Proteomics

Hi in this video, we're gonna be talking about proteomics. So proteomics is the analysis of a cell or a tissue or organisms. Um protein content. We also call this the protium you have to find below. So the proteome describes a complete set of proteins encoded by a genome. And so the just because the genome X. Has encodes for all these different proteins doesn't necessarily mean that those proteins will all be expressed at all the same times. Generally the cell responds to different environmental conditions and so different proteins are expressed at different times. And so when studying proteomics, you have to be able to understand that proteomics is very different depending on the condition or the timing that you're looking at the cell tissue or organism. And so another interesting thing is that due to protein processing things like alternative slicing or RNA editing, the proteome is actually much larger than the number of genes that code for proteins. Right? Because a single protein after it undergoes alternative splicing, you can have five or six different forms called ice A forms. And so that represents five or six different proteins. And that's encoded by one gene. So one gene doesn't necessarily encode one protein in humans through these different processing ways that can encode a bunch of different proteins. And so a few methods have been developed to isolate protein. So one is called gel electrophoresis and I talked about this a little bit in a cloning chopped in a cloning video. Um but I don't know if you've necessarily seen that yet, but I'll just mention it again here. So gel electrophoresis separates can separate proteins. It can also separate DNA and RNA. But this is a proteomics. So we're gonna talk about proteins. And so um there's a couple of different ways that this is done. One is SCS page which you can see actually here if I move out of the way and this is going to separate proteins by mass. So by their sides of their weight, things like that. But there's also other types of gel electrophoresis that can separate proteins by charge. And so when it's separating proteins by charge, we call it ice electric focusing the fancy term. And that protein migrates in the gel to the point where it charges zero. So you have a So essentially what this looks like when you draw it in the middle here, it's when you get it you get all these dots. So when you get the final product this is kind of what it looks like. There's so much different sizes and some have little tails after them. And this is what it looks like. And the reason that it looks like this is because you're separating by mass. So it's gonna be larger and smaller but you're also separated by charge. So positive and negative. And so if it's a negatively charged protein, it's going to want to move towards the positive side. And if it's a positive charge protein, it's gonna want to move towards the negative side. And so each one of these dots has eventually reached an ice electric focusing point where it charges zero because it's sitting in this spot where it no longer wants to move because it's already moved as far as its negative charge is or it's positive charges. So it's no longer being drawn to the other side anymore because its charges effectively zero. Now that it's moved that far. So typically protein shells kind of look like this, these these are called wells. Um and they you put the sample in that and it can run it this way, you can run it this way depending on how it's all set up. So like I said, you necessarily won't be tested on this in this class. But you may see this in a laboratory setting. Now there are a variety of different tech techniques used to identify what proteins exist in a certain sample. And so one of these is called mass spec or mass spectrometry. E. And this identifies proteins. So, an example of this would be tandem mass spec. And this separates proteins by mass and charge. So kind of like we did above. But the difference is that tandem aspect can actually identify what the amino acid sequence of the protein that you are looking at. Is. So generally you look at one protein or a sample with a variety of different proteins in it. And you say, you know what proteins are here, what their what's their amino acid sequence. A second type is called protein micro ray. And we've talked about micro rays in terms of D. N. A. Or RNA RNA micro rays but it's essentially the same for protein. So it detects proteins, it can also detect protein protein interactions an example. So say you have the cell, you want to know what proteins are being expressed in it while you take that sell you take out all the proteins and you run it on a protein micro array. And this array uses antibodies that have been fixed to this plate and you run the protein over it and the proteins that bind to the antibodies are in the samples. And the proteins that don't buy into the antibodies were never there to bind to begin with. So again, that's typically how microwave works. So here's an example of mass spec something you would get from aspect to be able to identify the sequence. So generally different amino acids, they have different chemical structure, meaning that they have a different size. They have a different charge and tandem aspect can actually detect those different sizes and charges and they represent them as peaks. And so each peak can represent a different amino acid. And so by knowing the peaks of the individual amino acids then you can say, okay, well this is definitely glycerine here. So that's the first sequence. This is veiling. So here we go. Now we have a losing. Now we have two failings and so you keep going reading the sequence and eventually you get a sequence or something that you think might be the sequence and then you can go back to the genome or whatever you are studying and say, okay, well, does this protein sequence exist? And what gene encodes for this protein sequence that we found on Mass Pet? Now you're probably going to do a lot more with mass Spec in a chemistry class. You don't typically do that in a genetics class or lab, but I do want to introduce it here because it is an important biological technique in addition to a chemistry technique. So with that, let's not move on.

SDS-PAGE is a method used to separate proteins by which of the following characteristics?

• A. Mass
• B. Length
• C. Charge
• D. Acidity

Which of the following methods would be best to identify an amino acid sequence of a protein?

• A. Protein microarrays
• B. ChIP
• C. Tandem Mass Spectrometry
• D. Reverse genetics