleavage technique, then it may not be possible toe order the fragments and to determine the sequence of our protein. And that's why typically, ah, minimum of at least two 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 re agents, it will generate different peptide fragments. And so those different peptide fragments we can actually align all of those different prag 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. And so 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 and so 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 two, and notice that it generates two different sets of peptide fragments. And then and step three over here we are essentially ordering all an overlapping the cleaved fragments from both of these, uh, cleavage techniques in order to determine the original protein sequence down below. And so, which will notice is that with this first box up here, this original protein, we're treating it with our first cleavage technique. Cleavage technique number one, which is with cyanogen bromide. And it generates these particular fragments that have shown these three fragments to die peptides and one try peptide, and you'll notice that these air the same exact fragments that air 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 two using a different re agent. This time, we're using chemo trips in. And so it generates all of these fragments here a die peptide ah, free loosen residue. Ah, free loosen amino acid. And then a tetra peptides shown here. And so in step number three 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 toe overlap in order the fragments. But for now, just to give you guys a quick little glimpse and insight to the strategy that we're gonna use, typically we're going to start with the longest fragment. And so the longest fragment is this tetra peptide here amongst all of the fragments. So this is the Tetra peptide. And so all we need to do is recognize that this tetra peptide here is the same one. Is this one over here so we can fill that in so notice it's m rmf so we can put an M r m f. And then what we can do is we can look for overlapping fragments, So notice that if we check this other fragment over here the RM that it overlaps perfectly with the RM here. So this RM fragment can fill in for the RM up here and then which will notice is that the three af from, uh, this fragment right here we'll overlap with the f from this fragment over here so we can put in fl down below right here because of the overlap. And then what you'll see is that we have an M here that also needs the overlap. And that overlaps with the M from the G g Y m fragment so we can put in G Y m. And of course, what you'll see is that we've got a g y fragment over here with the yellow that we can fill in so we can put in the G y here. And then. Of course, the loan losing over here will overlap with losing over here. And so, essentially, what we mean by these overlaps are these vertical overlap. So notice we have a glazing here confirming the end terminal amino acid residue down below in our original protein sequence as being glazing and so we can put glazing right here. And, uh, that is our first residue. Now moving on to the next set of overlap, we have the Tyra scene, which is confirming the second residue. So the overlapping fragments confirm a Tyra seen here and then applying the same strategy. What we have is overlapping Matthiasson in confirming the third residue asthma Thony. So we can put in Matthiasson in here and then what we have next is Argentine and then we have meth inning following that so we can put in Argentine and meth une And then, of course, what we have remaining our final Allan E and losing and so we can put in final Alan ing and losing. And so recall that when we used this cleavage technique all alone that we actually had to valid possibilities that we were not able to determine the order of these fragments. And that's exactly what we did in our previous lesson video. But when we introduce the second cleavage technique and we order the the overlapping fragments from these fragments generated from that cleavage technique with these fragments generated from a different cleavage technique that were actually able to determine the original protein sequence which we've confirmed down below here and so essentially, what we're saying is again that to orm or so two plus cleavage technique methods, cleavage methods are needed to order the fragments and reveal the sequence of the protein. And so again, we're gonna talk about an actual strategy for how toe overlap in order the fragments in our next lesson video. But before we get there, let's get a little bit of practice, so I'll see you guys in that practice video.