In this video, we're going to talk about tandem mass spectrometry. So tandem mass spectrometry, or tandem MS for short, is also known as just MSMS. And that's because it's a technique that literally uses two mass spectrometers, MS, that are hooked up in tandem, or essentially just taking two mass spectrometers and using them back to back. And so when we take two 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 an 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 tandem 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 tandem mass spectrometry. And also, when we use only one mass spectrometer, it was limited to relatively small proteins and peptides. And the reason it was limited to small proteins and peptides is because if you analyze the large protein, then, upon fragmentation, it's going to 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 order to limit the number of peaks on the spectrum, we're limited to using small proteins and small peptides when we only have one mass spectrometer. But when we use two 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 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 we're able to obtain a much cleaner and simpler mass spectrum that's a 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 follow 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 1, 2, 3, and 4. And what you'll notice is down below in our example image on the left-hand side, we have the same numbers, 1, 2, 3, and 4. And the numbers in the image actually correspond with the numbers in 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, we're 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 an 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 1, what you'll see is that we have this already purified protein, and this already purified protein is 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 can be used to fragment a protein. Or we could use a protease, which is an enzyme that can break down proteins. And there are also many different types of proteases. 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 said up in step number 1 up above, we're first going to fragment our protein. And so with step number 1, you can see that we have fragmentation here, and we're taking this large protein here and fragmenting it into a bunch of smaller pieces. And, really, that's it for step number 1. So in step number 2, we're taking all of these protein fragments and we're going to ionize those protein fragments and subject them to the first mass spectrometer, or the first MS here. And so this first mass spectrometer is different than the mass spectrometer that we're used to seeing, 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 spectrometry, normally we would do mass analysis. Those fragments would be deflected and hit a detector. But with mass spectrometry, with tandem mass spectrometry, the first mass spectrometer is acting as a filter to select collision or, a collision cell, collision or, a collision cell chamber. So let's take a look at our example down below to clear this up. So you can see we have our protein, our peptide fragments that were generated from step number 1. And we're gonna subject these peptide fragments to ionization and to the first mass spectrometer, MS 1 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, filtered away, these unwanted ions. Only one particular ion is selected to move forward and that's this selected peptide fragment here. And so this selected peptide fragment is going to enter this collision chamber. And that's exactly what we set up above in step 2. Now, in step number 3, what's going to happen is a noble gas, so a noble gas such as either helium or argon, is going to further fragment our selected peptide here into a bunch of smaller peptide fragments. And we know that, 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 our 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, the generated protein fragments, in step number 3, are going to enter 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 m/z 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, MS 2 here. And the second mass spectrometer, is going to 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, 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 numbered p 1, p 2, p 3, p 4, and p 5. And the protein of interest in this, case here is actually protein number 3. And so when we have a protein measure, we know that, tandem mass spectrometry allows us to analyze a single protein within a mixture which allows us to save a lot of time with having to purify this protein first. And so we can just take this protein mixture and, subject it 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 to act as a filter to select for one peptide for one protein to emerge at the end. And so notice that, p 1, p 2, p 4, and p 5, they are all being blocked here. They are all hitting a dead end. But protein number 3, 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 helium gas, and that's going to fragment protein number 3 into smaller peptide fragments. And then those peptide fragments, which are numbered, f 1, f 2, through f 5, which represent the fragments, are subjected to a second mass spectrometer, MS 2 here, which is going to essentially deflect all of these ratios and allow us to get a mass spectrum, where we can do mass analysis. And so here you can see how tandem mass spectrometry allows us to analyze an already purified protein or a single protein in a mixture. And so this here concludes our lesson on tandem mass spectrometry, and we'll be able to get some practice in our practice video. So, I'll see you guys there.
