Overview of Direct Protein Sequencing - Online Tutor, Practice Problems & Exam Prep

Direct protein sequencing involves understanding the primary structure, which includes amino acid composition and sequence. Techniques such as amino acid hydrolysis, chemical cleavage, and peptidases are essential for fragmenting large proteins into smaller peptides. Following cleavage, Edman degradation is employed for N-terminal sequencing. This process allows biochemists to predict protein structure and function, emphasizing the importance of accurate sequencing methods in proteomics. Techniques like HPLC are utilized for separation and analysis, ensuring precise identification of amino acid sequences.

Overview Of Direct Protein Sequencing

In this video, we're going to do an overview of direct protein sequencing. This video is quite unique because it is just an overview. 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 in more detail as we move forward in our course. In this video, I don't want you to get caught up in any one particular step or technique because I want you to focus on the overall big picture of direct protein sequencing. A lot of our confusion on these individual techniques will be cleared up as we move forward in our course. With that being said, let's get started.

First, I want you to recall that the primary level of protein structure is really important to biochemists because it dictates all the other levels of protein structure, as well as the functions of that protein. The primary level of protein structure includes both the amino acid composition or the number and types of amino acids, as well as the amino acid sequence. The amino acid sequence is also known as just the protein sequence, which is the particular order of amino acids from the N-terminal end of the protein to the C-terminal end of the protein. Biochemists can use the sequence of a protein to obtain more 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. It's really important for biochemists to have methods, different methods, for obtaining the protein sequence of an unknown protein. That's really what this video is all about.

It's important to keep in mind that most proteins in nature are quite large. They can vary from having several hundreds of amino acids to several thousand amino acids. It's really important that we cleave and fragment our large proteins down into smaller peptide fragments before sequencing them. The reason for why we need to cleave down our large proteins into fragments before sequencing them will be discussed later in our course in another video. For now, just know that in our example down below, we're going to be mentioning different techniques that are used to cleave and fragment our protein down into smaller pieces. Keep that in mind.

In our example below, what you'll notice is that we have a diagram, which you can use as a map that will guide you through the next set of lesson videos. The beginning of our map is over here, on the far left, and essentially what we're going to do is follow the arrows to the end of our map. This first portion of our map over here is really just a review. We know that when we're interested in sequencing a protein, we're going to need a source of that protein, typically cells. So here, we're performing cell lysis so that we can perform protein extraction and obtain all of our proteins, especially the proteins of interest.

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, blue proteins, and gray proteins, and they're all different colors and shapes. You can see we've got this orange protein right here, which is the particular protein of interest in this example. When we have a protein mixture, we know that we can perform a set of different protein purification techniques, such as all of the different types of chromatography that we already covered in our previous lesson videos, such as ion exchange chromatography, size exclusion, affinity chromatography, HPLC. Upon protein purification, we're able to take our particular protein of interest and isolate it. This is really where we're starting on our map. Nothing we've talked about from this point through this point is new information; it's all a review from our previous videos.

Once we have our isolated purified protein, we have two different paths we can take. We can take this path going up leading to a sequenced protein, or we can take the path to the right that also leads to a sequenced protein at the very bottom, shown here. For those of you that have already seen our videos on tandem mass spectrometry and peptide mass fingerprinting, you know that this upper path is a gold standard for sequencing proteins. If you haven't seen those videos on tandem mass spectrometry and peptide mass fingerprinting yet, we're going to talk about them very shortly. Don't be too worried. This path up here is the gold standard path, and it's a short path to get us the result 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 a tandem mass spectrometer? How do you perform this technique up here without a tandem mass spectrometer? You can't. We need a different technique to sequence our protein. That's what this right path, leading down into our diagram, is all about.

We can take our sample, our isolated purified protein, and we can split it into as many samples as we want. In this particular example, we're splitting our isolated purified protein into just three different samples: sample number 1, sample number 2, and sample number 3. Going down following the arrow, what you'll notice is that we first encounter the path for sample number 3, then sample number 2, and lastly sample number 1. You'll also notice that for sample number 3 and sample number 2 paths, they have dotted lines, which are essentially just to say that technically we could go directly to samples 3 and 2, but it's not really the best and most logical path. The best path is actually following the solid start with sample number 1, and that's exactly what we're going to do here. We're going to skip these dotted lines here. We'll come back to them later, but we're going to start with sample number 1.

With sample number 1, what we can do is take a little bit of our isolated purified protein here and put it in sample number 1. Then we can treat our isolated protein with a chemical known as FDNB. Don't get too caught up in exactly what it does, but briefly, FDNB is used for determining the N-terminal amino acid residue of the protein. That can be very useful for biochemists, and we'll talk about why in our later videos. After we treat our isolated purified protein sample with FDNB, we can subject it to our first cleavage technique. Remember, our large protein of interest here, most of our proteins, are going to be very large. Even though our protein is being shown to have only 8 amino acid residues, shown by these 8 orange dots, we can imagine that this protein is actually really large with several hundred to a thousand of amino acids.

Our first cleavage technique is called amino acid hydrolysis. We're going to talk about this in more detail later in our course. Essentially, it non-specifically cleaves all of the peptide bonds in our protein. All these dotted black lines that we see vertically going up and down are just referring to the cleavage of those peptide bonds. It results in free amino acids or amino acids that are not linked via covalent peptide bonds. After amino acid hydrolysis, we can subject these free amino acids to a technique such as HPLC. We can use HPLC to determine the amino acid composition. Recall that the amino acid composition only refers to the numbers or the abundance of amino acids, as well as the types of amino acids that are present. However, the composition does not tell us the order of amino acids or the sequence of amino acids.

Essentially, sample number 1 does not get us to where we need to be, which is obtaining the sequence of our proteins. But because it gives us the composition of our protein, we can use this composition to strategically select the appropriate reagents that we're going to use in sample number 2, as well as in sample number 3. That's why it's important to use sample number 1 here first in amino acid hydrolysis before we get to samples 2 and 3. Notice that the next step is to either follow this arrow through to step number 2 or follow the arrow and go to step number 3. It doesn't really matter which path we're going to take. First, we'll follow step 2, and then we'll move on to step 3.

In sample number 2, we can take another little bit of our isolated protein sample and move it down into our second step down below. We can then subject this isolated protein to a chemical reagent to perform chemical cleavage. Chemical cleavage essentially involves using very specific chemicals to cleave very specific peptide bonds. We're going to talk more about chemical cleavage as we move forward later in our course. There are a lot of different chemicals that we can use to perform chemical cleavage. Here, we're performing chemical cleavage that cleaves this one particular peptide bond. Not that it always cleaves one particular peptide bond, but chemical cleavage will only cleave very specific peptide bonds. This one position here generates two different fragments, a 3-residue fragment and a 5-residue fragment. We can see those down below, the 3-residue and the 5-residue peptide fragments.

All these arrows here are merging down to this one arrow down here. Before we continue forward, let's move over to sample number 3. In sample 3, we can take our last bit of our isolated purified protein sample and bring it down to this sample here. In this sample, we can subject our purified protein to a peptidase. Peptidases are enzymes, and they cleave very specific peptide bonds. In this particular example, the peptidase that's being used is cleaving at two different positions. It's cleaving at this peptide bond here and this peptide bond here. That generates three different fragments, a 2-residue fragment, and two 3-amino acid residue fragments. We can see those fragments down below here.

There are actually a lot of different types of peptidases. We're going to talk about all of these different peptidases in more detail as we move forward in our course. After we've generated all these peptide fragments from these cleavage techniques, we can separate all the peptide fragments using some kind of separation technique, such as HPLC. Once we've separated all the peptide fragments, we can subject each of these peptide fragments individually to peptide sequencing, sequencing the peptides, via techniques such as Edman degradation. Edman degradation requires that large proteins be cleaved down into fragments before they can actually be sequenced. The reason why we'll talk about in some of our later videos. But for now, what you need to know is that after we cleave our fragments down into our protein down into fragments, we'll separate the fragments, and then we'll subject them to sequencing. We can use the Edman degradation results essentially to obtain the end result, which is our sequenced orange peptide. You can see all the one-letter amino acid codes for those residues have been revealed, and we've successfully sequenced our protein.

This is really a map to the next set of lesson videos. We'll first start talking about FDNB moving forward, and then we'll talk about amino acid hydrolysis, followed by chemical cleavage, and then lastly, we'll end up with peptidases and Edman degradation. It's important to keep in mind that this is really just a map. This is where you are now, and this path here, going down is essentially the path that we're going to take as we move forward with our lesson videos. I would suggest you print off this map and have it side by side with you as we move forward in our lesson video, so you know exactly where we are in this map. This here, ends our initial lesson on the overview of direct protein sequencing, and we'll be able to get some practice in our next video. I'll see you there.

Which of the following is a protein sequencing technique?

• A. HPLC.
• B. Amino acid hydrolysis.
• C. Edman Degradation.
• D. Peptidase/chemical cleavage.

Overview Of Direct Protein Sequencing

So we already know that several different techniques are used to determine the sequence of a protein from our previous lesson video where we covered the map for the overview of direct protein sequencing. And so in this video, we're going to do a relatively quick overview of the techniques to sequence a protein in this chart format below. Notice that these techniques can be broken up into 2 general categories: protein cleavage techniques, as well as direct protein sequencing techniques. For our protein cleavage techniques, we have three that are listed: amino acid hydrolysis, chemical cleavage, and peptidases. And then for our protein sequencing technique, we only have one that is listed, and that is Edman degradation. Protein cleavage techniques are used to essentially take a large protein and break them down into smaller pieces. We're going to talk about the reagents that are used, the peptide bonds that are cleaved, and the result for each of these protein cleavage techniques. But again, we're going to talk more details about each of these cleavage techniques as we move forward in our course.

Now for our first technique here, amino acid hydrolysis, the reagent that is used is actually 6 molar hydrochloric acid or HCl. From our previous lesson video where we covered the map of direct protein sequencing, you may have picked up that amino acid hydrolysis is specifically used to cleave all of the peptide bonds. It non-specifically cleaves all of these peptide bonds. The result is that it generates a bunch of free amino acids. Free amino acids, recall, are not linked together via peptide bonds, and they're free and separate and independent from one another.

For our next protein cleavage technique, what we have is chemical cleavage, and the reagent is, of course, going to be a chemical. There are many different types of chemicals that can be used to cleave a protein. We're going to talk about a lot of those common chemicals that are used, later on in our course. The peptide bonds that are cleaved are very specific. So not all of the peptide bonds are cleaved, only very specific ones between very specific residues are cleaved. The result is going to be the generation of peptide fragments.

For peptidases, our last protein cleavage technique, peptidases ends in "ase". Anything that ends in "ase" is a good indicator that it's an enzyme. For the reagents here, we can state that it is indeed an enzyme for peptidases. The specific peptide bonds that are cleaved are very specific. Essentially, 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. There are lots of different peptidases that are out there, and moving forward, we're going to talk about some of the more common ones that your professor might expect you to know. The result of peptidase treatment is also going to be smaller peptide fragments.

We know that once we cleave our protein down into smaller fragments using protein cleavage techniques, we're able to follow it up using protein sequencing techniques such as Edman degradation. Edman degradation is really just N-terminal sequencing. Essentially, it sequences a protein starting at the N-terminal end and finishes sequencing the protein at the C-terminal end. We're going to talk a lot more details about exactly how Edman degradation works later on in our course. But for now, this is a good overview of the techniques that we're going to be talking about moving forward.

This here concludes our lesson on the overview of techniques to sequence a protein, and I'll see you guys in our practice videos.

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.

Alright. So this practice problem wants us to match each option on the left over here with each description on the right. It says that options may be used more than once. The four options are cyanogen bromide (CNBr), Edman degradation, elastase, and 6 molar hydrochloric acid (HCl). 6 molar HCl and Edman degradation should sound familiar to you guys from our previous lesson videos. But cyanogen bromide and elastase, we have not yet talked about. Just by looking at them a bit closely, cyanogen bromide is a chemical, and indeed, it's used for chemical cleavage. Now, elastase ends with "ase," indicating it's an enzyme. Elastase is indeed an enzyme, more specifically, a peptidase or an enzyme that cleaves peptide bonds. Looking at our first description, "an enzyme that cleaves specific peptide bonds," we know that multiple different reagents can be used to cleave specific peptide bonds, such as many different types of chemicals and peptidases. But because it specifically mentions an enzyme, we know it refers to our peptidase here, elastase. We can go ahead and assign option C for that description. The next description states, "nonspecifically cleaves all peptide bonds." The keyword here is "all" because that eliminates cyanogen bromide and elastase since they don't cleave all peptide bonds; they cleave very specific peptide bonds. We know that 6 molar hydrochloric acid is used to induce amino acid hydrolysis that will nonspecifically cleave all peptide bonds. For the next description, we assign option D. The subsequent description states, "a chemical that breaks specific peptide bonds." Because it specifies "specific peptide bonds," we know it cannot be 6 molar hydrochloric acid. And because it says "chemical," we know it cannot be elastase, since elastase is not a chemical, but an enzyme. Cyanogen bromide is the chemical used to cleave these specific peptide bonds. Thus, we assign option A for this description. The next description reads, "used as an N-terminal protein sequencing technique." There's only one protein sequencing technique listed—Edman degradation. We will talk more about Edman degradation later in our course, but for now, we assign option B for this description. The next has "used as a peptide cleavage technique." Cyanogen bromide is used as a peptide cleavage technique to cleave specific peptide bonds, and so is elastase. It's a peptidase used to cleave specific peptide bonds. Also, 6 molar hydrochloric acid, although it cleaves all peptide bonds, is still used as a peptide cleavage technique. Our answers for this one would be options A, C, and D. Next, the description says, "results in smaller peptide fragments." The term "fragments" implies that the peptide is broken into smaller pieces, yet not all peptides bonds are broken. It could be a type of peptidase, such as elastase, or a chemical, such as cyanogen bromide. Options A or C are appropriate here. Lastly, it states, "results in free amino acids." That implies all of the peptide bonds are cleaved. If it cleaves all peptide bonds nonspecifically, we can generate free amino acids. Thus, 6 molar hydrochloric acid would be the proper choice for this one, option D. That concludes this practice problem, and I'll see you guys in our next video.

Appropriately match each option to each reagent. Options may be used more than once.

a. Amino acid hydrolysis.                           _____ Hydrazine.

b. Chemical cleavage.                                _____ Trypsin.           

c. Peptidase.                                              _____ 6M Hydrochloric acid.

                                                                   ______ β-mercaptoethanol + iodoacetate.

                                                                   ______ Chymotrypsin.

So this practice problem wants us to appropriately match each option on the left-hand side to each reagent on the right-hand side, and it says that options may be used more than once. The three options are amino acid hydrolysis, chemical cleavage, and peptidase. Some of you might recall from your previous biology courses that both trypsin and chymotrypsin are relevant peptidases of our digestive systems, and they are used for breaking down large proteins into smaller peptide fragments. Although we have not yet talked about trypsin or chymotrypsin in much detail so far, we are going to focus on these two particular peptidases, as well as other peptidases that your professor might expect you guys to know. Because they are peptidases, we can mark them with the answer option c here. Now, what you'll also recognize is that beta-mercaptoethanol is a chemical that we discussed in our previous lessons for protein denaturation and the Anfinsen Experiment. We know that beta-mercaptoethanol is specifically used to cleave disulfide bonds, which are covalent bonds that form between the R groups of two cysteine residues. Because we know beta-mercaptoethanol is a chemical, and it's specifically used to cleave disulfide bonds, that automatically should give us a hint that using beta-mercaptoethanol with iodoacetamide is going to be a chemical cleavage process. So, we can put option b here. Even though we have not yet talked about iodoacetamide, later in our course, we're going to discuss what iodoacetamide is and how it can be used with beta-mercaptoethanol, and the effect that these two chemicals have together on an amino acid and on a protein. What we have left here is hydrazine and 6 molar hydrochloric acid. We know that 6 molar hydrochloric acid, or 6M HCl, is used specifically for amino acid hydrolysis, and it cleaves all of the peptide bonds nonspecifically and generates free amino acids. We're going to talk more about the details of amino acid hydrolysis moving forward in our course. But for now, we can put option a here for this particular reagent. Hydrazine, which we have not yet discussed, is a chemical that's specifically used for chemical cleavage. Because it's used for chemical cleavage, we can assign option b here. Again, we're going to discuss what hydrazine does and the impact it has on proteins later in our course, but for now, you can just note that it's used specifically to determine the C-terminal.

Here’s what students ask on this topic:

What is the primary structure of a protein and why is it important?

The primary structure of a protein refers to its amino acid sequence, which is the specific order of amino acids from the N-terminal end to the C-terminal end. This sequence is crucial because it dictates the protein's three-dimensional structure and, consequently, its function. The primary structure determines how the protein will fold into its secondary, tertiary, and quaternary structures. Any change or mutation in the amino acid sequence can significantly affect the protein's function, potentially leading to diseases or malfunctions. Therefore, understanding the primary structure is essential for biochemists to predict protein behavior and design targeted interventions.

Why is it necessary to cleave large proteins into smaller fragments before sequencing?

Cleaving large proteins into smaller fragments is necessary because most sequencing techniques, such as Edman degradation, are more effective on smaller peptides. Large proteins, which can consist of hundreds to thousands of amino acids, are challenging to sequence directly due to their complexity and size. By breaking them down into smaller, more manageable fragments, biochemists can accurately determine the sequence of each fragment. These smaller sequences can then be pieced together to reconstruct the entire protein sequence. This step is crucial for ensuring precise and efficient protein sequencing.

What are the main techniques used for protein cleavage in direct protein sequencing?

The main techniques used for protein cleavage in direct protein sequencing are amino acid hydrolysis, chemical cleavage, and peptidases. Amino acid hydrolysis uses 6 M HCl to non-specifically cleave all peptide bonds, generating free amino acids. Chemical cleavage employs specific chemicals to target and cleave particular peptide bonds, resulting in peptide fragments. Peptidases are enzymes that cleave specific peptide bonds between certain amino acids, also producing peptide fragments. These techniques are essential for breaking down large proteins into smaller pieces, facilitating subsequent sequencing steps.

How does Edman degradation work in protein sequencing?

Edman degradation is a method used for N-terminal sequencing of proteins. It sequentially removes one amino acid at a time from the N-terminal end of a peptide. The process involves treating the peptide with phenylisothiocyanate (PITC) under mildly alkaline conditions, which reacts with the N-terminal amino acid to form a phenylthiocarbamoyl derivative. This derivative is then cleaved to yield a phenylthiohydantoin (PTH)-amino acid, which can be identified through chromatography. The cycle is repeated to determine the sequence of the remaining peptide. This method is effective for sequencing peptides up to about 50 amino acids in length.

What role does HPLC play in direct protein sequencing?

High-Performance Liquid Chromatography (HPLC) plays a crucial role in direct protein sequencing by separating peptide fragments and free amino acids generated during protein cleavage. HPLC allows for the precise identification and quantification of these components based on their unique retention times. This separation is essential for subsequent sequencing steps, such as Edman degradation, as it ensures that each fragment can be individually analyzed. HPLC enhances the accuracy and efficiency of protein sequencing by providing a reliable method for isolating and characterizing the components of complex protein mixtures.