Mass Spectrometry

in this video, we're gonna talk about mass spectrometry. So most of you guys are already somewhat familiar with mass spectrometry from your previous organic chemistry courses. And in this video, we're going to do a review on what mass spectrometry is and how it typically operates. And so mass spectrometry, or M s, is a technique that could be applied toe a wide variety of molecules, including proteins and mass spectrometry. Ionized is quantifies and separates molecules based on their mass to charge ratio or their MZ ratios for short. And the M C ratio is really just a unique property that can be used to identify a molecule and to get structural and chemical information about that molecule. And since the Z here in the MZ ratio is the charge, and it's almost always equal to just one unit, that means that the MZ ratio is often considered to just be the mass of the molecule. And that's pretty much indicated by the mass and mass spectrometry. And so the rest of these bullet points here that follow are really just dedicated to telling us how a typical mass spectrometer operates and noticed that it's broken down into three general steps numbered 12 and three. And if we take a look at our example down below notice, we have an image of a typical mass spectrometer, and we also have the numbers 12 and three, and the numbers and the image correspond with the numbers and the text up above. So that's important to keep in mind. And so, for our first step here for how a typical mass spectrometer works, we haven't already purified peptide that first has to be converted into a gas and ionized in a vacuum. And the ionization occurs via controlled bombardment with electrons or with a noble gas such as helium. And the ionization typically leads to random fragmentation or breakdown of the peptide molecule. And usually the bonds that are being broken are going to be the peptide bonds that fragment the molecule. And later in our course, we're going to talk more details about how a peptide is fragmented during mass spectrometry. But for now, let's take a look at our example down below so we can apply our first general step here, and so notice on the far left here, what we have is an already purified peptide that is relatively small, and it's being entered into a vacuum where it's going to be converted into a gas and ionized with the use of electrons here, which are these blue dots being shown and noticed that, uh, after the first step is complete. We've generated all of these orange dots here, and all of these orange dots represent fragmented peptides that are in a gas ion form. And so moving on to step number two, these ionized gas peptide fragments are going to be exposed to an electric field or a magnetic field, and the electric field essentially deflects the ionized gas fragments. And, uh, the paths that these ionized gas fragments end up taking is a direct result of their MZ ratio. So fragments that have a smaller fragments that have a smaller MZ ratio are going to be deflected. Ah, lot mawr than fragments that have a larger MZ ratio because fragments that have a larger MZ ratio have a larger mass, and it requires more force to deflect them and then moving on the step number three here really quick. There's a detector that's going to read, measure the relative abundance as well as the MZ ratio of each ionized gas peptide fragment. So let's take a look at our image down below so that we can clear up steps two and three. And so again, we know after step one, we're resulting with these fragmented gas peptide ions. And so the fragmented gas peptide ions are being directed into a mass filter where there's an electric or a magnetic field that's applied, and so that ends up resulting in ion deflection. So notice that the paths, uh, that these ions air taking is, uh, they're being deflected and they're changing their path. And the path that they take is a direct result of their MZ ratio. Where, uh, molecules that have a small MZ ratio are deflected a lot mawr. And they end up hitting the detector at a different point than fragments that have ah larger MZ ratio. And it takes mawr to deflect those. And so they have off path that hits the detector, uh, further to the right here on our image. And so you can see that the detector is able to measure the abundance as well as the emcee ratio for each gas fragment ionized gas fragment that hits the detector. And so these numbers here are the MZ ratio, where to 90 represents the smallest MZ ratio Of these three fragments being shown for 30 is the intermediate, and then 5 70 is the largest fragment, uh, largest MZ ratio and largest fragment where it had the widest deflection path here. And so the detector is actually able to translate the information that it gathers and convert it into a data plot known as a mass spectrum. And we're gonna talk about mass spectrums on a lot more detail and our next lesson video. But for now, what we see here on the right is a mass spectrum which you guys are already somewhat familiar with from your previous organic chemistry courses. And notice that on the Y axis here, what we have is the relative abundance of each, uh, peak that's being shown. And then we have on the X axis the MZ ratio of each peak and notice throughout. We have all of these peaks which represent ionized gas peptide fragments, ionized gas peptide fragments, And so you can see Here are three of, uh, ionized gas peptide fragments that we showed in our image to the left, which have MZ ratios of 2 94 30 and 5 70 are being pointed out directly. And so even though in our image we only showed three peptide fragments in a typical mass spectrometry of a peptide, there's going to be a lot more peptide fragments that are being shown. And so you can see all of these different peaks that we see here represent different peptide fragments that were generated. And so one of the biggest difference is that you want to take note of here. Um, is the MZ ratio access here and the scaling of that access So typically in your previous organic chemistry courses, when you covered mass spectrometry, you were looking at relatively small molecules where a single peak might actually represent a single functional group with small amount of atoms. But here, with mass spectrometry of an entire peptide, essentially each of these, um, peaks represents an entire peptide fragment that has a whole bunch of atoms in it. And if it has a lot of atoms, it's gonna have a larger mass. And so that's why the MZ ratio has a larger scaling than what you're probably used to seeing in your previous organic chemistry courses, so you can see it ranges from 200 year up to units for the ratio. And so again, we're gonna talk. Ah, lot Maura, about mass spectrums in our next lesson video. But for now, this is our the conclusion of our review of mass spectrometry, and we'll be able to get a little bit of practice in our next video, so I'll see you guys there.