Strategy for Ordering Cleaved Fragments - Online Tutor, Practice Problems & Exam Prep

To effectively order cleaved protein fragments, follow a five-step strategy. Start by scanning for clues about composition and sequence. Next, recall the specific peptide bonds cleaved by reagents like trypsin and chymotrypsin. Identify N-terminal or C-terminal fragments based on cleavage recognition. Overlap these terminal fragments with others to reveal the original sequence. If stuck, use the longest fragment as a starting point for overlaps. This systematic approach aids in understanding peptide bond interactions and the importance of amino acid sequences in protein structure.

Strategy For Ordering Cleaved Fragments

In this video, we're going to talk about a strategy for ordering cleaved fragments. It turns out that there are actually multiple strategies to 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 only 5 different steps.

The first step, step number 1 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. What we'll find is that our professors like to give us these paragraph-style word problems with all of these sentences, and hidden within the sentences, there are clues. The clues could be the use of FDMV, or hydrazine, or some other chemical that we already covered in our previous videos. That's a really good first step: 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 reagent recognizes for cleavage. In our example down below, we'll be able to apply our first two steps in our strategy for ordering cleaved fragments. Trypsin is used to give 3 different peptide fragments, and then separately treated with chymotrypsin to give 4 peptide fragments.

Looking down below, what you'll notice in the left-hand chart, we have the trypsin fragments, and there are 3 trypsin fragments. In the right-hand chart, we have the chymotrypsin fragments, and there are these 4 chymotrypsin fragments shown below.

The example problem continues to say, identify the sequence of the 17 amino acid residues in the original starting peptide. In order to do this, we're going to need to order all of these cleaved fragments here. So we can apply our strategy for ordering cleaved fragments. And in our first step, step number 1, we're going to scan the problem for helpful clues. Looking at our problem, there's not a lot of helpful clues like using chemicals such as FDMV or hydrazine. But notice it does tell us our protein or our peptide has 17 amino acids, which reveals some of the amino acid composition there. That's going to be important moving forward.

Essentially that is it for step number 1, since there's not really a lot of helpful clues. Step number 1 is complete; we can give it a check. Now moving on to step number 2, notice that there is a step number 2 for the trypsin fragments, and there is also a step number 2 for the chymotrypsin fragments. In step number 2, all we need to do is recall which specific peptide bonds the reagent recognizes for cleavage.

Recall that trypsin does its splitting after a knight's sword. So, it cleaves the C-terminal peptide bonds of lysine and arginine amino acid residues. Chymotrypsin, the younger brother of trypsin, also cleaves the C-terminal peptide bonds, but it has a preference for which amino acids it recognizes for cleavage. The mnemonic that helps us memorize that is just "free your worries like me". We know that the L and the M here, the leucine and methionine, are cleaved at a much slower rate over longer periods of time.

Since there's no indication in our problem that leucine and methionine are actually going to be cleaved here, then we're going to assume that only the preferred amino acid residues of these aromatic residues, Phenylalanine, Tyrosine, and Tryptophan, are going to be cleaved. Essentially what we can do is say that trypsin is going to cleave Phenylalanine, Tyrosine, and Tryptophan residues. Essentially, that is it for step number 2. So, we can go ahead and give step number 2 a check over here and step number 2 a check over here. And now that we're done with step number 2, in our next video, we'll move on to step 3 in our strategy for ordering cleaved fragments. See you guys there.

Strategy For Ordering Cleaved Fragments

Alright. So now that we've covered the first two steps in our strategy for ordering cleave fragments, in this video, we're going to focus on our 3rd step. And in step number 3, we'll 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 reagent recognizes for cleavage. And so, we'll be able to see an example of this down below in our example. Now, another component of step number 3 is that we'll want to check if identified terminal fragments from different cleavage reagents 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. We'll be able to see an example of this component here, step number 3, up above in our example problem when we get there.

But first, let's cover this example down below. In this example, it's important to note that if the reagent cleaves the N terminal peptide bond, then we'll want to look to identify the N terminal amino acid fragment. And so, for example, we know that pepsin is a peptidase that cleaves the N terminal peptide bonds of Phenylalanine, Tyrosine, Tryptophan, and Leucine. Looking at this peptide down below, notice that it only has 2 amino acid residues that pepsin recognizes for cleavage and those are highlighted in red throughout our image. And so, we know that pepsin cleaves 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 our peptide into 3 different fragments. We have this fragment here on the far left, MAE, shown down below. We have the FERD 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 pepsin recognizes for cleavage. And so you can see the red terminal residues that are being recognized by Pepsin on all of the fragments, except for this one fragment over here. And that makes this fragment quite unique. And that uniqueness actually identifies this fragment as the N terminal fragment, simply because it does not have terminal amino acid residues that pepsin recognizes for cleavage. And so here it's pretty easy to see that this fragment is the N terminal fragment.

But even if we didn't have the original peptide sequence provided, and we were only given these peptide fragments below, we would still be able to identify this MAE fragment as the N terminal fragment, just because of this reasoning where it does not have terminal residues that pepsin recognizes for cleavage. And so applying the same logic to reagents that cleave C terminal peptide bonds, then we'll want to look to identify C terminal fragments. And so, for instance, we know that trypsin is a peptidase that cleaves the C terminal peptide bonds of lysine and arginine. And looking at our peptide below, which is the same peptide that we had up above here, notice that there are 3 residues that trypsin recognizes for cleavage, and that fragments our protein into 4 different fragments shown below. And so what you'll notice is that all of the fragments end with terminal amino acid residues that trypsin recognizes for cleavage shown in the green highlights here.

And only one of the fragments does not have terminal amino acid residues that trypsin 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 trypsin recognizes for cleavage. And so we'll be able to apply this, concept here to our example problem up above. And so this is the same exact example problem from our previous video. And with, again, what you'll notice is that there's a step number 3 for the trypsin fragments, and then there's also a step number 3 here for the chymotrypsin fragments. And of course, in step number 3, we'll want to identify either the N or the C terminal fragments. But because we previously identified in step number 2 that both of our reagents 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 trypsin fragments first, we know that trypsin recognizes lysine and arginine. And so notice that all of our fragments have a terminal amino acid residue of lysine and arginine, 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 as the C terminal fragment, simply because it does not have terminal amino acid residues that, trypsin recognizes for cleavage. And so if we do the same for chymotrypsin, we know that, chymotrypsin recognizes the aromatic amino acid residues. And so notice that we have aromatic amino acid residues on all of the fragments, 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 3, where we'll want to check if the identified terminal fragments from different cleavage reagents 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 trypsin fragments, and we have this C terminal fragment from the chymotrypsin fragments. And, when we check to see if they match, notice that we have a valine alanine cysteine with the trypsin fragments, and we also have valine alanine cysteine with the chymotrypsin fragments. And there is this overhang of a lysine residue, cysteine 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 3 of our strategy for ordering cleave fragments. And in our next video, we'll be able to tackle steps number 4 and steps number 5. So I'll see you guys there.

Strategy For Ordering Cleaved Fragments

Alright, so now that we've covered the first three steps in our 5-step strategy for ordering cleaved fragments, in this video, we're going to focus on the 4th and the 5th steps. In step number 4, we're going to use those terminal fragments that we identified back in step number 3, and overlap those terminal fragments with other peptide fragments from different cleavage techniques. We're going to continue to do that until we overlap all of the peptide fragments and until the sequence of the original peptide has been revealed. We'll be able to apply step number 4 in our example problem above. You'll notice that step number 5 is really only here as a backup in case you happen to get stuck for whatever reason. Hopefully, you won't get stuck, but it's always good to have a backup plan just in case. If for whatever reason you 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. 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 to overlap that peptide fragment with other peptide fragments until all of the peptide fragments have been overlapped and the sequence has been revealed. Again, if you happen to get stuck, all you need to do is move on to 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 4.

Recall in our previous video with step number 3, that we identified this circle fragment here, under the trypsin fragments, as the C-terminal fragment, and we identified this fragment over here as the C-terminal fragment for the chymotrypsin fragments. Down below, we can rewrite these fragments so that we can see how they overlap. Notice that we have the trypsin fragments that we can provide in this green space up above, and we have the chymotrypsin fragments that we can provide in the blue space. Then after we overlap the fragments, down below, we can provide the sequence of the original peptide. We're going to start with the terminal fragments, and we can start with the trypsin fragment here, which is the C-terminal fragment, valine, alanine, and cysteine. We've got cysteine, alanine, and we've got valine. Now we've got this valine, alanine, and cysteine, fragment rewritten down below, and we can overlap with the other C-terminal fragment under the chymotrypsin fragments. So we have cysteine here, we've got alanine, we've got valine, and of course, we've got this lysine that's hanging over. We've also got this lysine here. You'll see that we've got this cysteine overlapped, this alanine overlapped, and this valine overlapped.

Whenever we have overlapping fragments that have been confirmed, we can go ahead and start to cross off those residues from up above. We can cross off this entire fragment here because it's been completely overlapped. We can also cross off the portion of the chymotrypsin fragment that's been overlapped, which would be all of this portion here. The only portion that has not been overlapped yet is the lysine here. We're looking for a trypsin fragment that ends with lysine, so that it can have an overlap at this position. Now, we're going back and looking at the trypsin fragments, and looking for one that ends with a lysine. This fragment here ends with a lysine, and so this fragment here must be overlapping right here at this position. We've got the lysine here at this position, then we've got phenylalanine, glycine, alanine, serine, and alanine again.

Finally, we've got this lysine overlap here, so we can cross it off. We're looking for a chymotrypsin fragment that ends with phenylalanine, so that we can overlap that fragment here. There's only one fragment here that ends with phenylalanine. So we start to fill in this position here. We've got phenylalanine that's going to overlap here, glycine, alanine, serine, alanine. We've also got an overhang here that we also need to include, which is going to be arginine, cysteine, and methionine. Notice, we can essentially cross off the entire green fragment up above, so we can cross off these overlapping fragments here. Now, we're looking for some trypsin fragment that ends with arginine. There's only one trypsin fragment that remains, so that fragment must be the one that overlaps at this position. We've got arginine here, cysteine going backwards, methionine, phenylalanine, histidine, methionine again, tryptophan, and isoleucine.

Now that all of the fragments have been overlapped, there's really no need for us to move on to step number 5 because we did not actually get stuck. Remember, step number 5 is only there as a backup plan in case you happen to get stuck for whatever reason. But step number 5 is there, if you get stuck, you're going to 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. Now that we have all of the fragments overlapped, we can just essentially read these overlaps from left to right to reveal the sequence from the N-terminal end to the C-terminal end. You'll see that the sequence is going to be isoleucine, tryptophan, methionine, histidine, phenylalanine, methionine. So you get the point here; we're just going straight through this way, reading off the sequence. At this point, we're at cysteine, then we've got arginine, alanine, serine, alanine, glycine, phenylalanine, lysine, valine, alanine, and cysteine. Through overlapping these peptide fragments, we revealed the sequence of our original peptide with 17 amino acids. You can see here how this 5-step strategy is essentially a strategy that will allow us to overlap the fragments and reveal the sequence. It's a 4-step strategy if you don't get stuck. It's a good thing to have this as a backup, but again, it's only there in case you get stuck. That concludes this video, and we'll be able to get some practice utilizing this 5-step strategy in our practice problem. I'll see you guys there.

A sample of an unknown peptide was divided into two aliquots. One aliquot was treated with trypsin; the other was treated with cyanogen bromide. Given the following sequences of the resulting peptide fragments, deduce the sequence of the original peptide.

Trypsin treatment:                                                                         Cyanogen bromide treatment:                 
Asn—Thr—Trp—Met—Ile—Lys                                             Gln—Phe

Gly—Tyr—Met—Gln—Phe                                                     Val—Leu—Gly—Met

Val—Leu—Gly—Met—Ser—Arg                                            Ile—Lys—Gly—Tyr—Met

                                                                                                Ser—Arg—Asn—Thr—Trp—Met

Sequence: 

____-____-____-____-____-____-____-____-____-____-____-____-____-____-____-____-____

Alright. So this practice problem wants us to order the following cleaved fragments to reveal the sequence of the original peptide. And it says that a sample of an unknown peptide was divided into 2 aliquots where one was treated with trypsin and the other was treated with cyanogen bromide. Given the following sequences of the resulting peptide fragments, shown below with the trypsin fragments on the left and the cyanogen bromide fragments on the right, deduce the sequence of the original peptide. And since we know we need to order these cleaved fragments, we can use our 5 step strategy for ordering cleaved fragments. And notice in this box here, we have each of those steps listed. And recall that step number 1 is just to scan the problem for helpful clues that reveal either the sequence or composition information. And looking at our problem, there's not a lot of helpful clues like the use of FDNB or hydrazine, but we are provided the composition of these fragments, which allows us to determine the amino acid composition. And we know that there's a total of 17 amino acids, and we also know the number and the types of amino acids. And so that is part of, collecting the clues, and that's gonna be helpful moving forward. So now that step number 1 is essentially done, we can check it off and move on to step number 2. And in step number 2, all we need to do is recall the reagent cleavage site or the exact peptide bond and amino acid residues that the reagent recognizes for cleavage. And so recall that, trypsin does its splitting after a night's work, by its cleaves the C-terminal side of lysine and arginine amino acid residues. And cyanogen bromide, on the other hand, because of its abbreviation, CNBR, if we rotate that B 90 degrees counterclockwise, it tells us that it cleaves next to methionine residues, and so it cleaves the C-terminal side of methionine residues. And really, that is all step number 2 wanted us to do. So pretty easy, right? So we can go ahead and check it off and indicate step number 2 is done, and we can move on to step number 3. And in step number 3, we want to identify the terminal fragments. And because both trypsin and cyanogen bromide recognize the C-terminal peptide bond for cleavage, we want to identify the C-terminal fragments. And so remember that those are pretty easy to identify because they do not end with terminal amino acid residues that the reagent recognizes for cleavage. And so notice that because trypsin recognizes lysine and arginine, all of its peptide fragments end with a lysine or arginine, except for this peptide fragment here that ends with phenylalanine, and that's because it's actually the C-terminal peptide fragment. So we can go ahead and circle this fragment here and say that this is the C-terminal peptide fragment. Now similarly, with, cyanogen bromide over here, it recognizes methionine residues. So all of its peptide fragments end with methionine, except for one peptide fragment over here. And so this peptide fragment is easily identifiable as the C-terminal peptide fragment. And so that's it. That's really all step number 3 wanted us to do. So we've identified the, 2 C-terminal peptide fragments and now we can check to see if they match. So over here, we have glutamine and phenylalanine. And notice over here, we also have glutamine and phenylalanine. And even though we have this overhang over here, that's okay because, as long as we have the matching, C-terminal fragments, that is enough to say that we're on the right track and that we've probably identified the correct C-terminal fragments. So that's a good sign, and we can check off step number 3 and move on to our 4th step. And in this 4th step, we're going to overlap those terminal fragments with other peptide fragments until we reveal the sequence of our protein. And so looking down, notice that we can provide the trypsin fragments in these blank spots over here, and we can provide the cyanogen bromide fragments in these blanks here. And, up above, we can provide the sequence of the original peptide after we, performed the overlaps. And so we know that the cyanogen bromide C-terminal fragment is glutamine and phenylalanine, so we can go ahead and fill that in. We know that, phenylalanine will be here, glutamine will be here. And then for trypsin, we can go ahead and overlap this C-terminal fragment, which also has phenylalanine and glutamine over here, so phenylalanine and glutamine. And then, of course, it has the rest of this fragment that we need to continue to add on, so methionine, tyrosine, and glycine. And so what we can do is cross off the residues that have been overlapped, so we can cross off glutamine and phenylalanine from both of these fragments here. So now, this fagment is done and essentially, it's passed the baton onto this trypsin fragment which is down here. And we can look for now cyanogen bromide, fragments that will overlap. So, we're looking for a cyanogen bromide fragment that ends with methionine, but that also has tyrosine right next to it. And so notice that this fragment up here with the cyanogen bromide fragments has methionine, it ends with methionine and a tyrosine, and none of the other ones do. So that must mean this fragment is the one that overlaps here at this position. So we can go ahead and fill that in. So we've got methionine here, we've got tyrosine here, glycine, and then, of course, we've got the rest of the isoleucine and lysine. And so, essentially, now we can cross off these residues that have been overlapped, so we can cross off methionine, tyrosine, and glycine, so methionine, tyrosine, and glycine from both fragments. And so now we've passed the baton on to this fragment over here. This fragment right here, which has this overhang, and so now, we're gonna look for trypsin fragments that end with isoleucine and lysine so that they overlap. And so looking at the trypsin fragments, there are only two left and this fragment here ends with isoleucine and lysine, which is what we're looking for. So this fragment here must be the one that overlaps here at this position. So we can go ahead and fill that in. So we we've got lysine here, isoleucine, then we've got methionine, tryptophan, threonine, and asparagine. And again, that fragment is this fragment right up here. And so now we can cross off these overlapping residues of lysine and isoleucine from both of the fragments. So lysine and isoleucine can be crossed off here, as well as over here. So now, we're done with this blue fragment entirely. And we've passed the baton on to this fragment up here, this trypsin fragment, which is this one here. And now we can look for overlap. So we're looking for, cyanogen bromide fragments that essentially end with, a methionine and a tryptophan. And so looking at our, cyanogen bromide fragments, notice that this one ends with methionine and tryptophan, and the other one doesn't. So essentially, this fragment right here must be the one that overlaps at this position. So we can go ahead and fill that in. So we've got methionine, tryptophan, threonine, asparagine. And then, of course, we still have the serine and arginine overhang, so we have to include that, arginine, and serine. And so now we can go ahead and cross off the overlap, so we've got these overlaps here that we can cross off. And, and the methionine as well. So we can go ahead and cross those off, so we can cross off methionine, tryptophan, threonine, and asparagine, and we can do that for both residues. And so essentially, we've eliminated this entire trypsin fragment and passed the baton onto this fragment here, which again is this fragment here. And so now we've got these overhangs and we're looking for trypsin fragments that end with serine and arginine. And, of course, there's only one trypsin fragment left and itгs ends with serine and arginine. So this fragment here must be this remaining fragment. And so we can go ahead and fill that in. We've got arginine, serine, methionine, glycine, leucine, and valine. And so now we can eliminate these overhangs with serine and arginine, so we can cross those off from here, and we can also cross them off from this fragment here. So now, we're only got, one fragment that's left, and of course, this fragment must be the missing cyanogen bromide fragment that overlaps here. So we can go ahead and fill that in. We've got methionine, glycine, leucine, and valine. Alright. So now we've overlapped all of the peptide fragments, so we can go ahead and cross them all off from up above, and essentially, we've revealed the sequence of our peptide. So, all we need to do is fill it in up above, with these overlaps that are confirming. So, in our first position here, we have valine, and to save time, we'll use the one-letter code, so we have V. Next, we have leucine, then we have glycine, methionine, serine, arginine, asparagine, threonine, tryptophan, methionine, isoleucine, lysine, glycine, tyrosine, methionine, glutamine, and last but not least, phenylalanine. And so, essentially, what we've done is we've ordered the fragments here and we've revealed the sequence of our peptide. And so, this highlighted green portion here is the answer to our problem. And notice, we didn't even need to get to step number 5, because we never got stuck. And remember, step number 5 is only there if you guys happen to get stuck for whatever reason, if you're having trouble, you can move on to step number 5 and essentially do the same exact process, just using the longest peptide fragment. And so, this here concludes this practice problem, and I'll see you guys in our next video.

A peptide with 31 amino acid residues is independently treated with trypsin to give four fragments and separately treated with chymotrypsin to give six fragments (see chart below). FDNB treatment followed by amino acid hydrolysis resulted in DNP-Met and free amino acids. Identify the sequence of the 31 amino acid residues in the original unfragmented protein using one-letter amino acid codes.

Alright. So this practice problem wants us to overlap the following cleave fragments to identify the sequence of the 31 amino acid residues in the original unfragmented protein using one-letter amino acid codes. And down below, we have these blank spaces where we can provide the sequence of our peptide. And so we're going to need to use our 5 step strategy for ordering cleave fragments, and the first step is to scan our problem for helpful clues. And scanning our problem, notice we're treating our prop our peptide with FDNB, and from our previous lessons, we know that FDNB is a molecule that reacts to covalently label the free N-terminal amino acid residues of all polypeptide chains. And so after amino acid hydrolysis, which will not specifically cleave all of the peptide bonds, the amino acids are released as free amino acids. But the N-terminal amino acid residue is still labeled as a DNP. And so DNP methionine M here is indicating that the N-terminal amino acid residue is methionine. And so down below in our sequence, we can indicate that it is methionine. And for the chymotrypsin fragments at the same position, we can indicate methionine will be there as well. We can use the space up above to also represent the trypsin fragments. And so now that we're done with step 1, step 2 is all we need to do is recall exactly what peptide bonds are recognized for cleavage by the reagent. And so recall that trypsin does its splitting after a knight's sword. It cleaves the C-terminal peptide bonds of lysine and arginine amino acid residues. Chymotrypsin, trypsin's younger brother, also cleaves the C-terminal peptide bond. The mnemonic that helps us memorize the preference for which amino acid residues it recognizes for cleavage is free your worries like me, where leucine and methionine M here are cleaved at a much slower rate over longer periods of time. Since there's no indication in our problem that leucine and methionine are going to be cleaved, we're going to assume that only the aromatic amino acid residues are going to be recognized for cleavage by chymotrypsin. And so essentially, that is it for step number 2. In step number 3, we're going to identify either the N or C terminal fragments. And because both trypsin and chymotrypsin recognize the C-terminal peptide bond for cleavage, we're going to identify the C-terminal fragment. And that's pretty easy to identify because it does not end with a terminal amino acid residue that the reagent recognizes for cleavage. And so again, trypsin recognizes lysine and arginine. So, notice that all of its fragments end with either lysine or arginine, except for one fragment here that ends with valine, and that uniquely identifies it as the C-terminal fragment. Now applying the same logic to chymotrypsin, because it recognizes the aromatic amino acid residues, notice that all of its fragments end with aromatic amino acid residues, except for one fragment, which also ends with valine, and so that uniquely identifies it as the C-terminal fragment. And so now that we have 2 C-terminal fragments, we need to check to see if they match. This one has alanine isoleucine valine, and this one also has alanine isoleucine valine. So there is a match here, and it doesn't matter that we have this overhang over here. That's okay. So that lets us know that we're on the right track. And because we previously identified methionine as the N-terminal amino acid residue, we know that the N-terminal fragment has to begin with a methionine. And so looking at the trypsin fragments, notice that there's only one fragment that begins with a methionine. So that identifies this fragment here as the C-terminal fragment. I'm sorry, the N-terminal fragment. And so applying the same logic over here with the chymotrypsin, notice that we have 2 fragments that begin with methionine. But the fragment that's below MAF matches the MAF that we have over here. So because they match, that identifies this fragment here as the N-terminal fragment as well. So now that we've identified the N and the C terminal fragments, in our 4th step, we're going to use these N-terminal and C-terminal fragments for overlapping the other peptide fragments. So we can start over here with the trypsin N-terminal fragment, which is this sequence. So we can go ahead and write it down below. Methionine, alanine, phenylalanine, valine, isoleucine, alanine, valine, tyrosine, and lysine. And so, down below, what you can see is so that we can see where the overlap begins. We have, this N-terminal fragment down below, which is going to be methionine, alanine, phenylalanine. And, with the C-terminal fragments on the other side over here, we can start with C the C-terminal fragment over here with the trypsin. So we have, proline, we have valine, isoleucine, alanine, phenylalanine, proline, isoleucine, and glycine. And then, with the C terminal fragment over here, just so that we can see where the overlap begins, we have valine, isoleucine, and alanine. So now we have these overlaps, these overhangs here that we let us know where we can begin next. And we have overhangs over here as well, so it doesn't matter which side you decide to start from, I'm going to start from this side over here. So all we need to do is look for a chymotrypsin fragment that begins with valine. And so notice, looking at the chymotrypsin fragments, there's only one that begins with valine. It's this one here. So we can go ahead and fill that one in. So we've got valine, isoleucine, alanine, valine, and tyrosine. And that's this fragment here. So we still have this overhang right here. So, that means that we're looking for now a chymotrypsin fragment that overhangs with lysine. So we've got, this fragment here that ends with lysine, so we can go ahead and fill that in. So we've got lysine, cysteine, valine, alanine, isoleucine, leucine, valine, arginine, and tryptophan. And so now, we're looking for an overhang, for the trypsin CrossAxisAlignment:[TextDirection.rtl] fragments that begins with cysteine and valine. And so notice that there's one fragment here

The sequence of kassinin, a tachykinin dodecapeptide from the African frog Kassina senegalensis, was determined. A single round of Edman degradation identifies Asp as the N-terminus. A 2^nd sample of the peptide is treated with chymotrypsin, releasing two fragments with the following amino acid compositions: fragment 1 (G, T, M, V) and fragment 2 (D₂, Q, K, F, P, S, V). Next, a 3^rd sample of peptide is treated with trypsin, which results in two fragments with the following amino acid compositions: fragment 3 (D, P, K, V) and fragment 4 (D, Q, G, T, M, F, S, V). A 4^th sample was treated with CNBr, but the dodecapeptide was not cleaved.  A 5 ^th sample treated with elastase yields a single Gly residue & three fragments—fragment 5 (T, M), fragment 6 (D, K, P, S, V), and fragment 7, which was sequenced as: D—Q—F—V. What is the sequence of the dodecapeptide? 

Hint: Elastase cleaves C-terminal side of small neutral residues: G, A, V, L, I & S.

Sequence: _____-_____-_____-_____-_____-_____-_____-_____-_____-_____-_____-_____

Alright. So here's a really challenging practice problem that has a lot of different components to it. But really, all it's asking is what is the sequence of the dodecapeptide or peptide with 12 amino acid residues in it. And so notice that down below we have these blank spaces where we can provide the sequence of the dodecapeptide. And so really one of the things that makes this practice problem so challenging is the fact that we're not given a lot of sequenced peptide fragments, and instead we're given a lot of amino acid compositions of peptide fragments. And so what this means is that we're not going to be able to follow our 5 step strategy for ordering cleave fragments as strictly as we normally do, and we're going to need to get creative about how we solve this problem. And so notice that throughout the problem that we're going to treat our dodecapeptide with multiple different cleavage reagents, including chymotrypsin, trypsin, cyanogen bromide, and elastase. And so in these blank spaces, we can provide the sequence of these peptide fragments as we reveal them using the clues in the problem above. And so notice that this problem gives us a lot of information about this dodecapeptide. So we're only going to focus on the relevant pieces of information that reveal the sequence of the dodecapeptide. And so that being said, notice the second sentence says that a single round of Edman degradation identifies aspartic acid as the n terminal. And we know that Edman degradation is n terminal peptide sequencing, and it identifies 1 n terminal amino acid residue at a time. So a single round is going to identify the n terminal amino acid residue. And so because aspartic acid was identified and aspartic acid's one-letter amino acid code is D, down below we can indicate that the n terminal amino acid residue is aspartic acid. And so that means that aspartic acid is also going to be in this position for all of these fragments down below. And so already, we've started to reveal sequence about our dodecapeptide. So we're on the right track here, and we need to continue to read through our problem. And so this, the next sentence says that a second sample of the peptide is treated with chymotrypsin, releasing 2 different peptide fragments with the following amino acid compositions. And remember, compositions only include the number and the types of amino acids, but not the order or the sequence of amino acids. So the fragment 1 has a composition of Glycine, Threonine, methionine, and valine, but not necessarily in that particular order. And then the same applies for fragment number 2. And so what we'll need to recognize is that through combining the compositions of fragment 1 and fragment 2, we can reveal the entire composition of the dodecapeptide up above. And so I provided the composition in, of the amino acids, one-letter codes in alphabetical order, just so that we can make it easier to visualize if there are duplicates of the same type of amino acid. So we can see that there are 2 aspartic acids and there are 2 valines. But the rest of the of the amino acids are unique within this dodecapeptide. Now because we previously identified aspartic acid as the n terminal amino acid residue, we need to recognize that fragment number 1 over here does not have any aspartic acid residues. And fragment 2 does have aspartic acid residues, which means that it contains our n terminal amino acid residue, and this is going to be the n terminal fragment. And of course if fragment 2 is the n terminal fragment, that means that fragment 1 is going to be the c terminal fragment. And so essentially what we can, recall from our previous videos is that chymotrypsin is a peptidase that has a preference for which amino acids it recognizes for cleavage. And the mnemonic that helps us memorize that preference is free your worries like me. Where, the like me here is leucine and methionine which are cleaved at a much slower rate over longer periods of time. And since the problem does not indicate that leucine and methionine are going to be cleaved here, we're going to assume that chymotrypsin is only going to cleave its preferred aromatic amino acid residues here. And notice that our dodecapeptide composition only has one aromatic amino acid residue, which is a phenylalanine. And so we'll be able to determine the position of this single phenylalanine by comparing these fragments here. And so notice that, down below our, fragment 2 here is the n terminal fragment, and it has a total of 8 amino acid residues. And so it's going to contain the first 8 residues. And, of course, fragment number 1 is the c terminal fragment, so it's going to contain the last 4 residues. And so, essentially, what we can see is that there seems to be a peptide bond that's being broken right at this position. And so because chymotrypsin cleaves the c terminal peptide bonds of aromatic amino acid residues, and there's only one aromatic amino acid residue of a phenylalanine, that must mean that that phenylalanine must go at this position in order for its c terminal peptide bond to be cleaved by chymotrypsin. And so that means that phenylalanine has to go in this position for all of these residues here, and, at this position for all these fragments. And so that's great news. We've identified the position of another amino acid residue. So what we can do is start to cross off the amino acid residues that we've identified their position. So we can cross off phenylalanine, and we can also cross off one of the aspartic acids that we identified earlier. So we can continue to go through our problem here and move on to the next sentence, which says that a third sample of the peptide is treated with trypsin, which results in 2 peptide fragments again, but with the following amino acid compositions, fragment number 3 shown here, and fragment number 4 shown over here. And so notice that both fragment 3 and fragment 4 have aspartic acid residues, But what you'll also notice is that trypsin does its peptide bond splitting like a knight's sword, so it cleaves after lysine and arginine. And so even though both, fragments have aspartic acid residues, we can still determine which one is the N terminal fragment. So notice that if fragment number 4 were the n terminal fragment for the trypsin cleavage, then, and fragment 4 has 8 amino acid residues, then it would end here. And fragment 3 would have 4 amino acid residues and it would be over here. And so, essentially, since, trypsin cleaves the c terminal peptide bonds, it would cleave the same peptide bond that we got with chymotrypsin, and that would mean that we would need either lysine or arginine at this position here, but we already know that we have a phenylalanine here. So that must mean that this blue fragment, this fragment 4 here, is not the N terminal fragment. And instead, it's actually going to be the C terminal fragment. So up above, we can label as being the c terminal fragment. And, of course, that must mean that fragment 3 is going to be the n terminal fragment. And so, this is going to be important moving forward. And so, looking up our dodecapeptide composition, notice that trypsin only cleaves lysine and arginine. And there are no arginines, but there is one lysine, and so we can determine the position of this lysine down below. And since we know that fragment number 3 is the n terminal fragment, it's going to be the first four residues. And fragment, 4 is the c terminal fragment, so it's going to be the last 8 residues here. And so essentially what we're saying is that there's going to be a peptide bond that's broken right here at this position. And because again trypsin cleaves the c terminal peptide bond of lysine and arginine, and there's only 1 lysine in our composition, that means that that lysine has to go at this position here in order for, this peptide bond to be cleaved. And so what that means is we've revealed the position of the lysine, we can cross it off from our list up above, and we can also fill in that lysine is going to be in this position for all of the fragments, as well as for the sequence. And so, essentially, we can move on to our next portion of our problem. And so the next part says that a 4th sample was treated with cyanogen bromide, but the dodecapeptide was not cleaved. And we know that cyanogen bromide cleaves next to methionine residues. And so essentially, we know that it's going to cleave the c terminal peptide bond. And looking up at our dodecapeptide composition, notice that there's only one methionine amino acid residue. And so, because our dodecapeptide was not cleaved, there's only really one position where this methionine can go. Now, you might be thinking that proline amino acid residues are capable of blocking the cleavage. But we also know that, prolines typically block the cleavage of peptidases. And cyanogen bromide is not a peptidase. Cyanogen bromide is a chemical. And so what that means is that proline does not have the same blockage, effect that it has on cyanogen, that it has on peptidases. And so, proline can block peptidases, but it cannot block cyanogen bromide in the same way. And so that means that methionine residue here, the only methionine, has to be the c terminal amino acid residue. It has to be the very last residue because if it was at any other position, then we would expect cleavage. But we know that we did not get any cleavage. So because there was not any cleavage, we know that the methionine amino amino acid residue has to be the c terminal residue. So what that means is up above, we can go ahead and cross off methionine from our, composition list up above. And, essentially, what we can do is fill in methionines all the way across the board over here, for all of these other fragments. Alright. So now we're revealing even more information. And we can move on to our next sentence. And so the next sentence says that a 5th sample was treated with elastase, which yielded a single glycine residue and 3 peptide fragments with compositions of fragment 5 shown here, fragment 6, shown over here, and fragment 7, shown here, but fragment 7 was actually actually sequenced and revealed this sequence shown here. And so notice that we already have a phenylalanine position determined in our sequence shown here. And there's only 1 phenylalanine in our sequence. And so these residues that surround the phenylalanine here, valine, must be in the position to the right of the phenylalanine. So down below, we can indicate that valine goes into this position on the right of the phenylalanine. And then, on the left of the phenylalanine, we have aspartic acid and glutamine. And so on the left of the phenylalanine, we can put in aspartic acid and glutamine. And so let's go ahead and do that. So we have acid here, and, oops, glutamine here, so we can go ahead and fill that in all the way down. Alright. So now essentially what we can see is that we can cross off these residues from our amino acid composition up above. So we've got, the other aspartic acid, accounted for. We've got a glutamine accounted for, and we've got one of the valines accounted for. And so what we'll need to realize next is that, fragment 5 over here has a composition of threonine and methionine. And methionine, we know, is the last amino acid residue over here. And so the only way to get a fragment with threonine and methionine is if threonine is adjacent to the methionine. And so that means that threonine must go into this position over here. And so what we can do is fill in that threonine has to be in this position, next to the methionine, all the way through. All right. So now we're getting really close to determining the sequence, and we can go up above and cross it off our composition, so that we can keep track of things here. So now what we'll need to do is actually go back to our chymotrypsin fragments briefly. So remember that our chymotrypsin fragments, up above, we said that chymotrypsin generated 2 fragments. Chymotrypsin generated this first fragment here, which was the c terminal fragment, and it generated fragment 2, which was the n terminal fragment. And so we have the n terminal fragment was the first eight residues, and the c terminal fragment was the last 4 residues. And so because, we can see here that there is only one residue missing from the c terminal fragment, we can essentially determine what it is by crossing out the ones that we already know. So we already have a valine here, so we can cross off valine. We have a threonine, we can cross that off. And we have methionine, so we can cross that off too. And so the only one that we don't have accounted for is glycine, which means that glycine should go into this position here. And that means that it's going to go into the same position through all of our fragments here. And so that's great news. We've identif

Here’s what students ask on this topic:

What are the steps involved in ordering cleaved protein fragments?

The steps involved in ordering cleaved protein fragments are:

1. Scan the problem for clues about the composition and sequence of the protein.

2. Recall the specific peptide bonds cleaved by reagents like trypsin and chymotrypsin.

3. Identify the N-terminal or C-terminal fragments based on cleavage recognition.

4. Overlap these terminal fragments with other peptide fragments to reveal the original sequence.

5. If stuck, use the longest fragment as a starting point for overlaps.

This systematic approach helps in understanding peptide bond interactions and the importance of amino acid sequences in protein structure.

How does trypsin cleave peptide bonds?

Trypsin cleaves peptide bonds at the C-terminal side of lysine (K) and arginine (R) residues. This means that trypsin recognizes these amino acids and cuts the peptide bond immediately after them. This specificity is crucial for determining the sequence of cleaved fragments and helps in ordering them correctly when analyzing protein structures.

What is the role of chymotrypsin in protein cleavage?

Chymotrypsin cleaves peptide bonds at the C-terminal side of aromatic amino acids such as phenylalanine (F), tyrosine (Y), and tryptophan (W). It can also cleave at leucine (L) and methionine (M) but at a slower rate. This specificity helps in identifying and ordering cleaved fragments when analyzing protein sequences.

How do you identify N-terminal and C-terminal fragments in protein sequencing?

N-terminal fragments are identified by the absence of terminal amino acid residues that the cleavage reagent recognizes. For example, if a reagent cleaves at the N-terminal side, the fragment without the recognized terminal residue is the N-terminal fragment. Similarly, C-terminal fragments are identified by the absence of terminal residues recognized by C-terminal cleaving reagents. This identification is crucial for correctly ordering the fragments.

What should you do if you get stuck while ordering cleaved fragments?

If you get stuck while ordering cleaved fragments, use the longest peptide fragment as a starting point for overlapping other fragments. If there is a tie for the longest fragment, pick one and continue overlapping until the sequence is revealed. If you still encounter difficulties, move to the next longest fragment and repeat the process. This backup strategy ensures you can still determine the original sequence even if initial attempts are challenging.