Peptide Sequencing: Partial Hydrolysis - Online Tutor, Practice Problems & Exam Prep

Peptide sequencing involves partial hydrolysis using endopeptidases, which selectively cleave peptide bonds at specific amino acids, enhancing control over the process. Key enzymes include trypsin, chymotrypsin, and pepsin, each targeting distinct amino acids. Exopeptidases, like carboxypeptidase A and B, hydrolyze bonds at the C-terminus, with specificities for amino acids. Complete hydrolysis, achieved by boiling in hydrochloric acid, identifies amino acids and their concentrations but does not reveal sequences. This method complements partial hydrolysis to reconstruct peptide sequences effectively.

Peptide Sequencing: Partial Hydrolysis Concept 1

Hey, everyone. So with peptide sequencing, let's take a closer look at partial hydrolysis. Here, we're going to address partial hydrolysis with endopeptidases. Recall that before sequencing, peptides are partially hydrolyzed into smaller peptide chains, which we call fragments. We can use dilute acid, which hydrolyzes at random amino acids.

But if we want better control, we can use endopeptidases. These are enzymes that selectively hydrolyze peptide bonds at specific amino acids, providing not only better control but also a better outcome. We specifically target the hydrolysis of bonds at the carbonyl group or carboxyl group of the following non-terminal amino acids.

We’ll be using these endopeptidases to selectively hydrolyze specific amino acids. Now that we understand a little bit about what type of enzymes we're going to use, click on the next video and let’s explore more specifics when it comes to partial hydrolysis with these enzymes.

Peptide Sequencing: Partial Hydrolysis Concept 2

Hey, everyone. In this video, let's take a look at our different endopeptidases. So, here we're going to talk about enzyme catalyzed hydrolysis with the different enzymes in the form of trypsin, chymotrypsin, and pepsin. Now, trypsin, the location of bond cleavage, we're going to say resides at lysine and arginine. And our memory tool here is that lying and arguing will trip you up.

So remember, trip is trypsin, lye is lysine, and arguing is arginine. Next, we have chymotrypsin. With chymotrypsin, we're going to have here phenylalanine, we have here our tryptophan, and then here we have our tyrosine. And our memory tool here is that chowder is very aromatic.

We're going to say that these 3 amino acids have aromatic R groups. Then finally, we have pepsin, which has the same aromatic R groups, but also it has here leucine, and then we have aspartic acid, and we have glutamic acid. Our memory tool here is that pepsin breaks down aromatic lentils in acidic stomachs. So we're still dealing with our 3 aromatic amino acids. Lentils is for our leucine.

And then, aspartic acid and glutamic acid, they have acidic R groups. So acidic R groups, acidic stomach. So, that's how it all fits together. And we're going to say here that enzymes, this is important, will not cleave if proline is at a cleavage site. So, this is what they want to do, but this is an exception that resides.

If we have proline around, we can't do the cutting that we want to do. Alright. So just remember these memory tools and how these different types of enzymes cleave at these specific bond cleavage sites.

Peptide Sequencing: Partial Hydrolysis Example 1

Hey, everyone. So here it says, label locations of hydrolysis in the following peptide with its corresponding enzymes. So here we're gonna have t for trypsin, c for chymotrypsin, and p for pepsin. To be able to do this, we have to remember our memory tools. So if we're looking at trypsin first, remember that for trypsin, t, we're gonna say that lying and arguing will trip you up.

Remember, we're gonna cut or hydrolyze at the carboxyl end. Remember, when we're looking at an amino acid chain, this is the N-terminal, this is the C-terminal. We're cutting on the C-terminal end of the amino acid. And remember, we're cutting into fragments which means we cannot cleave at the ends because if we do, we'd have a lone amino acid. We want to have fragments that we're producing.

So we can't just have a lone amino acid there, which means that this is blocked and this is blocked. Even if it falls within the amino acid that a particular enzyme can cleave. Alright. So lysine and arginine. So here goes lysine.

So, trypsin could cut here, cleave here. And then we have arginine anywhere. Doesn't look like we do. Yeah. That would be the only one.

So, yeah. That's the only one. We don't have arginine anywhere else. Next, we go to chymotrypsin. So remember, chowder is very aromatic.

So for chymotrypsin, we're talking about chowder being aromatic. We're talking about the amino acids that have aromatic R-groups. So that would be phenylalanine, that would be tryptophan, and that would be tyrosine. If we take a look here, here goes phenylalanine, so this could cut there. Then we have tyrosine right here, So this could cut here.

And do we have tryptophan anywhere? No tryptophan anywhere. Alright. Then we have pepsin. Remember, pepsin also deals with these same aromatic ones.

So this could be chymotrypsin or pepsin doing it. And then it also has in addition to this, we have lentils which is leucine, and then we have acidic stomach. So that's talking about the acidic amino acids in the form of aspartic acid and glutamic acid. So glutamic acid is here, but remember if we cut here, we'd have a lone amino acid. We want fragments not lone amino acids, so that doesn't work.

And then we have aspartic acid here, so pepsin could cut here. If we look Where else do we have? We also have glutamic acid right here. That'd also be another one. So pepsin could cut here.

We wouldn't have cutting here. We wouldn't have oops, actually. And we wouldn't have cutting here. We have to go back. There would be no cutting here because remember, if there's a proline nearby, we can't cleave that bond.

So, actually, we cannot cut here. So that's not allowed. So the only places that we can have hydrolysis or cleaving are here, here, here, and here. So those would be our final answers.

How many peptide chains does the following peptide produce after being treated with chymotrypsin?

Thr-Lys-Cys-Tyr-His-Asp-Trp-Ile-Val-Phe

• A. 1
• B. 2
• C. 3
• D. 4

The following peptide is treated with pepsin and trypsin. Show where the enzymes will cleave the peptide.

Lys-Gly-Glu-Pro-Tyr-Gln-Asn-Ala-Ser-Leu-Gln-Phe-Ser-Cys-Trp

Hey, everyone. So in this practice question, it says, the following peptide is treated with pepsin and trypsin. Show where the enzymes will cleave the peptide. So, remember the memory tools that we have here is that with trypsin, we're going to say that lying and arguing will trip you up. So anywhere we have a lysine or an arginine is where we're going to cut, and that'll be on the C-terminus side. So if we take a look here, what do we have? We have a lysine here, but we don't want to cut here because we want to make fragments. We don't want alone amino acids. So no cut would happen there. Where else? We have arginine anywhere? Arginine? Nope. Nowhere else. And is there any other lysines around here? Nope. There are not. So we wouldn't have any cutting there. Now with pepsin remember, with pepsin, we have our aromatic amino acids. So we're talking about tryptophan, we're talking about tyrosine, we're talking about phenylalanine. In addition to this, we'd also

Peptide Sequencing: Partial Hydrolysis Example 2

Everyone. So let's take a look at peptide sequencing using fragments. Now, here we're going to say, after fragments are sequenced, they are fitted together to determine the full peptide sequence. In this example, we say that trypsin and chymotrypsin were used to partially hydrolyze a peptide. Propulsion sequence for the peptide from the following peptide fragments produced.

Alright. So here are our fragments, all these different fragments. And what we need to remember here is that when it comes to trypsin, it will cut anywhere we have lysine or arginine. With chymotrypsin, we're looking at the aromatic containing amino acids. So, here we're talking about Phenylalanine, we're talking about tryptophan, and we're talking about Tyrosine.

Right. Knowing this information will allow us to piece these things together. Right. So, if we take a look here, we have the different fragments and what we have to do here is we just kind of have to line them up with each other and figure out what this could look like. Alright.

So, here we're gonna start out with, Alright. So we have phenylalanine with lysine, then we have alanine, then we have tryptophan. Alright. So then here, we're trying to match the pieces together, and then look, we have here alanine and tryptophan.

So alanine, tryptophan, glycine, cysteine, tyrosine, and arginine. And what can fit with this portion here? Alright. So then we're gonna say here, look, we already had this Phenylalanine and Lysine here. So now we have these last fragments.

We're just trying to fit them together. So let's see how things can connect. We have here oh, look. We have here glycine. No.

We have here arginine, leucine, and valine. We have leucine and valine. And this part here is this part here. So we have how the pieces were cut in different places to give us our final structure. So our final sequence from this information, come down here.

We'd have this sequence to give us: Lysine, alanine, tryptophan, cysteine, tyrosine, arginine, leucine, and valine. So this would be our sequence from these different fragments connected together. We're seeing where things overlap because that's where enzymes are cutting and giving us these different fragments. We're just trying to piece them together.

Alright. So we're fitting them together to give us this full peptide sequence which is given below.

Acid-catalyzed partial hydrolysis of an unknown peptide produced the following peptide chains. Propose a sequence for the peptide.

Gly-IIe-Gly-Cys     Ile-Trp-Gly     Cys-Asn-Pro     Gly-Cys-Asn      Pro-Phe-Leu     Trp-Gly-Ile-Gly

Everyone, so here it says, as it catalyzes partial hydrolysis of an unknown peptide, produce the following peptide chains. Propose a sequence for the peptide. Alright. So we can see that we have these 6 different fragments, and what we need to do is we need to see where overlapping can occur. So if we look, we're seeing a lot of this amino acid overlapping in half of these fragments. So let's see how they fit together. So here, let's say we have here this first fragment and let's see how we can fit other ones together. So here we have it followed by glycine. So here, we'll do with this one here. So we have so let's see. So we have tryptophan as well. Alright. So we took out. Again, it's just playing around and seeing how things fit together. Oh, so here we have tryptophan, which overlaps here giving us this. Alright. So we're taking this one and taking this one. Then we have this fragment here that overlaps to give us this one. Look at that. Now, we need to find one that has a cysteine involved which has to be this one here. Right? So then we have here actually, is that the one oh, this one also has 16. We're trying to see how the pieces overlap with each other. So we have here we do this one, Gly, because it matches up with this. And now we have cysteine overlapping. And then finally we have this proline with phenylalanine and leucine. So we're seeing how pieces are overlapping. See these 2 overlap. These 3 overlap, these 2 overlap, these 2 overlap. This overlapping is how we're able to piece them together. Okay. So now we'll just bring all the fragments together. So we'd have this final chain where things overlap just combine. And and then pro. And this would represent our final chain. This will be our full peptide sequence from the fragments given to us.

Peptide Sequencing: Partial Hydrolysis Concept 3

Everyone, in our continued discussion of peptide sequencing, we're now going to take a look at partial hydrolysis when using exopeptidases. Here, we'll say that exopeptidases are enzymes that hydrolyze peptide bonds at the C-terminus ends of a peptide chain. Here, we have them broken down as carboxypeptidase A and B. For A, we're going to say it cleans off C-terminal amino acids with the exceptions of being arginine and lysine.

And with carboxypeptidase B, we're going to say it cleaves C-terminal arginine and lysines only. Right? Because if A doesn't do it, then B will step in and do it itself. Alright. So these are our two different types of exopeptidases that we're going to talk about.

And now that we've discussed the general idea behind the two, click on the next video and take a look at the example question where we have to use these different types of enzymes.

Peptide Sequencing: Partial Hydrolysis Example 3

Hey, everyone. In this example, it says that the peptide chain is reacted with carboxypeptidase A, and no reaction was observed. That tells me that it doesn't have, well, that means actually what it has at its end are either arginine or lysine. These are our end amino acids. And remember, carboxypeptidase A does not cleave these particular amino acids.

So, this peptide chain ends with one or both of these types of amino acids. Next, it was reacted with carboxypeptidase B. An unknown amino acid was released. And here it says reactions of the peptide chain with trypsin and pepsin produced the following fragments. What is the sequence of the peptide chain?

Alright. So remember, with trypsin, it's going to cut on the C-terminus ends if we're dealing with lysine or arginine. And with pepsin, pepsin will cleave if we have an aromatic amino acid. So we're talking about phenylalanine, tryptophan, or tyrosine. But it'll also cut if we see a leucine, and if we see one of the acidic amino acids in the form of aspartic acid or glutamic acid.

Alright. So, from this information, let's see what we can deduce. Alright. So, here we have our different fragments. These are the fragments that Trypson gave us and these are the fragments that glycine gave us.

Alright. So, if we look here, we have lysine and we have lysine. We can presume we will have lysine lysine, which overlaps with this next one here, which is lysine lysine, our amino acid, lysine phenylalanine. So we've taken care of these two.

Let's see. Next, we're going to see what continues to overlap. We have our amino acid, lysine phenyl.

Where does this overlap? Oh, right here. We have this one right here. So that can overlap right there.

Let’s see. Where else can we see an overlap happening? It's evident here with phenylalanine and arginine, and then we have an arginine here which connects with this one here. And then we have this last piece here, putting Y to N, and then lysine. So we’ve taken care of all the fragments here, and we’ve been able to fit them together where overlapping occurs. So then bring down everything.

And we can see, once we fit all the fragments together, look what it begins with and ends with. We can see that we have lysines. That's why the carboxypeptidase A didn't work. Remember, if we have arginine or lysine at the ends, nothing is going to happen with that particular exopeptidase. So, that's why there was no reaction initially.

This here would represent the entire sequence for my peptide chain. So this would be our final answer.

Peptide Sequencing: Partial Hydrolysis Concept 4

Hey everyone. So let's take a look at complete hydrolysis. Here, we're going to say that it's performed to confirm the identities of amino acids and their relative concentrations. And we're going to break it down as a, b, and c when talking about the overview of complete hydrolysis. With a, we're going to say our peptide chain is hydrolyzed by boiling in hydrochloric acid.

This cuts every single peptide bond, freeing all of our individual amino acids. So, now, we have a complete collection of amino acids. And, we're going to say with b, our amino acid solution is passed through what we call an ion exchange chromatography. So here, our amino acid solution passes through this machinery. And we're going to say what's important here is that we have our light source as it's passing through our photometer, and it's going to shine through this information which gives us our amino acid concentration versus time here.

And we're going to say for c, this is important. This does not provide the sequence of amino acids but serves as a reference for partial hydrolysis. The results now from this, we have identified different amino acids from this collection of individual amino acids. Again, we need to use it in unison with partial hydrolysis to see how these amino acids link up together and by fitting together these fragments, we can then figure out the full peptide sequence. But again, this is worked with in conjunction with partial hydrolysis.

Right? So this is what we talk about when we name complete hydrolysis.

Peptide Sequencing: Partial Hydrolysis Example 4

Hey everyone. So here you are tasked with proposing a peptide sequence from the following results of hydrolysis. You're going to have complete hydrolysis gives us these individual amino acids. Now, here treatment with carboxypeptidase A gives us a tyrosine. Remember, this is an exopeptidase, so it's cutting and cleaving on one of the ends of our peptide chain.

This tells me that tyrosine is the amino acid at the end of my peptide chain. If we look at our options, there are only 2 fragments that contain Tyrosine, this much longer fragment and then this dipeptide. So the longer fragment is what we're going to use. Remember, this dipeptide here is just here as a fragment that was created. If we look now, next up is arginine.

If we look, the only other fragment that has arginine in it is this one here. So this is going to fit right here. We're going to write the rest of it, so proline. And then we're going to have the remainder. So we've gotten this out.

Now we're just left with 3 other fragments. Let's see what else fits together. We have cysteine and phenylalanine. And look, cysteine and phenylalanine. Okay.

So then we have that. And then let's see what else fits together. We have lysine here. We have tryptophan here.

And then this dipeptide here would fit here. So from these fragments, we're able to fit them together to figure out what the final sequence will be. So if we come down here, we fit everything together. This would be the complete peptide sequence. So let's just write it out.

So this would be our final answer when we have to propose the peptide sequence. Alright. So this would be it in its entirety. Alright. So this would be our final answer.