Edman Degradation Sequenator and Sequencing Data Analysis
5. Protein Techniques
Edman Degradation Sequenator and Sequencing Data Analysis - Video Tutorials & Practice Problems
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Edman Degradation Sequenator and Sequencing Data Analysis
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Video transcript
In this video, we're gonna talk about the Edmond degradation sequinator and sequencing data analysis. So it turns out that the Edmond degradation sequinator. And so this sequinator can actually mix all of the different reagents including P ITC, trifluoroacetic acid, and aqueous acid, and it will also separate and identify all of the products and even record the data. And so the sequinator is so highly sensitive that just as little as 1 nanogram or 10 picomoles of an 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 sequinator shown here. And so the major takeaway here is that the Edmond degradation procedure can be automated and all, processed through a very advanced instrument called a sequinator. Now, down below, we're gonna talk about how the PTH amino acids, the final products of the Edmond degradation cycle, can be analyzed with HPLC. Now, we know that Edmond degradation is used in conjunction with high performance liquid chromatography, or HPLC. And that's because HPLC will identify the PTH amino acids like we said earlier. And so when these PTH amino acids are first generated, they're actually unknown PTH amino acids, and so we need to, identify these, unknown PTH amino acids through the use of HPLC. And the way that that works is that we compare them to the elution positions, relative to known amino acid controls. And so, if we have an unknown PTH amino acid that has a similar elution position control, then we're able to identify it. 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, mass spectrometry has a difficult time differentiating amino acids that have the same mass such as Leucine and Isoleucine. Since Leucine and Isoleucine are isomers of one another with the same exact chemical formula and the same mass. So, mass spectrometry cannot differentiate these two residues. And so, an advantage that Edmond degradation has over mass spectrometry sequencing is that it can more easily important, piece of information that you guys should note is that, in an HPLC chromatogram, the amino acid sequence is revealed from the left of the chromatogram to the right of the chromatogram in order from the n terminal end of the peptide to the c terminal end of the peptide. And we'll be able to, refresh our memories on how that works down below in our process. And so what you'll see is that we've got this HPLC chromatogram, which recall we covered, HPLC chromatograms way back in our previous lessons when we covered HPLC. And so notice what we have on the y axis is the absorbance or, which is in units of milliabsorbance. And then on the x axis, what we have is the elution time in minutes or how long it takes for these amino acids to, elute or come out of the HPLC column. And so the most important thing that I want you guys to take away, is that the HPLC chromatogram will actually reveal the peptide sequence from the left to the right of the chromatogram in order from the n 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 to identify the sequence. So notice that the first peak on the far left, we're going to be analyzing from the left to the right. So the first peak on the far left here is gonna be aspartic acid, then we have glutamic acid, asparagine, serine, threonine, glutamine, glycine, histidine, alanine, arginine, tyrosine, proline, methionine, we're valine, tryptophan, lysine, and, we have phenylalanine, isoleucine, and leucine. So phenylalanine, isoleucine, and leucine. So pretty easy, all we need to do is read the chromatogram, the HPLC chromatogram, 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 HPLC chromatograms reveal the sequence from the left to the right just like what we did here. But mass spectrums reveal 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.
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Problem
Problem
Separately determine the peptide sequences indicated by the HPLC chromatogram & mass spectrum below.
A) What is the peptide sequence indicated by the HPLC chromatogram?
B) What is the peptide sequence indicated by the mass spectrum (assuming prominent peaks correspond to y ions)?