In this video, we're going to talk about ordering cleaved fragments and how that even relates to Edman degradation sequencing. So from our previous lesson videos, we know that Edman degradation sequencing is limited to sequencing small peptides with less than 50 amino acid residues, which means that most proteins in nature, which are much larger than 50 amino acid residues, are going to need to be cleaved down into smaller peptide fragments before we can sequence them with Edman degradation. And so after fragmenting a large protein down into smaller peptide fragments, we know that we need to separate those peptide fragments and then sequence each of them separately via Edman degradation. And so you can imagine if we have a large protein, we're going to need to cleave it down into smaller peptide fragments, separate and then sequence each of those peptide fragments separately via Edman degradation. But then there's a question that arises and that is, how do we determine the order of these peptide fragments in the original protein? And so that's exactly what this question here is asking. It's asking how do we determine the order of the fragments in the original protein sequence. And so down below in this image, we're going to help across to the right of the diagram, and then we start with step 2 down below and move our way to the right again. And so, with this original protein here, notice what it has are question marks inside of the amino acid residues, which means that it has an unknown sequence. And just for, limitation on the amount of space I have on this page, I only have 7 amino acid residues in this original protein. But we know that most proteins in nature are much larger and have several hundreds to several thousands of amino acid residues. So I want you guys to imagine that this original protein here has many, many more amino acids, several 100 or even 1000. And so if we want to determine the sequence of this large original protein here, we know that we're going to need to first fragment it down into smaller peptide fragments so that we can sequence it with Edman degradation. And that's exactly what this first step is. It's to cleave our protein into fragments. And we've talked about many different protein cleavage techniques including chemical cleavage and proteases or peptidases. And so here we're using chemical cleavage with cyanogen bromide. And we know that once we take our original protein and fragment it down, that it's going to generate a bunch of protein or peptide fragments here. And so we have 2 dipeptide fragments and one tripeptide fragment. Notice that they still have these question marks. So after fragmenting our protein into fragments, we're going to need to separate those protein fragments and then sequence each of them via Edman degradation. And that's exactly what step number 2 is down below. So notice that we're going to separate each of these fragments over here, and then we are going to sequence each of the fragments separately via Edman degradation. And notice that we've changed all of these question marks into actual one-letter amino acid codes because we've revealed the sequence of the fragments. So now, this is exactly where this question comes into play. How do we determine the order of these fragments in the original protein sequence up above? So maybe this fragment here showed up at the very beginning, but maybe it showed up at the very end, or maybe it showed up over here. How do we determine exactly the order that these fragments came in? And so that's exactly what step number 3 is. It's what the order of the fragments is. It's what is the order of the fragments in the original protein in terms of being first, second, or third from the N terminal to the C terminal end. And so at this point, what we're going to do is a little bit of experimentation. So what that means is we're going to fill in these blanks here, but then later on, we might change them. So keep that in mind as we move along. And so, maybe the order of these fragments is pretty easy. Maybe it's literally in this exact order, where this is the first, fragment, and this FL here is the second fragment, and maybe this third fragment here is the 3rd fragment. I don't know. Let's check. So let's say that that is one possibility, and that seems to be what maybe this is trying to tell us over here, where here we have our original peptide, our original sequence, and we're trying to determine the possibilities for our original sequence. So maybe this RM fragment came first, just like what we said. And maybe this FL fragment came second. So, let's go ahead and put FL in for the second one. And then maybe this GYM fragment came 3rd. So, let's put that in a different color over here, GYM. So, maybe this is a possibility, but we have to remember that we cleaved our peptide, our protein, our original protein with cyanogen bromide, and cyanogen bromide cleaves next to methionine residues on the carboxyl side. And so that means that this methionine residue here, this peptide bond on the carboxyl side of it is going to be cleaved. And so if this were the original sequence, essentially this peptide bond would be cleaved. And this methionine residue doesn't have a peptide bond on its carboxyl side since it's literally the last residue of the peptide. And so that means that if this were the sequence, we would generate 1 dipeptide and then 1 pentapeptide upon cleavage with cyanogen bromide. But that's not the results that we actually got up above. We got 3 different peptide fragments, 2 dipeptides and one tripeptide. And so, it turns out that this here is not the correct sequence. So we can go ahead and get rid of that, and this is not the correct order over here. And so it turns out that the only way that we are able to generate 3 different fragments in this order is if the FL fragment is actually at the very end. And so if you don't understand that, it's okay. It's I don't really expect you guys to fully understand this process yet, but it turns out that this FL needs to go at the end. If it goes anywhere else, we won't be able to generate 3 fragments just because of how cyanogen bromide cleaves. And so we know that the FL fragment is going to be the 3rd, the last fragment at the very end, for sure. Otherwise, we wouldn't get 3 fragments. So now the question is, does the RM fragment come first, or does the RM fragment come second? And does the GYM fragment come first, or does it come second? And so notice over here with this first possibility that we have the RM fragment coming first. And so, if the RM fragment is coming first, that means that the GYM fragment must be coming second. And so, what we can do is put in the RM fragment over here. And so it turns out that both of these are valid possibilities because if we cleave with cyanogen bromide, notice that they are going to generate 3 different fragments. So it will cleave after the methionine residue here, and then also after the methionine dipeptides, and one tripeptide, just like what we saw up above. So possibility number 1 is a valid possibility. And then, looking at possibility number 2, it can cleave after this methionine residue here, and it will cleave after this methionine residue right here. And so that will generate 2 dipeptides and one tripeptide, just like what we have up here. So this sequence is different than the sequence possibility from number 1, and they are both valid sequences that seem to generate the same exact peptide fragments. So the question is asking, can you guys tell which sequence is correct? And the answer is actually no. You cannot tell which sequence is correct. So, what this means is, which of these sequences is correct with the provided amount of information. And so that actually presents an issue, that presents a problem that we have. And the problem is that by cleaving a protein with only one cleavage method, like we did up above here with cyanogen bromide, it's possible that the proper ordering of the fragments may not be possible. So it's possible that we may not be able to determine what the correct sequence is if we only cleave with 1 cleavage method. And so what that means is, typically in most scenarios, we're going to need to take our original protein and cleave it in multiple different ways. And we'll be able to understand that idea better when we get to our next video where we'll talk about how 2 or more different cleavage techniques are required to order the fragments properly and determine which of these possibilities is actually the correct possibility. And so, this here concludes our introduction to ordering cleave fragments, and how it relates to Edman degradation sequencing. And again, we'll be able to get some more practice as we move along in our next video. So I'll see you guys in those videos.
- 1. Introduction to Biochemistry4h 34m
- What is Biochemistry?5m
- Characteristics of Life12m
- Abiogenesis13m
- Nucleic Acids16m
- Proteins12m
- Carbohydrates8m
- Lipids10m
- Taxonomy10m
- Cell Organelles12m
- Endosymbiotic Theory11m
- Central Dogma22m
- Functional Groups15m
- Chemical Bonds13m
- Organic Chemistry31m
- Entropy17m
- Second Law of Thermodynamics11m
- Equilibrium Constant10m
- Gibbs Free Energy37m
- 2. Water3h 23m
- 3. Amino Acids8h 10m
- Amino Acid Groups8m
- Amino Acid Three Letter Code13m
- Amino Acid One Letter Code37m
- Amino Acid Configuration20m
- Essential Amino Acids14m
- Nonpolar Amino Acids21m
- Aromatic Amino Acids14m
- Polar Amino Acids16m
- Charged Amino Acids40m
- How to Memorize Amino Acids1h 7m
- Zwitterion33m
- Non-Ionizable Vs. Ionizable R-Groups11m
- Isoelectric Point10m
- Isoelectric Point of Amino Acids with Ionizable R-Groups51m
- Titrations of Amino Acids with Non-Ionizable R-Groups44m
- Titrations of Amino Acids with Ionizable R-Groups38m
- Amino Acids and Henderson-Hasselbalch44m
- 4. Protein Structure10h 4m
- Peptide Bond18m
- Primary Structure of Protein31m
- Altering Primary Protein Structure15m
- Drawing a Peptide44m
- Determining Net Charge of a Peptide42m
- Isoelectric Point of a Peptide37m
- Approximating Protein Mass7m
- Peptide Group22m
- Ramachandran Plot26m
- Atypical Ramachandran Plots12m
- Alpha Helix15m
- Alpha Helix Pitch and Rise20m
- Alpha Helix Hydrogen Bonding24m
- Alpha Helix Disruption23m
- Beta Strand12m
- Beta Sheet12m
- Antiparallel and Parallel Beta Sheets39m
- Beta Turns26m
- Tertiary Structure of Protein16m
- Protein Motifs and Domains23m
- Denaturation14m
- Anfinsen Experiment20m
- Protein Folding34m
- Chaperone Proteins19m
- Prions4m
- Quaternary Structure15m
- Simple Vs. Conjugated Proteins10m
- Fibrous and Globular Proteins11m
- 5. Protein Techniques14h 5m
- Protein Purification7m
- Protein Extraction5m
- Differential Centrifugation15m
- Salting Out18m
- Dialysis9m
- Column Chromatography11m
- Ion-Exchange Chromatography35m
- Anion-Exchange Chromatography38m
- Size Exclusion Chromatography28m
- Affinity Chromatography16m
- Specific Activity16m
- HPLC29m
- Spectrophotometry51m
- Native Gel Electrophoresis23m
- SDS-PAGE34m
- SDS-PAGE Strategies16m
- Isoelectric Focusing17m
- 2D-Electrophoresis23m
- Diagonal Electrophoresis29m
- Mass Spectrometry12m
- Mass Spectrum47m
- Tandem Mass Spectrometry16m
- Peptide Mass Fingerprinting16m
- Overview of Direct Protein Sequencing30m
- Amino Acid Hydrolysis10m
- FDNB26m
- Chemical Cleavage of Bonds29m
- Peptidases1h 6m
- Edman Degradation30m
- Edman Degradation Sequenator and Sequencing Data Analysis4m
- Edman Degradation Reaction Efficiency20m
- Ordering Cleaved Fragments21m
- Strategy for Ordering Cleaved Fragments58m
- Indirect Protein Sequencing Via Geneomic Analyses24m
- 6. Enzymes and Enzyme Kinetics13h 38m
- Enzymes24m
- Enzyme-Substrate Complex17m
- Lock and Key Vs. Induced Fit Models23m
- Optimal Enzyme Conditions9m
- Activation Energy24m
- Types of Enzymes41m
- Cofactor15m
- Catalysis19m
- Electrostatic and Metal Ion Catalysis11m
- Covalent Catalysis18m
- Reaction Rate10m
- Enzyme Kinetics24m
- Rate Constants and Rate Law35m
- Reaction Orders52m
- Rate Constant Units11m
- Initial Velocity31m
- Vmax Enzyme27m
- Km Enzyme42m
- Steady-State Conditions25m
- Michaelis-Menten Assumptions18m
- Michaelis-Menten Equation52m
- Lineweaver-Burk Plot43m
- Michaelis-Menten vs. Lineweaver-Burk Plots20m
- Shifting Lineweaver-Burk Plots37m
- Calculating Vmax40m
- Calculating Km31m
- Kcat46m
- Specificity Constant1h 1m
- 7. Enzyme Inhibition and Regulation 8h 42m
- Enzyme Inhibition13m
- Irreversible Inhibition12m
- Reversible Inhibition9m
- Inhibition Constant26m
- Degree of Inhibition15m
- Apparent Km and Vmax29m
- Inhibition Effects on Reaction Rate10m
- Competitive Inhibition52m
- Uncompetitive Inhibition33m
- Mixed Inhibition40m
- Noncompetitive Inhibition26m
- Recap of Reversible Inhibition37m
- Allosteric Regulation7m
- Allosteric Kinetics17m
- Allosteric Enzyme Conformations33m
- Allosteric Effectors18m
- Concerted (MWC) Model25m
- Sequential (KNF) Model20m
- Negative Feedback13m
- Positive Feedback15m
- Post Translational Modification14m
- Ubiquitination19m
- Phosphorylation16m
- Zymogens13m
- 8. Protein Function 9h 41m
- Introduction to Protein-Ligand Interactions15m
- Protein-Ligand Equilibrium Constants22m
- Protein-Ligand Fractional Saturation32m
- Myoglobin vs. Hemoglobin27m
- Heme Prosthetic Group31m
- Hemoglobin Cooperativity23m
- Hill Equation21m
- Hill Plot42m
- Hemoglobin Binding in Tissues & Lungs31m
- Hemoglobin Carbonation & Protonation19m
- Bohr Effect23m
- BPG Regulation of Hemoglobin24m
- Fetal Hemoglobin6m
- Sickle Cell Anemia24m
- Chymotrypsin18m
- Chymotrypsin's Catalytic Mechanism38m
- Glycogen Phosphorylase21m
- Liver vs Muscle Glycogen Phosphorylase21m
- Antibody35m
- ELISA15m
- Motor Proteins14m
- Skeletal Muscle Anatomy22m
- Skeletal Muscle Contraction45m
- 9. Carbohydrates7h 49m
- Carbohydrates19m
- Monosaccharides15m
- Stereochemistry of Monosaccharides33m
- Monosaccharide Configurations32m
- Cyclic Monosaccharides20m
- Hemiacetal vs. Hemiketal19m
- Anomer14m
- Mutarotation13m
- Pyranose Conformations23m
- Common Monosaccharides33m
- Derivatives of Monosaccharides21m
- Reducing Sugars21m
- Reducing Sugars Tests19m
- Glycosidic Bond48m
- Disaccharides40m
- Glycoconjugates12m
- Polysaccharide7m
- Cellulose7m
- Chitin8m
- Peptidoglycan12m
- Starch13m
- Glycogen14m
- Lectins16m
- 10. Lipids5h 49m
- Lipids15m
- Fatty Acids30m
- Fatty Acid Nomenclature11m
- Omega-3 Fatty Acids12m
- Triacylglycerols11m
- Glycerophospholipids24m
- Sphingolipids13m
- Sphingophospholipids8m
- Sphingoglycolipids12m
- Sphingolipid Recap22m
- Waxes5m
- Eicosanoids19m
- Isoprenoids9m
- Steroids14m
- Steroid Hormones11m
- Lipid Vitamins19m
- Comprehensive Final Lipid Map13m
- Biological Membranes16m
- Physical Properties of Biological Membranes18m
- Types of Membrane Proteins8m
- Integral Membrane Proteins16m
- Peripheral Membrane Proteins12m
- Lipid-Linked Membrane Proteins21m
- 11. Biological Membranes and Transport 6h 37m
- Biological Membrane Transport21m
- Passive vs. Active Transport18m
- Passive Membrane Transport21m
- Facilitated Diffusion8m
- Erythrocyte Facilitated Transporter Models30m
- Membrane Transport of Ions29m
- Primary Active Membrane Transport15m
- Sodium-Potassium Ion Pump20m
- SERCA: Calcium Ion Pump10m
- ABC Transporters12m
- Secondary Active Membrane Transport12m
- Glucose Active Symporter Model19m
- Endocytosis & Exocytosis18m
- Neurotransmitter Release23m
- Summary of Membrane Transport21m
- Thermodynamics of Membrane Diffusion: Uncharged Molecule51m
- Thermodynamics of Membrane Diffusion: Charged Ion1h 1m
- 12. Biosignaling9h 45m
- Introduction to Biosignaling44m
- G protein-Coupled Receptors32m
- Stimulatory Adenylate Cyclase GPCR Signaling42m
- cAMP & PKA28m
- Inhibitory Adenylate Cyclase GPCR Signaling29m
- Drugs & Toxins Affecting GPCR Signaling20m
- Recap of Adenylate Cyclase GPCR Signaling5m
- Phosphoinositide GPCR Signaling58m
- PSP Secondary Messengers & PKC27m
- Recap of Phosphoinositide Signaling7m
- Receptor Tyrosine Kinases26m
- Insulin28m
- Insulin Receptor23m
- Insulin Signaling on Glucose Metabolism57m
- Recap Of Insulin Signaling in Glucose Metabolism6m
- Insulin Signaling as a Growth Factor1h 1m
- Recap of Insulin Signaling As A Growth Factor9m
- Recap of Insulin Signaling1m
- Jak-Stat Signaling25m
- Lipid Hormone Signaling15m
- Summary of Biosignaling13m
- Signaling Defects & Cancer20m
- Review 1: Nucleic Acids, Lipids, & Membranes2h 47m
- Nucleic Acids 19m
- Nucleic Acids 211m
- Nucleic Acids 34m
- Nucleic Acids 44m
- DNA Sequencing 19m
- DNA Sequencing 211m
- Lipids 111m
- Lipids 24m
- Membrane Structure 110m
- Membrane Structure 29m
- Membrane Transport 18m
- Membrane Transport 24m
- Membrane Transport 36m
- Practice - Nucleic Acids 111m
- Practice - Nucleic Acids 23m
- Practice - Nucleic Acids 39m
- Lipids11m
- Practice - Membrane Structure 17m
- Practice - Membrane Structure 25m
- Practice - Membrane Transport 16m
- Practice - Membrane Transport 26m
- Review 2: Biosignaling, Glycolysis, Gluconeogenesis, & PP-Pathway3h 12m
- Biosignaling 19m
- Biosignaling 219m
- Biosignaling 311m
- Biosignaling 49m
- Glycolysis 17m
- Glycolysis 27m
- Glycolysis 38m
- Glycolysis 410m
- Fermentation6m
- Gluconeogenesis 18m
- Gluconeogenesis 27m
- Pentose Phosphate Pathway15m
- Practice - Biosignaling13m
- Practice - Bioenergetics 110m
- Practice - Bioenergetics 216m
- Practice - Glycolysis 111m
- Practice - Glycolysis 27m
- Practice - Gluconeogenesis5m
- Practice - Pentose Phosphate Path6m
- Review 3: Pyruvate & Fatty Acid Oxidation, Citric Acid Cycle, & Glycogen Metabolism2h 26m
- Pyruvate Oxidation9m
- Citric Acid Cycle 114m
- Citric Acid Cycle 27m
- Citric Acid Cycle 37m
- Citric Acid Cycle 411m
- Metabolic Regulation 18m
- Metabolic Regulation 213m
- Glycogen Metabolism 16m
- Glycogen Metabolism 28m
- Fatty Acid Oxidation 111m
- Fatty Acid Oxidation 28m
- Citric Acid Cycle Practice 17m
- Citric Acid Cycle Practice 26m
- Citric Acid Cycle Practice 32m
- Glucose and Glycogen Regulation Practice 14m
- Glucose and Glycogen Regulation Practice 26m
- Fatty Acid Oxidation Practice 14m
- Fatty Acid Oxidation Practice 27m
- Review 4: Amino Acid Oxidation, Oxidative Phosphorylation, & Photophosphorylation1h 48m
- Amino Acid Oxidation 15m
- Amino Acid Oxidation 211m
- Oxidative Phosphorylation 18m
- Oxidative Phosphorylation 210m
- Oxidative Phosphorylation 310m
- Oxidative Phosphorylation 47m
- Photophosphorylation 15m
- Photophosphorylation 29m
- Photophosphorylation 310m
- Practice: Amino Acid Oxidation 12m
- Practice: Amino Acid Oxidation 22m
- Practice: Oxidative Phosphorylation 15m
- Practice: Oxidative Phosphorylation 24m
- Practice: Oxidative Phosphorylation 35m
- Practice: Photophosphorylation 15m
- Practice: Photophosphorylation 21m
Ordering Cleaved Fragments - Online Tutor, Practice Problems & Exam Prep
![](/channels/images/AIStars.png)
To determine the sequence of a protein, it is essential to cleave it into smaller peptide fragments using techniques like Edman degradation. However, using a single cleavage method may not reveal the correct order of fragments. Employing at least two different cleavage techniques allows for overlapping peptide fragments, which helps reconstruct the original protein sequence. This process highlights the importance of understanding peptide bonds and the role of specific reagents, such as cyanogen bromide and chymotrypsin, in generating distinct fragments for analysis.
Ordering Cleaved Fragments
Video transcript
Ordering Cleaved Fragments
Video transcript
So in our last lesson video, we presented the problem that if we treat our protein with only one cleavage technique, then it may not be possible to order the fragments and to determine the sequence of our protein. And that's why typically a minimum of at least 2 different cleavage techniques are required in order to properly order the fragments. And so the reason it works like this is because when we treat the same protein separately with different cleavage techniques or different reagents, it will generate different peptide fragments. And so those different peptide fragments, we can actually align all of these different peptide fragments so that the overlapping peptide fragments will actually reveal the original order of the fragments and the actual sequence of the original protein.
Let's take a look at our example down below so that we can better understand this overlapping idea and the ordering of the cleaved fragments. Notice in our diagram what we have in the first box up here is one particular cleavage technique on our original protein, and then down below, in the second box here, we have the same exact original protein as up above, except we're treating it with a different cleavage technique, cleavage technique number 2. Notice that it generates 2 different sets of peptide fragments. Then, in step 3 over here, we are essentially ordering all and overlapping the cleaved fragments from both of these cleavage techniques in order to determine the original protein sequence down below.
Notice in this first box up here, this original protein, we are treating it with our first cleavage technique, cleavage technique number 1, which is with cyanogen bromide, and it generates these particular fragments that are shown: these three fragments, 2 dipeptides and one tripeptide. And you'll notice that these are the same exact fragments that are generated from our previous lesson video in the example. And now, down below here, we're treating the same exact original protein with a second cleavage technique, cleavage technique number 2, using a different reagent. This time, we're using chymotrypsin. And it generates all of these fragments here: a dipeptide, a free leucine residue, a free leucine amino acid, and then a tetrapeptide shown here.
In step number 3, we're seeing how we can overlap and order the fragments in order to reveal the original protein sequence. Now in our next lesson video, we're going to talk about an actual strategy for how to overlap and order the fragments. But for now, just to give you guys a quick little glimpse and insight into the strategy that we're going to use, typically we're going to start with the longest fragment. So the longest fragment is this tetrapeptide here amongst all of the fragments. So this is the tetrapeptide, and all we need to do is recognize that this tetrapeptide here is the same one as this one over here. We can fill that in. So notice it's MRMF, so we can put in MRMF. Then what we can do is look for overlapping fragments. Notice that if we check this other fragment over here, the RM, that it overlaps perfectly with the RM here. This RM fragment can fill in for the RM up here. And what you'll notice is that the F from this fragment right here will overlap with the F from this fragment over here. We can put in FL down below right here because of the overlap. And what you'll see is that we have an M here that also needs to overlap, and it overlaps with the M from the GIGYM fragment. We can put in GYM. And, of course, what you'll see is that we've got a GY fragment over here with the yellow that we can fill in. So we can put in the GY here. And then, of course, the lone leucine over here will overlap with the leucine down below. Essentially what we mean by these overlaps are these vertical overlaps. Notice we have a glycine here confirming the N-terminal amino acid residue down below in our original protein sequence as being glycine. So we can put glycine right here, and that is our first residue. Now moving on to the next set of overlap, we have Tyrosine which is confirming the second residue. So the overlapping fragments confirm a tyrosine here. And then applying the same strategy, what we have is overlapping methionine, confirming the 3rd residue as methionine, so we can put in methionine here. And then, what we have next is arginine, and then we have methionine following that, so we can put in arginine and methionine. And then of course, what we have remaining are phenylalanine and leucine. So we can put in phenylalanine and leucine.
Recall that when we use this cleavage technique all alone, we actually had 2 valid possibilities that we were not able to determine the order of these fragments, order the overlapping fragments from the order the overlapping fragments from these, fragments generated from that cleavage technique, with these fragments generated from a different cleavage technique, that we're actually able to determine the original protein sequence, which we've confirmed down below here. Essentially what we're saying again is that 2 or more cleavage techniques, cleavage methods, are needed to order the fragments and reveal the sequence of the protein. Again, we're going to talk about an actual strategy for how to overlap and order the fragments in our next lesson video. But before we get there, let's get a little bit of practice. I'll see you guys in that practice video.
Overlap, align & order the following peptide fragments to reveal the sequence of the original protein.
Fragments from cleavage method #1:
Fragments from cleavage method #2:
Problem Transcript
Here’s what students ask on this topic:
What is Edman degradation and how is it used in protein sequencing?
Edman degradation is a method used to sequence amino acids in a peptide. It involves selectively removing the N-terminal amino acid of the peptide, which is then identified through chromatography or electrophoresis. This process is repeated to determine the sequence of the peptide. However, Edman degradation is limited to small peptides with fewer than 50 amino acids. For larger proteins, the protein must first be cleaved into smaller fragments using chemical or enzymatic methods before sequencing each fragment individually. This technique is crucial for understanding protein structure and function in proteomics.
![](/channels/images/AIStars.png)
Why are multiple cleavage techniques necessary for protein sequencing?
Multiple cleavage techniques are necessary for protein sequencing because using only one method may not provide enough information to determine the correct order of peptide fragments. Different cleavage techniques generate different sets of peptide fragments, which can be aligned based on overlapping sequences. This overlapping helps to reconstruct the original protein sequence accurately. For example, using cyanogen bromide and chymotrypsin together can produce complementary fragments that reveal the protein's primary structure. This approach ensures a more reliable and complete sequencing of larger proteins.
![](/channels/images/AIStars.png)
How does cyanogen bromide cleave proteins?
Cyanogen bromide (CNBr) cleaves proteins specifically at methionine residues. It targets the peptide bond on the carboxyl side of methionine, resulting in the cleavage of the protein into smaller peptide fragments. This specificity makes CNBr a valuable tool in protein sequencing, as it allows for predictable fragmentation patterns. After cleavage, the resulting peptide fragments can be separated and sequenced individually, aiding in the reconstruction of the original protein sequence.
![](/channels/images/AIStars.png)
What role does chymotrypsin play in protein sequencing?
Chymotrypsin is a protease that cleaves peptide bonds at the carboxyl side of aromatic amino acids such as phenylalanine, tyrosine, and tryptophan. In protein sequencing, chymotrypsin is used as a secondary cleavage technique to generate different peptide fragments from those produced by other methods like cyanogen bromide. The fragments generated by chymotrypsin can be aligned with those from other cleavage techniques based on overlapping sequences, helping to accurately determine the original protein sequence. This complementary approach is essential for sequencing larger proteins.
![](/channels/images/AIStars.png)
What is the importance of overlapping peptide fragments in protein sequencing?
Overlapping peptide fragments are crucial in protein sequencing because they allow for the accurate reconstruction of the original protein sequence. When a protein is cleaved using different techniques, each method produces unique fragments. By aligning these fragments based on their overlapping sequences, researchers can determine the correct order of amino acids in the protein. This method ensures that the entire sequence is covered and helps to resolve ambiguities that may arise from using a single cleavage technique. Overlapping fragments provide a more complete and reliable protein sequence.
![](/channels/images/AIStars.png)