Strategy for Ordering Cleaved Fragments

in this video, we're gonna talk about a strategy for ordering cleaved fragments. So it turns out that there's actually multiple strategies toe order, cleaved fragments. So if you already have your own strategy that works for you, then fantastic. You've got nothing to worry about. But if you don't have a strategy and you're looking for one, here's a solid strategy that I came up with that's proven to be effective and involves Onley five different steps. And so the first step step number one in our strategy for ordering cleaved fragments is to just scan the problem for helpful clues that reveal either composition or sequence information about our protein. And so what we'll find is that our professors like to give us these paragraphs style word problems with all of these sentences and hidden within the sentences. There are clues, and so the clues could be the use of F, d, N B, or hydrazine or some other chemical that we already covered in our previous videos. And so that's a really good first step is to collect all of the clues from our practice problem. Now, after we do that in the second step, all we need to do is recall which specific peptide bonds the re agent recognizes for cleavage, or that the re agent Cleaves and so in our example down below will be able to apply our first two steps and our strategy for ordering cleaved fragments. And so, in the example problem, it says, a peptide with 17 amino acids is treated with trips in to give three different peptide fragments and then separately, treated with chemo trips in to give four peptide fragments. And so looking down below, which will notice is in the left hand chart. We have the trips and fragments, and there are three trips and fragments and then in the right hand chart. We have the chemo trips and fragments, and there are these four chemo trips and fragments Joan Below. And so the example. Problem continues to say, identify the sequence of the 17 amino acid residues in the original starting peptide. And so, in order to do this, we're going to need toe order all of these cleaved fragments here so we can apply our strategy for ordering Cleef fragments. And in our first step step number one, we're going to scan the problem for helpful clues. Now, looking at our problem, there's not a lot of helpful clues, like using chemicals such as F, D, N, B or hydrazine, but notice that it does tell us our protein, or peptide, has 17 amino acids, which reveals some of the amino acid composition there. And so that's going to be important moving forward. And so essentially that is it for step number one, Since, uh, there's not really a lot of helpful clues, so step number one is complete. We can give it a check now moving on to Step number two. Notice that there's a step number two for the trips and fragments, and there's also a step number two for the chemo trips and fragments. And so in step number two, all we need to do is recall which specific peptide bonds the re agent recognizes for cleavage and so recall that trips and does it split in after a night sword. So it cleaves the C terminal peptide bonds of lysine and arginine, amino acid residues and Kimo trips in the younger brother of trips, and also Cleaves the C terminal peptide bonds. But it has a preference for which amino acids it recognizes for cleavage and the pneumonic that helps us memorize. That is just free your worries like May. And so we know that the l and the M here the losing and man thinning are cleaved at a much slower rate over longer periods of time. And so, since there is no indication in our problem that losing and thinning are actually gonna be cleaved here, then we're going to assume that Onley the preferred amino acid residues of these aromatic residues Final Alan, entire scene and trip to fan are going to be cleaved. And so, essentially, what we could do is say that trips and is going to cleave final Alimi tire scene and trip to fan residues. And essentially, that is it for step number two. So we can go ahead and give step number two a check over here and step number two a check over here. And so now that we're done with step number two and our next video, we'll move on to step three in our strategy for ordering clean fragments. So see you guys there

All right. So now that we've covered the first two steps in our strategy for ordering cleave fragments in this video, we're gonna focus on our third step and in step number three will want to identify either the N Terminal or the C terminal peptide fragments Now, Usually, these peptide fragments are pretty easy to identify because they usually do not have terminal amino acid residues that the re agent recognizes for cleavage. And so we'll be able to see an example of this down below and our example now another component of step number three is that will want to check if identified. Terminal fragments from different cleavage re agents actually match each other. And we'll want to do that just to make sure that we're ordering the fragments correctly and that we're on the right track and we'll be able to see an example of this component here. Step number three up above and our example problem when we get there. But first, let's cover this example down below, and in this example, it's important to note that if the re agent cleaves the end terminal peptide bond, then we'll want toe look to identify the end terminal amino acid fragment. And so, uh, for example, we know that Pepsi is a pep today's that cleaves the end terminal peptide bonds of fennel, Alan entire scene trip to fan and losing. And looking at this peptide down below notice that it only has two amino acid residues that peps and recognizes for cleavage. And those are highlighted and read throughout our image. And so we know that Peps in Cleaves, the End Terminal peptide bond and the N terminal peptide bond fragmentation is shown with these green lines here, next to the residues. And, of course, that fragments are peptide into three different fragments. We have this fragment here on the far left make shown down below. We have the Ferg fragment right here in the middle, and then we have the C terminal fragment on the end over here. And so what you'll notice is that of all of these peptide fragments and with terminal amino acid residues that peps and recognizes for cleavage and so you can see the red terminal residues that are being recognized by Pepsi in on all of the fragments, except for this one fragment over here, and that makes us fragment quite unique, and that uniqueness actually identifies this fragment as the end terminal fragment simply because it does not have terminal amino acid residues that Pepsi in recognizes for cleavage. And so here it's pretty easy to see that this fragment is the end terminal fragment. But even if we didn't have the original peptide sequence provided and we were on Lee given these peptide fragments below, we would still be able to identify this. Make a fragment as the n terminal fragment. Just because of this reasoning, where does not have terminal residues that peps and recognizes for cleavage? And so applying the same logic to re agents that cleave c terminal peptide bonds, Then we'll want to look to identify C terminal fragments. And so, for instance, we know that Trips in is a pep today's that does it split in after a night's ward. So it cleaves the C terminal peptide bonds of Licensing and our Ginny and looking at our peptide below, which is the same peptide that we had up above here, notice that there are three residues that trips and recognizes for cleavage, and that fragments are protein into four different fragments shown below. And so what you'll notice is that all of the fragments and with terminal amino acid residues that trips and recognizes for cleavage shown in the green highlights here and on Lee, one of the fragments does not have terminal amino acid residues that trips and recognizes for cleavage, and that makes it unique and easy to identify as the C terminal fragment. Simply because again, it does not end with terminal amino acid residues that trips and recognizes for cleavage. And so we'll be able to apply this, uh, concept here to our example problem up above. And so this is the same exact example problem from our previous video. And again, which will notice is that there's a step number three for the trips and fragments. And then there's also a step number three here for the chemo trips and fragments. And, of course, in step number three will want to identify either the end or the C terminal fragments. But because we previously identified and step number two that both of our re agents recognize the C terminal peptide bond, we know that we're going to look to identify the C terminal fragment and of course, if we take a look at our trips and fragments first. We know that trips and recognizes licensing and Argentine. And so notice that all of our fragments have a terminal amino acid residue of licensing and Argentine, except for one fragment. And that one fragment is pretty easily identified as the C terminal fragment and so we can go ahead and circle this fragment. Here is the C terminal fragment, simply because it does not have terminal amino acid residues that trips in recognizes for cleavage. And so we do the same for chemo trips. And we know that kind of trips and recognizes the aromatic amino acid residues. And so notice that we have aromatic amino acid residues on all of the fragments. Uh, terminal amino acid residues on all of the fragments, except for just one fragment, which is this fragment right here. And so that identifies this fragment as the C terminal fragment. And so recall that down below we said that there's another component to step number three where we'll want to check if the identified terminal fragments from different cleavage re agents actually match one another so that we can make sure we're on the right track. And so what you'll notice is we have this C terminal fragment from the trips and fragments, and we have this C terminal fragment from the chemo trips and fragments. And when we check to see if they match, noticed that we have availing Allen and Sistine with the trips and fragments. And we also have veiling Alan insisting with the chemo trips and fragments, and there is this overhang of a listen residue. But that's okay. And so because they have the same matching veiling Alan insisting fragments, that shows us that we're moving on the right track and that we've identified the C terminal fragments correctly. And so this concludes our video on step number three of our strategy for order include fragments, and in our next video, we'll be able to tackle out steps number four and steps number five. So I'll see you guys there

All right, So now that we've covered the first three steps in our Five Steps, strategy for order include fragments and this video. We're going to focus on the fourth and the fifth steps. And so, in step number four, we're going to use those terminal fragments that we identified back in step number three and overlapped those terminal fragments with other peptide fragments from different cleavage techniques. And we're gonna continue to do that until we overlap all of the peptide fragments and until the sequence of the original peptide has been revealed and we'll be able to apply Step number four up in our example problem above. Now what you'll notice is that step number five is really only here as a backup in case you guys happen to get stuck for whatever reason. Now, hopefully you guys won't get stuck. But it's always good to have a backup plan, just in case. And so, if for whatever reason you guys happen to get stuck, all you need to do is use the longest peptide fragment as a starting point for overlapping all of the other peptide fragments. Now, if there's ever a tie for the longest peptide fragment. All you need to do is pick one of them. Doesn't matter which one and continue toe overlap that peptide fragment with other peptide fragments until all of the peptide fragments have been overlapped and the sequence has been revealed. Now again, if you happen to get stuck, all you need to do is move onto the next largest fragment and again continue to do the same and overlap that peptide fragment with all of the peptide fragments until the original sequence has been revealed. So let's take a look at our example problem above so we can apply Step number four and five. And so what you'll see is with this practice or this example, problem with Step number four. All we need to do is overlap those terminal fragments that we identified back and step number three and essentially overlap them like a puzzle. And we do that until we reveal the sequence. And so recall in our previous video was Step number three that we identified this circle fragment here under the trips and fragments as the C terminal fragment, and we identified this fragment over here as the C terminal fragment for the kinda trips and fragments and so down below. We can rewrite these fragments so that we can see how they overlap and notice that we have the trips and fragments that we can provide in this green space up above. And we have the chemo trips and fragments that we can provide in the blue space. And then after we overlap the fragments down below, we can provide the sequence of the original peptide. And so again, we're going to start with the terminal fragments and we can start with the trips and fragment here, which is the C terminal fragment, veiling, aligning and Sistine. And so, essentially, what we can do is we can write that in here. So we've got 16 Al Inning and we've got veiling. So now we've got this veiling Alan ing and Sistine Fragment rewritten down below. And we can overlap with the other C terminal fragment under the chemo trips and, uh, fragments. And so essentially all we need to do is overlap. So we have Sistine here. We've got Alan. I mean, we've got veiling, and of course, we've got this lie scene that's hanging over, and so we also going to take that into account here. We've got this. Listen here. And so now what you'll see is that we've got this Sistine overlapped, this challenging overlapped and this veiling overlapped. And whenever we have overlapping fragments that have been confirmed, we can go ahead and start to cross off those residues from up above. And so essentially waken cross off this entire fragment here because it's been completely overlapped. And so we can also cross off the portion of the kind of trips and fragment that's been overlap, which would be all of this portion here and the Onley portion that has not been overlap yet is the lice in here? So essentially, what we're looking for is a trips and fragment that ends with license so that it can have an overlap at this position. So now we're going back and looking at the trips and fragments, and we're looking for one that ends with a lie scene. And so you'll notice that this fragment here ends with a lie scene. And so this fragment here must be overlapping right here at this position and so we can go ahead and start to fill that in. So we've got the lie seen here in this position. Then we've got a fetal Alan ing we've got glazing, Alan. I mean, Serigne and Alan and again. And so essentially, that would be this peptide fragment just rewritten down below. And so now that we've got this license overlapped right here, we can go ahead and cross off that fragment those residues from up above. So now, finally, we've got this blue licensing, uh, overlap so we can cross it off. And we've got the licensing in this green fragment overlap. And so, essentially, what we're looking for now is a Kimo trips and fragment that ends with fennel Allen in so that we can overlap that fragment here. And so, looking at the chemo trips and fragments, notice that there's Onley. There's to hear that end with fennel Alimi. There's this fragment here and there's also this fragment. So that means that we need to go back one more residue and look at, uh, this one. So now we're looking for one that ends with final outing, but also has a glazing right next to it. And so this one ends with phenylalanine, but it has a history in right next to it. so that's not gonna be the one that fits at this position. Instead, we have this one that has a final outing and a glazing, and so that's gonna be the fragment right here that's going to align at this position. So we're gonna start to fill in this position here. So we've got a final Allan E. That's gonna overlap here. We've got glazing, we've got Alan. I mean, searing Alan. I mean, then we've got essentially this overhang right here that we also need to include. So it's gonna be Argentine Sistine and meth signing. And so, essentially, what you'll see is now we've got all of these overlaps here, and these overlaps we can cross off from up above, so notice we can essentially cross off the entire green fragment up above, Uh, so we can cross off this entire fragment that's been overlapped, and we can also cross off the portions of the blue fragment that have been overlapped, which would be essentially all of this up until the Argentine. So now we're gonna look for some trips and fragment that ends with Argentine. And so, of course, there's Onley. One trips and fragment that remains so that fragment must be the one that overlaps at this position. So all we need to do is fill that in. So we've got Argentine here. We've got Sistine going backwards. We've got my thinning. Then we've got fennel, Allan E histamine, Mutthiah ning again trip to fan. And we've got eso losing. And so now that we've got these overlapping portions here, we can go ahead and cross those off from up above. And so we can cross off the Argentine Sistine Meth inning portion with the blue and also with green fragment. We can cross off everything up until the final Allan E. So now we're looking for Kimo trips and fragments that are going to end with a final Allen and so that we can overlap here. And so notice that there's this fragment here that has final outing and we can go ahead and overlap that one. So we've got phenylalanine, histamine and meth, I ning, and so essentially we can cross off these overlapping fragments here, and we can cross off these as well. And then, of course, we only have is a losing and trip to fan left. And of course, that's gonna be this last portion of the chemo trips and this last fragment here so we can go ahead and write that in a swell. So we've got is a losing and trip to fan. And so, essentially, what you'll see is now we've got all of the fragments overlap so we can cross them all up from above. And now that all of the fragments have been overlapped, there's really no need for us to move on to step number five, because we were not. Actually, we did not actually get stuck. So remember, Step number five is on. Lee. There is a back up plan in case you guys happen to get stuck for whatever reason. But step number five is there. Uh, if you get stuck, you're gonna use the longest peptide fragment as a starting point for overlapping and essentially do exactly what we did here looking for overlaps between different peptide fragments from different cleavages. And so now that we have all of the fragments overlap, weaken, just essentially read these overlaps from left to right toe reveal the sequence from the n terminal end to the C terminal end. And so what you'll see is the sequence is gonna be is a losing trip to fan meth fining histamine fino, Alan ing meth I need So you guys get the point here? We're just going straight through this way of reading off the sequence. So at this point, we're at 16. Where then We've got Argentine Alan Een searing Alan een glazing Fino, Allan E, Lie Scene, Valeant, Alan Een and Sistine. And so, essentially here, through overlapping these peptide fragments, we revealed the sequence of our original peptide with 17 amino acids. And so you can see here how this five step strategy is essentially a strategy that will allow us to overlap the fragments and reveal the sequence. And really, it's a four step strategy if you don't get stuck. So it's a good thing to have this as a backup, but again, it's on Lee there in case you guys get stuck. So that concludes this video, and we'll be able to get some practice utilizing this five step strategy and our practice problems. So I'll see you guys there