Overview of Direct Protein Sequencing - Video Tutorials & Practice Problems
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Overview Of Direct Protein Sequencing
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and this video, we're going to do an overview of direct protein sequencing, and this video is actually quite unique because of the fact that it is just an overview. And so we're going to mention a bunch of different techniques that we have not yet talked about. But we will cover each of those techniques and more detail as we move forward in our course. And so in this video, I don't want you guys to get caught up in any one particular step or technique, because I want you guys to focus on the overall big picture of direct protein sequencing. And I want you to remember that a lot of our confusion on these individual techniques is gonna be cleared up as we move forward in our course. And so that's so that being said, let's get started. So first, I want you guys to recall that the primary level of protein structure is actually really important to biochemists because it dictates all the other levels of protein structure as well as the functions of that protein. And the primary level of protein structure actually includes both the amino acid composition or the number and types of amino acids, as well as the amino acid sequence in the amino acid sequence, is also known as just the protein sequence, which is really just the particular order of amino acids from the n terminal end of the protein to the C terminal end of the protein and biochemist can use the sequence of a protein tow. Obtain mawr information about that protein because they're able to use the sequence of the protein to predict both the overall three dimensional structure of that protein, as well as the functions of that protein just by having the protein sequence. And so it's really important for biochemist tohave methods, different methods for obtaining the protein sequence of unknown protein. And that's really what this video is all about now. One thing that's important to keep in mind about our overview of direct protein sequencing is that most proteins in nature are actually quite large, and they can vary from having several hundreds of amino acids to several thousands of amino acids. And it's actually really important that we cleave and fragment our large proteins down into smaller peptide fragments before prior toe, actually sequencing them. And so the reason for exactly why we need to cleave down our large proteins into fragments before sequencing them. We'll talk about that reason later on in our course in another video. But for now, I just want you to know that in our example down below, we're gonna be mentioning a bunch of different techniques that are used just to cleave and fragment our protein down into smaller pieces. So keep that in mind. And in our example below, what you'll notice is that we have a diagram. And literally you guys can use this diagram as a map that will guide you guys through the next set of lesson videos. And so the beginning of our map is over here on the far left. And essentially what we're gonna do is follow the arrows, uh, to the end of our map. And this first portion of our map over here is really just review. And so we know that when we're interested in sequencing of protein, we're gonna need a source of that protein and typically the source they're going to be cells. So here we're performing sell license so that we can performed protein extraction and obtain all of our proteins, especially the proteins of interest. So here we have a protein mixture where you can see we have a bunch of different types of proteins. We've got these green proteins, black proteins and blue proteins and great proteins. And they're all different colors and shapes. And you can see we've got this orange protein right here, which is the particular protein of interest in this example. And so when we have a protein mixture, we know that we can perform a set of ah, uh, different protein purification techniques such as all of the different types of chromatography that we already covered in our previous lesson. Videos such as, um ion exchange, chromatography size, exclusion, affinity, chromatography H PLC, and so upon protein purification were able to take our particular protein of interest and were able to isolate that particular protein of interest. And so over here we have our isolated, purified protein. And so, uh, this is really where you guys are on our map. So again, nothing that we've talked about from this point through to this point is new information. It's all review from our previous videos. And so what you can see here is that once we have our isolated, purified protein. We have these two different paths that we could take. We could take this path going up leading to a sequence protein, or we could take the path to the right that leads to the bottom of our diagram. That also leads to a sequence protein at the very bottom down below. Show him here. So we have these two different paths that we can take. And so, for those of you guys that have already seen our videos on Tanna Mass spectrometry and peptide mass fingerprinting, you know that this path up here is a a gold standard for sequencing proteins. But if you haven't yet gotten to those videos on Tanna mass spectrometry and peptide mass fingerprinting, we're going to talk about those videos very shortly. So don't be too worried. And essentially, what you guys should know is that this path up here is really the gold standard path, and it's a really short path to get us the result that we want, which is a sequenced orange protein here, the protein of interest. But what happens if your lab does not have access to a mass spectrometer or tandem mass spectrometer? How do you perform this technique up here? Uh, without a tan. A mass spectrometer? Well, you can't. And so we need a different technique to be able to sequence our protein. And really, that's what this right path leading down into our diagram is all about. And so what you'll see is that we can take our sample, are isolated, purify it proteins and weaken, split it into X number of samples. Meaning however many samples we want essentially and, uh, in this example in particular, we're splitting our isolated purified protein into just three different samples. Number sample number one, sample number two and sample number three. And so going down following the arrow, which will notice, is that we encounter the path for sample number three here, uh, first and then we encounter sample number two, and then last we encounter sample number one. But which will also notice is that for sample number three and sample number two past that they have dotted lines, and those dotted lines are essentially just saying that technically, we could go directly into sample three and sample too. But it's not really the best and the most logical path and so the best path is actually following the solid line over and starting with sample number one. And that's exactly what we're gonna do here. We're gonna essentially skip these dotted lines here. We'll come back to them later, but we're going to start with sample number one. So essentially with sample number one. What we could do is take a little bit of our isolated, purified protein here, and we can take it and put it in sample number one. And then what we can do is we can treat our isolated, purified protein here with a chemical known as F D N B. And again, this chemical is gonna be a chemical that will talk more about as we move forward in our course. And, uh, don't get too caught up in exactly what it does, but very briefly here. Essentially, what its use for is determining the end terminal amino acid residue of the protein, and that could be very useful for biochemists. And we'll talk about why in our later videos now, after we treat our isolated, purified protein sample here with F, d and B, we can, uh, subjected to our first cleavage techniques. So remember our large are protein of interest here. Most of our proteins are going to be very large. And so even though our protein is being shown toe have Onley eight amino acid residues shown by these eight orange dots, We can imagine that this protein right here is actually really large, with several hundreds to thousands of amino acids. Now, our first cleavage, uh, technique is called amino acid, Hydraulics, ISS. And again, we're gonna talk more details about this technique as we move forward in our course. But essentially what it does, is it non specifically cleaves all of the peptide bonds in our protein. And so all these dotted black lines that we see vertically going up and down like this are just referring to the cleavage of those peptide bonds and it results and free amino acids, or amino acids that are not linked via co violent, um, peptide bonds. And so, uh, after amino acid hydraulics is weaken. Subject these free amino acids here to a techniques such as H PLC, and we can use the H P L. C to determine the amino acid composition and recall that the amino acid composition is on Lee, referring to the numbers or the abundance of amino acids, as well as the types of amino acids that are present. But the composition does not tell us the order of amino acids or the sequence of amino acids. And so essentially, sample number one does not get us toe where we need to be, which is obtaining the sequence of our proteins. But because it does give us the composition of our sequence, uh, the composition of our protein. We can use this composition to strategically select the appropriate re agents that we're going to use in sample number two as well as in sample number three. So that's why it's important to use sample number one here first and amino acid hydraulics, ISS before we actually get to sample to and sample three. It's just to be able to strategize exactly what re agents we should use. So notice that the next step here is to essentially, uh, follow this arrow through, and we can essentially either go to step number two or we could follow the arrow and go to step number three, and it really doesn't matter which path that we're gonna take. So first we're going to follow Step two. And then after we followed up to we'll move on to step three. So in sample number two, essentially, what we have here is we could take another little bit of our isolated protein sample, and we can move it down into our second step down below. And we could have subject this isolated protein, uh, here to a chemical re agent to perform chemical cleavage. And so chemical cleavages. Essentially, when we're used very specific chemicals to cleave very specific peptide bonds. And again, we're gonna talk Maura about chemical cleavage as we move forward later in our course. And there are a lot of different chemicals that we can use to perform chemical cleavage. And here we're performing chemical cleavage that cleaves this one particular peptide bond. So recall that with amino acid hydrologists, we cleaved all of the peptide bonds. But with chemical cleavage here, we're only cleaning this one particular peptide bond. Now, uh, it's not that it always cleans one particular peptide bond, but chemical cleavage will Onley cleave very specific peptide bonds, so sometimes it can cleave at multiple places. But here it's on leak leaving at one position. And so this one position here generates two different fragments, this three residue fragment and this five residue fragments, and we can see those down below the three residue and the five residue peptide fragments. Now, what you'll notice is that all of these arrows here are merging down to this one arrow down here. So before we continue forward, let's move over to sample number three. And so with sample three, essentially, what we could do is we could take our last bit of our isolated, purified protein sample and we can bring it down to, uh, this sample three here. And so in this sample three weaken subject are purified protein to a pep today's. And this is essentially our third peptide cleavage technique, our third protein, cleavage technique. And so peptide aces are enzymes because they end in A S e and their enzymes that cleave very specific peptide bonds. And this particular example with this particular pep today's that's being used, uh, is cleaving at two different positions. It's cleaving at this peptide bond here in this peptide bond here, and that generates three different fragments, a two residue fragment and 23 amino acid residue fragments and we could see those fragments down below here. Now, there are actually ah lot of different types of peptide aces, and again, we're gonna talk about all of these different peptide aces in more detail. Aziz, we move forward in our course now, after we've generated all of these peptide fragments from these different cleavage techniques, what we could do is we can separate all of the peptide fragments using some kind of separation technique such as H p l C. And once we've separated all the peptide fragments, we can subject each of these peptide fragments individually to, uh, peptide sequencing to sequencing the peptides via techniques such as Edmund Degradation. And so Edmund Degradation requires that large proteins be cleaved down into fragments before they can actually be sequenced. And again, the reason why we'll talk about and some of our later videos. But for now, what we need to know is that after we cleave are fragments down in tow, our protein down into fragments will separate the fragments and then we'll subject them to sequencing, and then we can use this. Edmund Degradation results essentially to obtain the end result, which is our sequenced orange peptides so you can see all of the one letter amino acid codes for those residents have been revealed and we've successfully sequence are protein. And so essentially here. What you can see is that this is really a map to the next set of lesson videos. And so it will first start talking about F d N B moving forward. And then we'll talk about amino acid, hydraulics, ISS, followed by chemical cleavage. And then lastly, we'll end up with Pep Today's is and Edmund degradation. And so that's important to keep in mind that this is really just a map. This is where you guys are now, And, uh, this path here, uh, going down is essentially the path that we're gonna take as we move forward with our lesson videos. So I would suggest you guys print off this map and have it side by side with you as we move forward in our lesson video. So you guys know exactly where we are in this map. So this here, uh, ends our initial lesson on the overview of direct protein sequencing, 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 is a protein sequencing technique?
A
HPLC.
B
Amino acid hydrolysis.
C
Edman Degradation.
D
Peptidase/chemical cleavage.
3
concept
Overview Of Direct Protein Sequencing
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So we already know that several different techniques are used to determine the sequence of a protein from our previous lesson video, where recovered the map for the overview of direct protein sequencing. And so in this video we're gonna do a relatively quick overview of the techniques to sequence of protein in this chart format below and so notice that these techniques can be broken up into two general categories. And those are protein cleavage techniques, as well as direct protein sequencing techniques. And so notice for our protein cleavage techniques. We have three that are listed, and those are amino acid. Hydraulics is chemical cleavage and pep, today's is. And then for our protein sequencing technique, we only have one that is listed, and that is Edmund Degradation. Now for our protein cleavage techniques recall that those air used to essentially take a large protein and break them down into smaller pieces. And so we're going to talk about the re agents that are used, the peptide bonds that air cleaved and the result for each of these protein cleavage techniques. But again, we're gonna talk mawr details about each of these cleavage techniques as we move forward in our course Now for our first technique here. Amino acid, hydraulics, ISS. The result, the re agent that's used is actually six Mohler hydrochloric acid or six Mohler HCL. And from our previous lesson video where recovered the map of direct protein sequencing you may have picked up that amino acid hydraulics. ISS is specifically used to cleave all of the peptide bonds. So the peptide bonds that air cleaved are all and it non specifically cleaves all of these peptide bonds, and the result is that it generates a bunch of free amino acids and free amino acids. Recall are not co violently linked together via peptide bonds, and they're free and separate and independent from one another. Now, for our next protein cleavage technique, what we have is chemical cleavage, and the re agent is, of course, going to be a chemical. And so there are many different types of chemicals that could be used to cleave a protein, and we're gonna talk about ah, lot of those common chemicals that are used later on in our course. But for the peptide bonds that air cleaved, what we're going to put is that it cleaves very specific peptide bonds. So not all of the peptide bonds are cleaved Onley Very specific ones between very specific residues are cleaved. And again we're gonna talk mawr details about these chemicals and the bond that they cleaved later in our course. And the result is going to be the generation of peptide fragments, so we could put peptide fragments in here now for the pep. Today's is our last chemical, our last protein cleavage technique, pep. Today's ends in a S E. And anything that ends in a C. It's a good indicator that it's going to be an enzyme. And so for the re agents here, we can put that it is indeed an enzyme for the pep today's is, and the specific peptide bonds that are cleaved are actually going to be very specific peptide bonds. And so, essentially, what that saying is that it does not cleave all of the peptide bonds and it doesn't cleave random peptide bonds. It cleaves very specific peptide bonds between very specific residues. And again, there are lots of different peptides is that are out there and moving forward. We're gonna talk about some of the more common ones that your professor might expect you guys know and then the result of pep. Today's treatment is also going to be peptide fragments, smaller peptide fragments. So we know that once we cleave, are protein down into smaller fragments using protein cleavage techniques were able to follow it up using protein sequencing techniques such as Edmund Degradation. And so Edmund Degradation is really just n terminal. It's defined as end terminal sequencing and terminal protein sequencing. And so, essentially, what that means is that it sequences a protein starting at the end terminal end and finishes sequencing the protein at the C terminal end. And again, we're gonna talk a lot more details about exactly how Edmund Degradation works later on in our course. But for now, this is a good overview of the techniques that we're gonna be talking about moving forward. And so this here concludes our lesson on the overview of techniques to sequence of protein, and I'll see you guys in our practice videos
4
Problem
Problem
Appropriately match each option to each description. Options may be used more than once.
a. Cyanogen bromide (CNBr). _____ An enzyme that cleaves specific peptide bonds.
b. Edman Degradation. _____ Nonspecifically cleaves all peptide bonds.
c. Elastase. _____ A chemical that breaks specific peptide bonds.
d. 6M HCl. _____ Used as an N-terminal protein sequencing technique.
_____ Used as a peptide cleavage technique.
_____ Results in smaller peptide fragments.
_____ Results in free amino acids.
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Problem
Problem
Appropriately match each option to each reagent. Options may be used more than once.
