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5. Protein Techniques

Tandem Mass Spectrometry


Tandem Mass Spectrometry

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in this video, we're gonna talk about Tana Mass spectrometry. So tandem mass spectrometry or tandem M s for short is also known as just m s m s. And that's because it's a technique that literally uses to mass spectrometers m s, that air hooked up in tandem or essentially just taking to mass spectrometers and using them back to back. And so when we take to mass spectrometers and use them back to back like we do with tandem mass spectrometry that actually provides several key advantages. And collectively, those key advantages make tandem mass spectrometry one of the gold standards for sequencing proteins. And so some of these advantages include the fact that tandem mass spectrometry can be used to analyze and already purified protein or a single protein within a mixture of other proteins, which means that we can skip most of the protein purification process when it comes to analyzing a single protein with tandem mass spectrometry and that will save a boatload of time. And so no wonder why Tana Mass spectrometry is a gold standard for sequencing proteins. It saves so much time with having to purify a protein because we can analyze protein mixtures now recall in our previous lesson videos, when we talked about general mass spectrometry with only one mass spectrometer that it was limited to an already purified protein. So this is an actual advantage that's unique to Tanna mass spectrometry and also when we're used on Lee one mass spectrometer. It was limited to relatively small proteins and peptides, and the reason it was limited to small proteins and peptides. It's because if you analyze the large protein, then upon fragmentation, it's gonna generate a whole bunch of peptide fragments. The larger the protein, the more peptide fragments. And if you have too many peptide fragments, that's going to lead to too many peaks on the mass spectrum. Too many peaks on a mass spectrum can make it really difficult to analyze. And so, in orderto limit, the number of peaks on the spectrum were limited to using small proteins and small peptides when we Onley have one mass spectrometer. But when we use to mass spectrometers back to back like we do with tandem mass spectrometry, we are not limited to small proteins or peptides. We can actually analyze much, much larger proteins, and so that is a very important advantage, and the reason that we're allowed to do that with tandem mass spectrometry is because tandem mass spectrometry allows for the filtering of unwanted ions or unwanted peptide fragments. And so if we're able to filter away unwanted fragments, then we're able to manage the number of fragments that show up as peaks on the spectrum. So we're essentially managing the number of peaks on the spectrum and were able to obtain ah, much cleaner and simpler mass spectrum. That's, ah, lot easier to analyze and a lot easier to get sequencing data from. And so that's another reason why tandem mass spectrometry is a gold standard for sequencing proteins. Now the rest of these bullet points that followed down below are essentially dedicated to telling us how tandem mass spectrometry works, and we've broken it down into four general steps, and those steps are numbered 123 and four. And what you'll notice is down below in our example image. On the left hand side, we have the same numbers 123 and four, and the numbers and the image actually correspond with numbers and the text up above. So that's important to keep in mind now, one of the main differences between the image that's on the left hand side over here and the image that's on the right over here is what the starting material is. So notice on the left were starting with an already purified protein, and on the right, we're starting with a protein mixture, so we're really pointing out the advantage. That tandem mass spectrometry can analyze both a protein and already purified protein and a protein mixture, allowing us to save time with the protein purification process. So first we're going to analyze the image on the left hand side over here, and then, after we're done analyzing the image on the left, we'll take a look at the image on the right, which has essentially the same or similar steps to the image on the left. Okay, so let's get started with our steps up above. And so for step number one, which will see, is that we have this already purified protein and this already purified protein as first going to be fragmented. It's going to be fragmented, using either some type of chemical, so there are many different types of chemicals that could be used to fragment of protein. Or we could use a Proteus, which is an enzyme that can break down proteins. And there are also many different types of Proteus is. So if we take a look at our example down below on the left hand side notice we're starting with our already purified protein and, like we set up in step number one up above were first going to fragment our protein. And so, with step number one, you can see that we have fragmentation here and we're taking this large protein ear and fragmenting it into a bunch of smaller pieces. And really, that's it for step number one. So in step number two, we're taking all of these protein fragments, and we're going to ionized those protein fragments and subject them to the first mass spectrometer or the first M s here. And so this first mass spectrometer is different than the mass spectrometer that we're used to seeing, uh, in our previous lessons, because this first mass spectrometer here actually acts as a filter. And so we have one single peptide fragment that's actually going to be filtered and selected for to emerge at the end and continue forward in our process. So with our previous knowledge of mass spectrometry, normally we would do mass analysis. Those fragments would be deflected and hit a detector, but with mass spectrometry with Tana Mass spectrometry tandem mass spectrometry. The first mass spectrometer is acting as a filter to select for one fragment to emerge at the end. And then that selected protein fragment that we're interested in is going to enter a collision or a collision sell chamber. So let's take a look at our example down below to clear this up. So you can see we have our protein or peptide fragments that were generated from step number one. And we're going to subject these peptide fragments to ionization and to the first mass spectrometer m s one here. And so notice that all of these peptide fragments are being deflected. But we're not so much interested in those peptide fragments. So they're being filtered for, and they're being, uh, filtered away these unwanted ions. And on Lee, one particular ion is selected to move forward. And that's this selected peptide fragment here. And so this selected peptide fragment is going toe enter this collision chamber and That's exactly what we set up above. And step two now. And step number three, what's gonna happen is a noble gas. So a noble gas such as either helium or argon is going to further fragment are selected peptide here into a bunch of smaller peptide fragments. And we know that, uh, it's usually going to break at peptide bonds from our previous lesson videos. And so if we take a look down at our example, we can see we have are selected peptide. It enters the collision chamber, and it's going to be bombarded with a noble gas such as helium or argon. And that's going to further fragment our peptide of interest. Now, in the last and final step here, uh, the generated protein fragments, uh, in step number three are going to enter a second, a second mass spectrometer. And in that second mass spectrometer, that is where the detector is that it's going to allow us to measure all of the MZ ratios and so we can see that we're taking all of these peptide fragments that are generated, and we're subjecting them to a second mass spectrometer, Uh, m s to here and the second mass spectrometer eyes going to direct all of the peptide fragments to a detector. And we're going to be able to essentially get a mass spectrum and do mass analysis of the selected peptide fragment here. And so really, when you think about this, we're essentially taking a small little piece of this purified protein here we're pulling it out, and then we're fragmenting this small little protein into a bunch of other smaller fragments. And then we're analyzing this, uh, these smaller fragments here, and that is really how tandem mass spectrometry works. Now, over here on the right hand image notice what we have is a protein mixture that we're starting with, and we have a bunch of proteins number p one p two p three p four mp five and the protein of interest in this case here is actually protein number three. And so when we have a protein measure, we know that, uh, tandem mass spectrometry allows us to analyze a single protein within a mixture which allows us to save ah, lot of time with having to purify this protein first. And so we can just take this protein mixture and subjected to tandem mass spectrometry where it's going to. All of these proteins are gonna be ionized and subjected to the first mass spectrometer where, of course, recall that the first mass spectrometer is going toe act as a filter to select for one peptide for one protein to emerge at the end. And so notice that p one p two p four and p five. They're all being, ah, blocked here. They're all hitting a dead end. But protein number three, which is right here, is selected to continue forward in the process and it enters a collision chamber where it's going to be bombarded with a noble gas. Here, it's a helium gas and that's going to fragment protein number three into smaller peptide fragments. And then those peptide fragments which are numbered uh, F one F two through F five, which represent the fragments, are subjected to a second mass spectrometer M s to here, which is going to essentially deflect all of these fragments to a detector. And that detector's gonna measure all the MZ ratios and allow us to get a mass spectrum where we could do mass analysis. And so here you can see how tandem mass spectrometry allows us to analyze and already purified protein or a single protein in a mixture. And so this year concludes our lesson on Tanna Mass spectrometry, and we'll be able to get some practice in our practice video, so I'll see you guys there.

Tandem mass spectrometry combines which of the following devices?


In your tandem mass-spectrometry of a pure protein, you focused a fragment with an m/z of 1,268 through the process & into the second mass-spec and found y-ion peaks of 1,137 and 1,022. The mass in Daltons for the possible relevant amino acids are provided: Y (163), N (114), W (186), D (115), G (57), L (113) and M (131). What is the order of the first two amino acid residues in the 1,268 fragment from N-terminal to C-terminal?