Diagonal Electrophoresis - Online Tutor, Practice Problems & Exam Prep

Diagonal electrophoresis is a technique used to isolate and identify disulfide-linked proteins, crucial for determining the positions of disulfide bonds formed between cysteine residues. This method distinguishes between interchain and intrachain disulfide bonds based on their migration patterns in SDS-PAGE. Interchain disulfides result in smaller, faster-traveling fragments, while intrachain disulfides cause slower migration due to shape changes. Ultimately, this technique aids biochemists in sequencing peptides to reveal disulfide bond positions, enhancing our understanding of protein structure and function.

Diagonal Electrophoresis

In this video, we're going to talk about diagonal electrophoresis. So diagonal electrophoresis is another type of electrophoresis technique, which means that it uses an electric field to separate proteins. Now more specifically, diagonal electrophoresis is used by biochemists to isolate and identify disulfide-linked proteins, and they use it to determine the positions of original disulfide bonds. Recall from our previous lessons that disulfide bonds are covalent bonds that link the r groups of any 2 cysteine residues, and these 2 cysteine residues could be present either on the same polypeptide chain and form a disulfide bond or they could be present on separate polypeptide chains and covalently link those separate polypeptide chains via a disulfide bond. We're going to discuss how diagonal electrophoresis works below in our example.

For the results of diagonal electrophoresis, it turns out that the peptides that do not have disulfide bonds, or the peptides without disulfide bonds, are going to align diagonally or align on a diagonal line due to unchanged mobility. What you should know is that any proteins that lie on that diagonal line, are going to not have any disulfide bond. Those are going to be the peptides without disulfide bonds. Peptides with disulfide bonds are going to lie off of the diagonal, because they are going to have changed mobility.

In our example of diagonal electrophoresis, notice that on the far left we have our native protein. This native protein has a very particular shape to it. It has its native protein structure. We know when counting amino acids, we consider amino acids from the N-terminal end to the C-terminal end. This native protein here has three cysteine residues, located at position number 36, position number 54, and position number 72. We can clearly see that cysteine 36 and cysteine 54 form a disulfide bond, while cysteine 72 does not form a disulfide bond.

The very first step in diagonal electrophoresis is to fragment the protein or cleave the protein into fragments. This can be done by treating the native protein with either an enzyme such as a peptidase, which breaks down proteins, or a chemical that will cleave the protein into fragments. After we cleave the protein into fragments, the next step is to separate all these protein fragments via SDS PAGE, which separates proteins based on their molecular size.

After separating our protein fragments in SDS PAGE, the third step is to expose all of the proteins inside of our gel to performic acid vapors. Performic acid vapors will diffuse into the gel and interact with the proteins, cleaving all of the disulfide bonds. In the fourth step, we take our SDS PAGE gel and turn it sideways, running SDS PAGE a second time but in the perpendicular direction. When we run SDS PAGE in the perpendicular direction, we finally see the diagonal line we were expecting. The peptides that align diagonally do not have disulfide bonds, while the peptides that lie off of the diagonal line do have disulfide bonds.

The protein bands that lie off of the diagonal line are actually disulfide-linked. These are the proteins that the biochemist is interested in if they are trying to determine the position of the disulfide bonds. In the fifth and final step, these disulfide-linked peptides are isolated from the gel and subjected to sequencing. Sequencing is the technique that reveals the disulfide bond positions. This shows how diagonal electrophoresis can be a very useful tool to help biochemists narrow down which fragments actually have a disulfide bond and which do not.

After sequencing, the biochemist would be able to confirm that it's cysteine 36 and cysteine 54 that form a disulfide bond, and cysteine 72 does not form a disulfide bond. That concludes our lesson on diagonal electrophoresis, and we'll be able to get some practice in our next video, so I'll see you guys there.

Which of the following techniques is used specifically to help determine the site of a disulfide bond?

• A. Edman degradation.
• B. Affinity chromatography.
• C. SDS-PAGE.
• D. Diagonal electrophoresis.
• E. MALDI-TOF Mass spectrometry.

Diagonal Electrophoresis

So in our last lesson video, we said that diagonal electrophoresis can isolate peptide fragments that are disulfide-linked, and it can be used to help determine the original position of disulfide bonds. Now in this video, we're going to talk about how diagonal electrophoresis can distinguish between interchain disulfide bonds as well as intrachain disulfide bonds. It turns out that diagonal electrophoresis results actually differ a little for interchain disulfide bonds, which are disulfide bonds that form between cysteine residues found on separate polypeptide chains, as well as intrachain disulfide bonds, which are disulfide bonds that form between cysteine residues found on the same polypeptide chain. Upon cleavage of only the interchain disulfides using performic acid, the peptide fragments get smaller and ultimately travel faster in the gel. Upon cleavage of only the intrachain disulfides using performic acid, the peptide fragments will change their shape and travel slower in the gel. Let's take a look at our example below of the interchain versus intrachain disulfides in diagonal electrophoresis to clear up this idea.

We know that the very first step in diagonal electrophoresis is to cleave our native protein down into a bunch of different fragments. When we cleave our peptide into fragments, all of the disulfide bonds remain intact, and nothing happens to them. The second step is running our protein fragments using PAGE, and we separate the protein fragments based on their size. That SDS-PAGE keeps all of the disulfide bonds intact. You can see we have a bunch of different protein fragments being shown here, from p1 all the way through p7. You'll notice that all of the disulfide bonds remain intact for the first gel electrophoresis of diagonal electrophoresis. The first two disulfide bonds that are present are color-coded in yellow because they are interchain disulfides that form between separate polypeptide chains. You can see that p1 is forming a disulfide bond with p2, and p4 is forming a disulfide bond with p5, and those are interchain disulfides. Whereas with p6 here, protein fragment 6, we have an intrachain disulfide because it's forming within the same polypeptide chain.

After the first direction of electrophoresis, the third step of diagonal electrophoresis is to expose all of these peptide fragments to performic acid, which cleaves all of the disulfide bonds. On this axis is the second direction of electrophoresis after the disulfide bonds have been cleaved. You'll notice that inside our gel, there are no more disulfide bonds. When we run our second direction of electrophoresis, the peptide fragments that do not have any disulfide bonds, such as peptide fragment 3 and peptide fragment 7, align diagonally on a diagonal line. That's exactly what we see in our diagonal electrophoresis gel, that peptide fragment 3 and peptide fragment 7, which are the only two that do not have any disulfides, align diagonally on this line.

Any peptide fragments that do not align on the fragment, such as all of these other peptide fragments listed here, they do contain some kind of disulfide bond. So now to distinguish between the interchain and the intrachain disulfide bonds, notice that the peptide fragments containing interchain disulfides, these here and those over there, get smaller and ultimately travel faster in the gel. Because they travel faster in the gel, you'll notice that they are below the diagonal line. These fragments, because they contained disulfide bonds interchained, became smaller and traveled faster, and that's why we find them below the diagonal line. With intrachain disulfide bonds, such as the one with peptide fragment 6, when the intrachain disulfide bond is cleaved, peptide fragment 6 changes its shape and travels slower through the gel than it did the first time through electrophoresis, showing up above the diagonal line. Essentially, the way we distinguish between interchain and intrachain disulfides in diagonal electrophoresis is by looking at the migration of the peptides relative to the diagonal line that forms. Peptides that migrate faster have some kind of interchain disulfide bond, while peptide fragments that migrate slower have an intrachain disulfide.

This concludes our lesson here on the difference between interchain and intrachain disulfides in diagonal electrophoresis results, and we'll get a bit of practice in our next video. I'll see you guys there.

In the plot below, circle the point(s) representing peptides with intrachain disulfides.

Alright. So this practice problem says, in the plot below, circle the point or points representing peptides with intrachain disulfides. Below, we have a diagonal electrophoresis plot where the first direction of electrophoresis on the x-axis runs from left to right. Then, on the y-axis, we have electrophoresis after performic acid exposure, and we know that performic acid cleaves all of the disulfide bonds. With diagonal electrophoresis, we know a diagonal line forms, representing peptides or proteins. The peptides that fall onto this diagonal line are peptides that have no disulfide bonds. This means that any points on this diagonal line can be ignored since we are looking to circle points representing peptides that have disulfide bonds, specifically intrachain disulfides. Recall from our previous lessons that there are two types of disulfide bonds: interchain disulfides, which are between separate polypeptide chains, and intrachain disulfides, which occur within the same polypeptide chain. Upon cleavage of interchain disulfides, the peptide chains get smaller and, therefore, travel faster through the gel. Upon cleavage of intrachain disulfides, the polypeptide chain changes shape and travels slower through the gel. We are looking for peptides with intrachain disulfides, meaning peptides that travel slower after exposure to performic acid. The direction of the second electrophoresis goes from the bottom to the top. Proteins that are larger or change shape and travel slower remain towards the bottom of the plot, while proteins that travel faster go towards the top of the plot. The diagonal line represents peptides that did not change their mobility from the first to the second electrophoresis. Peptides on one side of the diagonal line traveled faster and had interchain disulfides. Those on the other side had intrachain disulfides. Since we are looking to circle peptides with intrachain disulfides, we identify the ones on the side of the diagonal line that indicates slower travel through the gel post-exposure to performic acid. This concludes our answer to the problem, and I'll see you guys in our next video.

A gene encoding a protein with a single disulfide bond undergoes a mutation that changes a serine residue into a cysteine residue. You want to find out whether the disulfide pairing in this mutant is the same as in the original protein. Briefly layout a diagonal electrophoresis experiment to determine the answer.

So this practice problem says, a gene encoding a protein with a single disulfide bond undergoes a mutation that changes a serine residue into a cysteine residue, and we know that cysteine residues are capable of forming disulfide bonds. And so you want to find out whether or not the disulfide pairing in this mutant is the same as in the original protein. And it says to briefly lay out a diagonal electrophoresis experiment to determine the answer. And so notice down below, I've provided a little image of our protein. So on the left, we have the original protein and on the right, we have the mutant protein. We know that the original protein only has a single disulfide bond, and so it's shown in red right here at this position. What we also know is that our original protein has a serine residue. And so notice that we've shown the serine residue over here in blue. Now with the mutant protein on the right, we know that the serine residue was on the original protein was mutated into a cysteine residue as it says up above. And so notice we have an additional cysteine shown in green. And so we want to find out whether the disulfide pairing in the mutant is the same as in the original. So we want to know, are these same 2 cysteines forming a disulfide like the original, or are there different cysteines forming a disulfide? And so, we know that diagonal electrophoresis is capable of isolating peptide fragments that are disulfide linked, and we can use it to help us determine the position of a disulfide bond. So we want to lay out the process of a diagonal electrophoresis experiment. So we know that the first step is going to be to cleave the peptides into fragments. So we want to be able to cleave the 2 peptides into fragments. And so, that will be the first step. Once we've cleaved them into fragments, we know that their disulfide bonds are going to remain intact. Wherever they are, they're going to remain intact. And so the second step, step number 2, is going to be to run SDS-PAGE. And so when we run SDS-PAGE, we're going to separate the fragments based on their molecular size. But again, the fragments where the disulfide bonds are included are going to migrate together because those fragments are going to be covalently linked. And so the third step involves treating the gel with performic acid, and we know that performic acid is going to cleave disulfide bonds. And once we've cleaved those disulfides, we can move on to our fourth step, which is going to be, of course, to run SDS-PAGE in the perpendicular direction. And so, what that allows us to do is to separate out the fragments that were disulfide-linked, that contained disulfides. And so if the original and the mutant protein generate the same exact pattern in this second SDS-PAGE gel, that would be reason to conclude that perhaps they have the same exact disulfide bonds. But if they generated different fragments, then that would say that the mutant has a different disulfide pairing than the original protein. And so, after we look at that band pattern in the second SDS-PAGE gel, we can, of course, take our, separated fragments that lie off of the diagonal sequencing. So we can isolate and sequence the disulfide-linked peptides. And so, that would allow us to determine the actual positions of the disulfide bonds, which cysteine residues were forming those disulfides. And so this is a very brief layout of the diagonal electrophoresis experiment, but make sure that you understand the general process of all of these steps. This concludes the answer to our practice problem and I'll see you guys in our next video.

Here’s what students ask on this topic:

What is diagonal electrophoresis and how does it work?

Diagonal electrophoresis is a technique used to isolate and identify disulfide-linked proteins, crucial for determining the positions of disulfide bonds formed between cysteine residues. The process involves several steps: first, the protein is fragmented using an enzyme or chemical. These fragments are then separated by SDS-PAGE based on their molecular size. Next, the gel is exposed to performic acid vapors, which cleave the disulfide bonds. The gel is then turned 90 degrees and run through SDS-PAGE again. Peptides without disulfide bonds align diagonally, while those with disulfide bonds lie off the diagonal due to changes in mobility.

How does diagonal electrophoresis distinguish between interchain and intrachain disulfide bonds?

Diagonal electrophoresis distinguishes between interchain and intrachain disulfide bonds based on their migration patterns in SDS-PAGE. Interchain disulfides, which form between cysteine residues on separate polypeptide chains, result in smaller fragments that travel faster and appear below the diagonal line. In contrast, intrachain disulfides, which form within the same polypeptide chain, cause the fragments to change shape and travel slower, appearing above the diagonal line. This difference in migration helps biochemists identify the type of disulfide bond present.

What are the steps involved in diagonal electrophoresis?

The steps involved in diagonal electrophoresis are: 1) Fragment the protein using an enzyme or chemical. 2) Separate the fragments by SDS-PAGE based on molecular size. 3) Expose the gel to performic acid vapors to cleave disulfide bonds. 4) Turn the gel 90 degrees and run SDS-PAGE again in the perpendicular direction. 5) Identify peptides without disulfide bonds that align diagonally and those with disulfide bonds that lie off the diagonal. 6) Isolate and sequence the disulfide-linked peptides to determine the positions of the disulfide bonds.

Why are disulfide bonds important in protein structure?

Disulfide bonds are covalent bonds that form between the sulfur atoms of two cysteine residues. They play a crucial role in stabilizing the three-dimensional structure of proteins by linking different parts of the polypeptide chain or different polypeptide chains together. This stabilization is essential for the protein's proper folding, stability, and function. Disulfide bonds are particularly important in extracellular proteins, where they help maintain structural integrity under varying environmental conditions.

What role does performic acid play in diagonal electrophoresis?

Performic acid plays a critical role in diagonal electrophoresis by cleaving disulfide bonds within the protein fragments. After the initial separation of protein fragments by SDS-PAGE, the gel is exposed to performic acid vapors. These vapors diffuse into the gel and interact with the proteins, breaking the disulfide bonds. This cleavage allows the fragments to be separated based on their actual sizes in the second round of SDS-PAGE, helping to identify which fragments were originally disulfide-linked.