Edman Degradation Sequenator and Sequencing Data Analysis

in this video, we're gonna talk about the Edmund degradation sequin ater and sequencing data analysis. So it turns out that the Edmund degradation procedure can actually be an automated process that's carried out in a machine called a sequin ator. And so this sequin ater can actually mix all of the different re agents, including P I. T. C. Try floor acetic acid and acquis acid, and it will also separate and identify all of the products and even record the data. And so the sequin ator is so highly sensitive that justus little as one nanogram or 10 p camels oven amino acid can be detected. And that's a very, very small amount. And so down below. What you'll see is we have an image of an advanced protein sequence later shown here. And so the major take away here is that the Edmund degradation procedure can be automated and all process through a very advanced instrument called a sequin ator. Now, down below, we're gonna talk about how the p th amino acids, the final products of the Edmund Degradation cycle, can be analyzed with H p l C. Now we know that Edmund degradation is used in conjunction with high performance liquid chromatography, or H P l C. And that's because H PLC will identify the P th amino acids like we said earlier. And so when these p th amino acids are first generated, there actually unknown p th amino acids. And so we need to identify these unknown p th amino acids through the use of H PLC. And the way that works is that we compare them to the illusion positions relative to known amino acid, uh, controls. And so, if we have an unknown p th amino acid that has a similar allusion position as a known control, then we're able to identify and so down below there are two important pieces of information that you guys should note moving forward. And so recall from our mass spectrometry lessons that, uh, mass spectrometry has a difficult time differentiating amino acids that have the same mass, such as losing and is a losing since losing and is a losing our ice summers of one another with the same exact chemical formula in the same mass. So mass spectrometry cannot differentiate these two residues, and so an advantage that Edmund Degradation has over mass spectrometry sequencing is that it can mawr easily differentiate amino acids of the same mass such as losing and I solution. Now, the second important piece of information that you guys should note is that, uh, in an H PLC chromatic graham. The amino acid sequence is revealed from the left of the chromatograph to the right of the chromatic graham in order from the N terminal end of the peptide to the C terminal end of the peptide, and we'll be ableto refresh our memories on how that works down below and our example for the H PLC chromatograph of P t H amino acids that are generated in the Edmund degradation process. And so what you'll see is that we've got this h PLC cream Mata Graham, which recall we covered, uh, H PLC chromatograph way back in our previous lessons when we covered H p l C. And so notice what we have on the why access is the absorb INTs or, uh which is in units of Millie absorb INTs and then on the X axis. What we have is the illusion time in minutes, or how long it takes for these amino acids to ah loot or come out of the H PLC column. And so, the most important thing that I want you guys to take away eyes that the H PLC chromatic Graham will actually reveal the peptide sequence from the left to the right of the chromatograph. Um, in order from the end terminal end to the C terminal end of the peptide. And so what that means is, all we need to do is look at these peaks and, uh, to identify the sequence. So notice that the first peak on the far left we're gonna be analyzing from the left to the right. So the first peak on the far left here is gonna be a Spartak acid. Then we have glue tannic acid, a spare Jean searing three inning uh, glutamine glazing pista Dean Alan in Argentine tire scene throwing meth I ning, we're availing trip to fan placing and we have female Alan and I solution and losing so fennel Allan E s a loosen and loosen. So pretty easy. All we need to do is read the chromatograph, um, the H PLC chromatograph from the left to the right. Now, this is actually very different from the mass spectrums of mass spectrometry. Because, remember, with the mass spectrums of mass spectrometry, we read the sequence from the right to the left. So that's something important to note that H PLC chromatic grams reveal the sequence from the left until the right, just like what we did here. But mass spectrums revealed the sequence backwards from the right to the left. So this concludes our lesson here, and we'll be able to get some practice in our next video, so I'll see you guys there.