in this video, we're gonna talk about Edmund Degradation reaction, efficiency. So in our previous videos, we've said that Edmund Degradation can Onley be used on small peptides. And here we're just reinforcing that same idea with a little bit more detail by saying that Edmund Degradation is Onley practical for small peptides with less than about 50 amino acid residues. Now that might seem like a really limiting factor about Edmund degradation, since most proteins in nature have way mawr than just 50 amino acid residues. But that's exactly why most proteins need to be cleaved down into smaller peptide fragments with less than 50 amino acid residues each in order for those proteins to be sequenced via Edmund Degradation. But the question here is actually why? Why is it that Edmund Degradation is Onley practical for small peptides with less than 50 amino acid residues? Well, it turns out the answer has to do with the reaction efficiency per cycle for Edmund degradation and the reaction efficiency per cycle for most modern Edmund degradation. Sequin haters is about 99% which means that in each Edmund degradation cycle, 99% of the reactions, or 99% of the peptides are going to react successfully and successfully released there in terminal amino acid residue, where it will be successfully identified. And so, let's face it, a 99% reaction. Efficiency is a really high success rate. And if you guys were to get a 99% on your next test, I wouldn't be complaining. You wouldn't be complaining. We all be pretty happy and satisfied. But with Edmund Degradation, 99% reaction efficiency means that in each Edmund degradation cycle, they're still going to be about 1% of the reactions, or 1% of the peptides that are going to fail to release their end terminal amino acid residue in the correct cycle. And so although 99% reaction efficiency seems really, really high, we have to remember that this is the reaction efficiency per cycle. And one Edmund degradation cycle reveals Onley, one amino acid residue. And so if we have 50 amino acid residues in the protein, then we need 50 Edmund degradation cycles. And again, with each Edmund degradation cycle, 1% of the peptides failed to release their amino acid, and so we get an accumulation of 1% of failed products with each cycle. And so this accumulation of failed products with each cycle is really the reason why Edmund Degradation is limited to small peptides with less than amino acid residues. So just to clear up that idea, let's take a look at our example down below and notice. In this example, we have a pool of identical peptides on the left hand side, and these are Decca peptides because they have 10 amino acid residues, and they have an amino and on the far left and a car box hill and on the far right. And notice that the N terminal amino acid residue is highlighted here in gold. And that's because after one round or one cycle of Edmund degradation, this and terminal amino acid residue highlighted in gold is the one that's going to pop off of the chain and be identified as a P. T. H amino acid residue. And so notice that the first cycle of Edmund degradation can be initiated with fennel is a diocesan eight or P I. T. C. To initiate the first reaction, and then we can treat it with try floor acetic acid or cf three c h to initiate the second reaction. And then, of course, we treat the released amino acid derivative with acquis acid, or H 30 plus to initiate the third reaction that generates that P. T. H amino acid final product that we identify. And so we know, uh, this from our previous lessons. And the result is that most of the peptides are going to release their end terminal amino acid residue, and we can see that down below. Most of the peptides here have indeed release. They're in terminal amino acid residue, and that's, uh, indicated by these check marks here. But notice that not all of the peptides release they're in terminal amino acid residue. So this peptide here failed to release its and terminal amino acid residue. Now, in this diagram, it might seem that one out of four or 25% of the peptides are going to fail to release their amino acid residue. But in reality it's on Lee 1% of the peptides that fail, and so it's not as bad as 25% but even with 1% that's still capable of limiting Edmund degradation to small peptides. And the real reason is because notice that this peptide here is saying whoops, guess I'll just release it in the next cycle. And that's really the issue here. The fact that the next cycle is supposed to identify the second amino acid residue, not the first amino acid residue. And so if this peptide releases its N terminal residue and the next cycle, it's really just contaminating the second cycle with unwanted P th amino acids. And so, essentially, these unwanted P th amino acid side products will accumulate with each Edmund degradation cycle. And so if you have enough Edmund degradation cycles, you'll get, ah, lot of side products that accumulate. And ultimately, thes side products are going to obscure the results and make it really, really difficult to determine the sequence of the protein. And so it's important to note that larger proteins with Maura amino acid residues are going to require mawr Edmund degradation cycles. And with Mawr Edmund degradation cycles, there are going to be mawr side products that accumulate and again mawr side products. Accumulating means that the results are going to be obscured and mawr difficult to interpret. And so again, We know that most proteins in nature are exist, Aziz being naturally long, so they have lots and lots of amino acid residues, and they range from having several hundreds up to several thousands of amino acid residues. But that means if we were just try to sequence those large proteins with Edmund degradation, we would need several hundreds to several thousands of Edmund degradation cycles. And again, that's a lot of Edmund degradation cycles, where each one is going to produce Mawr and Mawr side products that accumulate. And so that's too many side products that accumulate, and that obscures the results. And that's exactly why the solution to sequencing long proteins in nature is to cleave down those large proteins into smaller fragments before Edmund Degradation. And that's exactly why we talked about all of those protein cleavage techniques in our previous lessons, such as, um, amino acid, hydraulics, ISS, chemical cleavage and pep. Today's is, and so this year, uh, concludes our lesson on Edmund degradation, reaction, efficiency and our next lesson video. We're going to talk about a cumulative yield and how the reaction efficiency can be used to calculate the cumulative yield. So I'll see you guys in that video

in this video, we're gonna talk about how to use the Edmund degradation reaction efficiency to calculate the cumulative yield, which is something that your professors likely gonna want you guys to Dio. And so we already know that cumulative yield can be calculated from the reaction efficiency. And really, all the cumulative yield is is the relative amount of a very specific product that's obtained in a chemical reaction. And so with Edmund Degradation, the very specific final product that's obtained and analyzed is the p th amino acid. And so notice what this equation shown below that it expresses the relationship between the Edmund degradation, reaction, efficiency, the number of Edmund degradation cycles and the cumulative yield. And so the reaction efficiency per cycle raised to the power of the number of Edmund degradation cycles is equal to the cumulative you. And so, just for some context, accurate protein sequencing typically requires a high cumulative yield, usually greater then about 60% and so a cumulative yield of 60% just suggest that 60% of the products of that Edmund cycle are actually the correct P th amino acid, and the remainder of the percentage essentially 40% are going to be unwanted p th amino acid side products. And so recall from our previous lesson video that it's these unwanted p th amino acid side products that can obscure the results. And so essentially, the goal is to keep this percentage of unwanted P th amino acid side products as low as possible and to keep the cumulative yield percentage as high as possible. And so a biochemist can expect accurate protein sequencing with any combination of reaction, efficiency and number of Edmund degradation cycles. That gives a cumulative yield greater than 60%. But any combination of reaction, efficiency and number of Edmund cycles that yields a cumulative yield lower than 60%. Ah, biochemist should be really cautious because inaccurate protein sequencing is at risk. And so that's something important to keep in mind. And so, with our example down below, will be able to get a better idea of how this works. And so it says. Let's say each reaction cycle of the Edmund Degradation procedure has a reaction efficiency of 99% where 1% of each reaction cycle produces unwanted P th amino acid side products calculate the total cumulative yield of the correct p th amino acid immediately after the 50th Edmund degradation cycle. And so because it's asking us to calculate the cumulative yield, all we need to do is use our equation up above and so notice that were given the reaction efficiency of 99% and 99% as a decimal 0.99 And so, if we take the reaction efficiency and raise it to the power of the number of Edmund Degradation cycles, which is 50 in our example problem, then we can get our cumulative yield and the cumulative yield weaken abbreviate with see why here for cumulative yield. And so, if you take your calculator and you do 0.99 raised to the power of 50 you'll get an answer of 0.605 And this is our cumulative yield in a decimal format. So if we want to convert it to a percentage, all we gotta do is multiply it by 100% and that is going to equal 65%. And so this is the answer to our example problem, 60.5% is the cumulative yield here, and so notice that 60.5% is greater than 60% which means that with a reaction efficiency of 99% at the 50th Edmund Degradation Cycle, we can expect to get accurate protein sequencing. And so what you'll notice is that this is just barely making the threshold of 60%. It's literally just 600.5% higher. And so, if we were toe, add just one more Edmund Degradation cycle by essentially changing our value of the Edmund cycles here to ah value of 51. What you would see is that this here would generate a cumulative yield of about 59.9% and this is a cumulative yield below our 60% threshold. And so what that means is that with 51 Edmund cycles that already we're starting to risk accurate protein sequencing. And it's possible that we may not get accurate protein sequencing with a 51st residue here. And so that's why typically, biochemist like to use a threshold of 60% to make sure that the correct p th amino acid is in high abundance. And so this here concludes our lesson on how to use reaction efficiency to calculate the cumulative yield and in our next couple of practice, videos will be able to get more practice, so I'll see you guys there.