Edman Degradation Sequenator and Sequencing Data Analysis - Online Tutor, Practice Problems & Exam Prep

The Edman degradation procedure utilizes a sequinator for automated sequencing and analysis of amino acids. This highly sensitive instrument can detect as little as 1 nanogram of an amino acid. The resulting PTH amino acids are analyzed using high-performance liquid chromatography (HPLC), which identifies them based on their elution positions compared to known controls. Unlike mass spectrometry, which reads sequences from right to left, HPLC reveals the peptide sequence from left to right, providing a clear method for amino acid analysis.

Edman Degradation Sequenator and Sequencing Data Analysis

In this video, we're going to talk about the Edman degradation sequinator and sequencing data analysis. So it turns out that the Edman degradation sequinator can mix all of the different reagents including phenylisothiocyanate (PITC), trifluoroacetic acid, and aqueous acid, and it will also separate and identify all of the products and even record the data. The sequinator is so highly sensitive that just as little as 1 nanogram or 10 picomoles of an amino acid can be detected, which is a very small amount. Down below, what you'll see is we have an image of an advanced protein sequinator shown here. The major takeaway here is that the Edman degradation procedure can be automated and processed through a very advanced instrument called a sequinator.

Now, down below, we're going to talk about how the phenylthiohydantoin (PTH) amino acids, the final products of the Edman degradation cycle, can be analyzed with high-performance liquid chromatography (HPLC). Edman degradation is used in conjunction with HPLC because HPLC will identify the PTH amino acids as mentioned earlier. When these PTH amino acids are first generated, they are unknown PTH amino acids, so we need to identify these unknown PTH amino acids through the use of HPLC. This is achieved by comparing them to the elution positions relative to known amino acid controls. If we have an unknown PTH amino acid that has a similar elution position control, then we're able to identify it. 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, mass spectrometry cannot differentiate these two residues. An advantage that Edman degradation has over mass spectrometry sequencing is that it can more easily distinguish between these.

The important piece of information that you 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. You'll see we've got this HPLC chromatogram. On the y-axis is the absorbance, which is in units of milliabsorbance, and on the x-axis is the elution time in minutes. The most important thing to take away is that the HPLC chromatogram will reveal the peptide sequence from left to right in order from the N-terminal end to the C-terminal end of the peptide. All we need to do is to look at these peaks to identify the sequence. Notice that the first peak on the far left is aspartic acid, followed by glutamic acid, asparagine, serine, threonine, glutamine, glycine, histidine, alanine, arginine, tyrosine, proline, methionine, valine, tryptophan, lysine, phenylalanine, isoleucine, and leucine. Reading the HPLC chromatogram from left to right is actually very different from the mass spectra of mass spectrometry, where we read the sequence from right to left. It's important to note that HPLC chromatograms reveal the sequence from left to right, unlike mass spectra which reveal the sequence backwards from right to left. This concludes our lesson here, and we'll be able to get some practice in our next video. I'll see you guys there.

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)?

So this practice problem is broken up into 2 parts. We have part a up above and part b down below. And it says to separately determine the peptide sequences indicated by the HPLC chromatogram, which is in part a, and then the mass spectrum, which is down in part b. And so first, we're going to focus on part a and the HPLC chromatogram. Part a asks, what is the peptide sequence indicated by the HPLC chromatogram? And what you'll notice is we've got this peptide sequence space over here, where of course the N-terminal residue goes on the far left and the C-terminal residue goes on the far right. And the HPLC chromatogram has absorbance on the y-axis and the elution time on the x-axis. And we know from our previous lesson video that the HPLC chromatogram reveals the amino acid sequence, from the left of the chromatogram to the right of the chromatogram. And so all we need to do is read the residues at the top of the peaks from the left to the right. First, we have valine, so we could put valine here first, then we have isoleucine, so we can put isoleucine next, then we have leucine. And so you guys see the pattern here. Next, we have tyrosine, and then last but not least, we have phenylalanine. And essentially, that is it. We've revealed the peptide sequence by analyzing the HPLC chromatogram here. So pretty easy, read it from left to the right. Now, down below in part b, we have a mass spectrum. It says, what is the peptide sequence indicated by the mass spectrum, assuming that the prominent peaks correspond to y ions? In our mass spectrum below, notice we have the relative abundance on the y-axis and the m/z ratio on the x-axis. We know that the y-ion peaks are the more stable ions that are going to generate the most prominent peaks, and they are going to be read from the right of the mass spectrum to the left of the mass spectrum in order to reveal the peptide sequence from the N-terminal end on the far left to the C-terminal end on the right. So essentially, with the mass spectrum, we're going to be reading it backwards, whereas with the HPLC chromatogram above, we read it from left to right. So that's a big difference to take into account here. We know that the difference in mass between the corresponding appropriate y-ion peaks will reveal amino acid residues. Reading it from right to left, what we have is arginine first, then we have alanine, next is glycine, and then we have alanine again. Cysteine is next, then we have glycine, alanine, valine, and last but not least is alanine. And so that is essentially it for this practice problem. We have revealed the amino acid sequence from the mass spectrum, as well as from the HPLC chromatogram up above. And you could see and compare the differences between analyzing these two different data plots. So that concludes this practice problem, and I'll see you guys in our next video.

Here’s what students ask on this topic:

What is Edman degradation and how does it work?

Edman degradation is a method used to sequence amino acids in a peptide. It involves the stepwise removal of one amino acid at a time from the N-terminal end of the peptide. The process uses phenylisothiocyanate (PITC) to label the N-terminal amino acid, which is then cleaved off and identified as a phenylthiohydantoin (PTH) derivative. This cycle is repeated to determine the sequence of amino acids in the peptide. The procedure can be automated using a sequinator, which mixes reagents, separates products, and records data, making it highly efficient and sensitive.

How does high-performance liquid chromatography (HPLC) assist in Edman degradation?

High-performance liquid chromatography (HPLC) is used in Edman degradation to identify the PTH amino acids generated during the sequencing process. HPLC separates these PTH derivatives based on their elution times, which are compared to known amino acid controls. By matching the elution positions of unknown PTH amino acids with those of known standards, the specific amino acids can be identified. This method provides a clear and accurate way to determine the sequence of amino acids in a peptide, from the N-terminal to the C-terminal end.

What are the advantages of Edman degradation over mass spectrometry for protein sequencing?

Edman degradation has several advantages over mass spectrometry for protein sequencing. One key advantage is its ability to differentiate between amino acids with the same mass, such as leucine and isoleucine, which are isomers. Mass spectrometry struggles with this differentiation due to their identical masses. Additionally, Edman degradation provides a clear sequence of amino acids from the N-terminal to the C-terminal end, whereas mass spectrometry reads sequences from the C-terminal to the N-terminal end. This makes Edman degradation particularly useful for precise and sequential identification of amino acids in a peptide.

What is the role of a sequinator in Edman degradation?

A sequinator is an advanced instrument used in Edman degradation to automate the sequencing process. It mixes all necessary reagents, including phenylisothiocyanate (PITC), trifluoroacetic acid, and aqueous acid, and performs the sequential removal and identification of amino acids. The sequinator is highly sensitive, capable of detecting as little as 1 nanogram of an amino acid. It also records the data, making the process efficient and accurate. By automating the procedure, the sequinator significantly enhances the speed and reliability of amino acid sequencing.

How is the sequence of amino acids read in an HPLC chromatogram during Edman degradation?

In an HPLC chromatogram used during Edman degradation, the sequence of amino acids is read from left to right, corresponding to the N-terminal to the C-terminal end of the peptide. The y-axis of the chromatogram represents absorbance, while the x-axis shows the elution time in minutes. Each peak on the chromatogram corresponds to a specific PTH amino acid. By analyzing these peaks and comparing their elution positions to known standards, the sequence of amino acids can be determined. This method provides a clear and systematic way to identify the peptide sequence.