Diagonal Electrophoresis - Video Tutorials & Practice Problems
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concept
Diagonal Electrophoresis
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In this video, we're gonna 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. And so 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 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. And so down below in our example of diagonal electrophoresis, we're gonna talk about how diagonal electrophoresis works. But just to give you guys an a heads up on what to expect, 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 actually going to align diagonally or align on a diagonal line due to unchanged mobility. And so again, this doesn't make a lot of sense right now, but it will once we get to our example. And so what I want you guys to know is that you're gonna be looking for some kind of diagonal line, and any proteins that lie on that diagonal line, that align on the diagonal line, are going to not have any disulfide bond. Those are gonna be the peptides without disulfide bonds. And so what that means is that peptides that do have disulfide bonds, so peptides with disulfide bonds, are actually going to lie off of the diagonal. So that means that they will not be on this diagonal line and that is because they are going to have changed mobility. And so let's take a look at our example down below of diagonal electrophoresis to clear up this idea. And so notice that on the far left up here, what we have is our native protein. And notice that our native protein has this very particular shape to it. It has its native protein structure. And we know that it's gonna have an n terminal end, and it will also have a c terminal end on the opposite end. And so we know that when we're counting amino acids, that we count them and we consider amino acids from the n terminal end to the c terminal end. And so, we can count amino acids and give them a particular position. So notice that this particular native protein here has a total of 3 cysteine residues, and they're located at position number 36, position number 54, and position number 72. And so we can clearly see from this image that it's cysteine 36 and cysteine 54 that form a disulfide bond, which is indicated here. And cysteine 72 does not form a disulfide bond. But let's say for, just for the sake of this example, that the biochemist performing this experiment does not know which of these 3 cysteines are disulfide bonded. And that's why the, biochemist would perform diagonal electrophoresis to figure out which of these 3 cysteines are disulfide bonded if any of them are. And so the very first step in diagonal electrophoresis is going to be to fragment the protein or cleave the protein into fragments. And so the biochemist can do that by treating the native protein with either an enzyme such as a peptidase, which breaks down proteins, or the biochemist could use some kind of chemical that will cleave the protein down into fragments. And so of course, after we cleave the protein into fragments, that's going to result in a bunch of protein fragments. And so notice that we have a protein fragment over here that has, cysteine 72, which again, we know does not have a disulfide bond. And then we have another fragment over here, but notice that this fragment has a disulfide bond between cysteine 36 and cysteine 54. And so these two fragments, even though their backbones are separated, they're still covalently linked via the disulfide bond. And so after we cleave our protein into fragments, the second step is going to be to separate all of these protein fragments via SDS PAGE. And we know that SDS PAGE separates proteins based on their molecular size. And so notice that all of these protein fragments have different molecular sizes. So, SDS page is able to separate all of these proteins based on their molecular size. Now notice that the protein that contains the, disulfide bond here is going to migrate together. So these 2 peptide fragments are gonna migrate together because they're disulfide linked, and that means that they're gonna show up as a single band in the s d s page gel, even though there are 2 fragments. And so that's important to keep in mind as we move along through our process. So after we separate our protein fragments, in SDS PAGE, the third step, which is down below here, is to expose all of the proteins that are inside of our gel over here to performic acid vapors. And the performic acid vapors will diffuse they're literally a gas that will diffuse into the gel gel and interact with the proteins. And they, when they interact with the proteins, they're going to cleave all of the dis sulfide bonds. And so, essentially, you can think of performic acid as having the same or a similar effect as beta mercaptoethanol, which also cleaves disulfide bonds. And down below, what we have is an image of the performic acid structure, so we can label it as performic acid. And so after we expose our gel and the protein fragments to performic acid, what's gonna happen is all of the disulfide bonds are gonna be cleaved, which means that our fragment up here, which had a disulfide bond, is no longer going to have a disulfide bond linking those two chains together. And so, in the 4th step, all we need to do is take our s d s page page gel at the top up here and we can take it and literally just turn it sideways. And when we turn it sideways, we can run sds page a a second time, but this time, we run SDS page in the perpendicular direction. And so what you'll see is that when we run stspage in the perpendicular direction, we finally see that diagonal line that we knew that we were going to, see earlier. We were expecting to see a diagonal line, and there it is. This is our diagonal line. And so recall up above, we said that with the diagonal line that we see, it's the peptides that do not have dis sulfide bonds that align diagonally. So all of the peptide fragments that are in this diagonal, all of these do not have disulfide bonds, but the ones that lie off of the diagonal line do have disulfide bonds. That would be these two fragments here. And so the reason that all of these peptides align diagonally is because their mobility 1st gel to the second gel. And so, those peptides from the 1st gel to the 2nd gel, and so they run the same distance and they all, form a diagonal line. However, the peptides that lie off of the diagonal line, they are migrating differently from the 1st SDS page to the 2nd SDS page, and that's why they lie off of the diagonal. And the reason that they run differently from the first to the second SDS is because of this third step that's in between where we added performic acid, which cleaved the disulfide bonds. So now that they don't have that disulfide bond linking them, they will run according to their actual sizes. So you've got this smaller fragment that will move faster and another fragment here that's also gonna move, faster. And so that's why they lie off the line. So an important takeaway is that it's the protein bands that lie off of the diagonal line that are actually disulfide linked. So it's these proteins that the biochemist is gonna be interested in if they're trying to determine the position of the disulfide bonds. And so it makes it pretty easy to see visually that these are the ones that are interested in because all of the other ones lie on this diagonal. So we can take these 2 disulfide linked proteins and we can move them into our 5th and final step. And the 5th and final step is to take these disulfide linked peptides, isolate them from the gel, and then subject them to sequencing. And the sequencing technique is what really reveals the disulfide positions. But it would have been it's the, diagonal electrophoresis that allowed us to obtain the disulfide linked peptides in the first place. And so up above, what we can say is that the very 5th and final step is that peptides that are found to be disulfide linked can be isolated, sequenced to determine the disulfide bond positions. And so you can see here how diagonal electrophoresis can be a really useful tool to help, bio chemists narrow down which fragments actually have a disulfide bond and which fragments do not. And so after sequencing the biochemist would be able to reveal that it's cysteine 36 and cysteine 54 that form a disulfide bond, and cysteine 72 would not form a disulfide bond. And so 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.
2
Problem
Problem
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.
3
concept
Diagonal Electrophoresis
Video duration:
6m
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Video transcript
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. And so it turns out that diagonal electrophoresis results actually differs a little bit 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. And so it turns out that upon cleavage of only the interchain disulfides using performic acid leads to the peptide fragments getting smaller and ultimately traveling faster in the gel. And so upon cleavage of only the intra chain disulfides using performic acid, the peptide fragments will actually change their shape and travel slower in the gel. And so let's take a look at our example down below of the interchain versus the interchain dis disulfides in diagonal electrophoresis to clear up this idea. And so we know that the very first step in diagonal electrophoresis is to cleave our native protein down into a bunch of different fragments. And so when we cleave our peptide into a bunch of fragments, all of the disulfide bonds remain intact and, nothing happens to them. And so, the second step, we run our protein fragments using PAGE, and we separate the protein fragments based on their size. And so, that SDS PAGE, keeps all of the disulfide bonds intact. And so what you can see is we have a bunch of different protein fragments that are being shown here, and they go from p 1 all the way through p 7. And what you'll notice is that all of the disulfide bonds are remaining intact for the first gel electrophoresis of diagonal electrophoresis. And so, what you'll notice is the first two disulfide bonds that are present, we are color coding in yellow because they are inter chain disulfides that form between separate polypeptide chains. And so you can see that p 1 is forming a disulfide bond with p 2, and p 4 is forming a disulfide bond with p 5, and those are inter chain disulfides. Whereas with p 6 here, protein fragment 6, we have an intra chain disulfide because it's forming within the same polypeptide chain. Now after the first direction of electrophoresis, we know that the 3rd step of diagonal electrophoresis is to expose all of these peptide fragments to performic acid, and performic acid cleaves all of the disulfide bonds. So we can see that, what we have on this axis is the second direction of electrophoresis after the disulfide bonds have been cleaved. And so what you'll notice is inside of our gel, there are no more disulfide bonds that are found. And so when we run our second direction of electrophoresis, we know that the peptides, fragments that do not have any disulfide bonds, such as peptide fragment 3 and peptide fragment 7, are going to align diagonally on a diagonal line. And that's exactly what we see in our diagonal electrophoresis gel, that peptide fragment 3 and peptide fragment 7, which are the only 2 that do not have any disulfides, they align diagonally on this line. And any of the peptide fragments that do not align on the fragment, such as all of these other peptide fragments that are listed here, they do contain some kind of disulfide bond. So now to distinguish between the interchain and the intra chain disulfide bonds. So notice that what we said previously is that the peptide fragments that contain interchain disulfides, such as this one here and this one over here, those peptide fragments will get smaller and ultimately travel faster in the gel. And because they travel faster in the gel, notice that they are below the diagonal line. These fragments, because they contained, disulfide bonds, inter chained disulfide bonds, they became smaller and traveled faster, and that's why we find them below the, diagonal line. Now with intra chain disulfide bonds, such as the one with peptide fragment in And that's what we exactly what we see down below that, when the intra chain disulfide bond is cleaved, peptide fragment 6 changes its shape, and it changes its shape in such a way that it travels slower through the gel than it did the first time, through electrophoresis. And so that's why it shows up above the diagonal line, it shows up above. So, essentially, what we're saying is that the way that we distinguish between interchain and intrachain disulfides and diagonal electrophoresis is by looking at the migration of the peptides relative to the diagonal line that forms. And so peptides that migrate faster, which are these peptides here, are going to, have some kind of interchain disulfide bond, and peptide fragments that migrate slower are going to have an intrachain disulfide. And so this concludes our lesson here on the difference between inter chain and inter chain disulfides in diagonal electrophoresis results, and we'll be able to get a little bit of practice in our next video. So I'll see you guys there.
4
Problem
Problem
In the plot below, circle the point(s) representing peptides with intrachain disulfides.
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5
Problem
Problem
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.