Hey, guys. In this video, we're going to talk about indirect protein sequencing via genomic analyses. So up until this point in our course, we've only focused on direct protein sequencing methods such as tandem mass spectrometry or Edman degradation. Now direct protein sequencing is used on already extracted or isolated proteins. And direct protein sequencing is able to directly identify the sequence of unknown proteins in a sample. However, direct protein sequencing does not account for how biochemists obtain most of their protein sequencing data. And so most of the protein sequencing data is actually derived indirectly from genomic analyses or translating the nucleotide sequences of genes into amino acid sequences. And so this brings up the question, why is most of the protein sequencing data obtained via genomic analyses? Why would we obtain most of our protein sequencing data this way? Well, it turns out that it actually saves a boatload of time. It saves a lot of time. Working with DNA is actually easier than working with proteins in a lab and that's because we know that proteins are really sensitive to lots of conditions, and they can be pretty easily denatured if the temperature is off or if the pH is different. And DNA is more resistant to essentially decomposing and breaking apart. And so because DNA is more stable, it's easier to work with, and so that allows us to essentially work with DNA faster. And it turns out that DNA sequencing is actually significantly faster, cheaper, and more efficient and informative than direct protein sequencing since direct protein sequencing only allows us to obtain the amino acid sequence, but DNA sequencing allows us to obtain the nucleotide sequence. And then from that nucleotide sequence, we can derive derive the amino acid sequence using the genetic code. And so essentially, overall, genomic analyses allows us to collect more data and more protein sequencing data faster. And so that begs the question, why do we even need direct protein sequencing if genomic analysis is the best way that allows us to obtain more protein sequencing data faster? Why do we even need direct protein sequencing if indirect genomic analysis is the best at that? Well, it turns out that we can't just scrap direct protein sequencing because direct protein sequencing has its own sets of advantages. And some of those advantages include the fact that genomic analyses are not able to identify an unknown protein sample on its own. And so, because it cannot do this, that's something that direct protein sequencing is easily able to do. And that's because when we're working with genomic analyses, we're going to need a DNA sample. And so, if we only have an unknown protein or just protein, then we're not able to perform genomic analyses on these proteins. So, it's not, that's not a good thing about genomic analyses. Now, in addition to that, unlike genomic analyses, direct protein sequencing via tandem mass spectrometry can actually reveal chemically modified amino acid residues. And that allows us to identify, essentially, proteins that are genes And so genomic analyses does not reveal chemically modified amino acid residues, but direct protein sequencing can. So that's another advantage of direct protein sequencing and another reason for why we can't just scrap all of the direct protein sequencing techniques. So the rest of this video here is going to refresh our memories on how the genetic code works, which allows us to perform genomic analyses. So recall from our previous videos that the genetic code actually reveals the connection between the codons of nucleic acids and the amino acids of proteins. And so in our example below, we're going to use the genetic code to reveal the peptide sequence in the example shown over here on the right. And so, what you'll see is on the left here we have the genetic code. And recall that the genetic code is essentially reading the codon of the mRNA, and the codons have 3 nucleotides. So with this genetic code, we have the first base of the codon on the left, we have the second base of the codon, so the second base of the codon, on the top here, and then we have the third base of the codon over here on the right. And so recall that the first base of the codon limits us to one particular row here. The second base of the codon limits us to one particular column. And then the 3rd particular codon limits us to a specific position in a box. And so, what you'll see here is that we have a DNA coding sequence that's provided, and you can see that it has a 5 prime end and a 3 prime end. And so we know that this DNA coding sequence can be converted into an mRNA sequence through the process of transcription that's shown here, represented by this arrow. And mRNA sequence is going to be exactly the same as the DNA coding sequence up above, except the fact that all of the threonines, are going to be converted into U's, or uracils, because mRNA only has uracils. And so these two threonines here are going to be converted into uracils in our RNA sequence. And so, now that we have our mRNA sequence, we know that the genetic code breaks down and reads the mRNA sequence in codons, which are sets of 3 nucleotides. So our first codon are these first three nucleotides, AUG. And so again, the first base of our codon is A. And so because it's A, it limits us to this column. I'm sorry, this row. The second base of our codon is u, so we can see that here, u. And so in the second base of our codon, it limits us to one particular column. So the overlap between these two is this box right here. And then the 3rd codon is, I'm sorry, the 3rd base of our codon is g, and so that limits us to this particular position within the box, which is a u g. An AUG codon corresponds with a methionine amino acid residue, which is why we have methionine as our first residue on the n terminal end of our peptide. So moving on to our next codon, we have GCU. And so GCU corresponds with this, first residue here in this column, I'm sorry, this row. Then we have C, which limits us to this column. So now we're in this box. And then U limits us to this one particular position, GCU, which is an alanine amino acid residue. So over here, we can put an A for alanine in that position. And so essentially what we can do is continue through this process here and move on to our next codon. So the next codon is GGC, and GGC, G is here in this row. G, the second one is G, so that limits us to this column, so now we're in this box. And then C here limits us to a GGC, which is glycine, so glycine is our next residue. And now you guys are probably remembering how this works here, and so what we can do is fill out the rest of these codons here. So we have, after GGC, we have CGG, then we have AGC, and then last but not least, we have AAA. And so CGG corresponds with an arginine, so this is an arginine, CGG. And then, AGC corresponds with a serine, and then of course AAA corresponds with a lysine. And so, what we can see is that the amino acid sequence of our peptide is actually revealed through genomic analysis. We obtain the DNA sequence and we sequence that DNA, And then, through the process of transcription and translation, the genetic code, we are able to obtain the sequence of our peptide. And so this is an indirect method to be able to sequence our peptides. And that's exactly how, indirect sequencing via genomic analyses works. And so in our next couple of videos, we'll be able to get some practice utilizing the genetic code and indirect protein sequencing. So I'll see you guys in those practice videos.
- 1. Introduction to Biochemistry4h 34m
- What is Biochemistry?5m
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- Review 1: Nucleic Acids, Lipids, & Membranes2h 47m
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- Review 4: Amino Acid Oxidation, Oxidative Phosphorylation, & Photophosphorylation1h 48m
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- Practice: Amino Acid Oxidation 12m
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- Practice: Oxidative Phosphorylation 35m
- Practice: Photophosphorylation 15m
- Practice: Photophosphorylation 21m
Indirect Protein Sequencing Via Geneomic Analyses - Online Tutor, Practice Problems & Exam Prep
Indirect protein sequencing through genomic analyses is more efficient than direct methods like tandem mass spectrometry. Working with DNA is easier due to its stability, allowing faster and cheaper sequencing. While genomic analyses can derive amino acid sequences from nucleotide sequences, they cannot identify unknown proteins or detect chemically modified residues, which direct sequencing can. Understanding the genetic code is crucial for translating mRNA codons into amino acids, revealing peptide sequences. This synthesis of genomic and direct methods enhances our ability to analyze proteins effectively.
Indirect Protein Sequencing Via Genomic Analyses
Video transcript
Use the genetic code above & the coding DNA sequence below to determine the protein sequence.
Problem Transcript
Suppose the sequence below is a template DNA sequence. What is the corresponding protein sequence?
Problem Transcript
Even when the sequence of nucleotides for a gene is available and genomic analyses can be performed, direct chemical techniques on the physical protein are still required to determine:
Here’s what students ask on this topic:
What is indirect protein sequencing via genomic analyses?
Indirect protein sequencing via genomic analyses involves determining the amino acid sequence of a protein by first sequencing the DNA that encodes it. This method leverages the stability and ease of working with DNA compared to proteins. By sequencing the DNA, we can obtain the nucleotide sequence, which can then be translated into the corresponding amino acid sequence using the genetic code. This approach is faster, cheaper, and more efficient than direct protein sequencing methods like tandem mass spectrometry or Edman degradation.
Why is DNA sequencing preferred over direct protein sequencing?
DNA sequencing is preferred over direct protein sequencing because it is faster, cheaper, and more efficient. DNA is more stable and easier to work with in the lab compared to proteins, which are sensitive to conditions like temperature and pH. Additionally, DNA sequencing provides more comprehensive data, allowing researchers to derive the amino acid sequence from the nucleotide sequence. This makes genomic analyses a more practical and informative approach for obtaining protein sequencing data.
What are the limitations of genomic analyses in protein sequencing?
Genomic analyses have several limitations in protein sequencing. Firstly, they cannot identify unknown protein samples without a corresponding DNA sequence. Secondly, genomic analyses cannot detect chemically modified amino acid residues, which are important for understanding protein function and regulation. These limitations necessitate the use of direct protein sequencing methods, such as tandem mass spectrometry, which can identify unknown proteins and reveal post-translational modifications.
How does the genetic code facilitate indirect protein sequencing?
The genetic code facilitates indirect protein sequencing by providing a set of rules for translating nucleotide sequences (mRNA codons) into amino acid sequences. Each codon, a sequence of three nucleotides, corresponds to a specific amino acid. By using the genetic code, researchers can convert the mRNA sequence derived from DNA into the corresponding peptide sequence. This process involves reading the codons in the mRNA and matching them to their respective amino acids, thus revealing the protein's sequence.
What are the advantages of direct protein sequencing methods?
Direct protein sequencing methods, such as tandem mass spectrometry and Edman degradation, have several advantages. They can identify unknown protein samples without needing a corresponding DNA sequence. Additionally, direct methods can detect chemically modified amino acid residues, which are crucial for understanding protein function and regulation. These capabilities make direct protein sequencing essential for comprehensive protein analysis, complementing the data obtained from genomic analyses.