So, last lecture was just a very brief introduction to mass spectrometry base proteomics and explaining to you a little bit about what it can measure. This lecture we're going to go into more of the technical details of how Tandem Mass Spectrometry actually works and what, what are the different components that make up a Mass Spectrometer. So, if you remember we went over this general work flow for a Tandem Mass Spectrometry set up last lecture. So, we'll just review it briefly here. You're starting with a sample. Either from a tissue or, cell culture, where you've, isolated proteins and typically one separates and enriches, those proteins for, based on the types of things one is looking for. And this separation and enrichment can actually be quite extensive. One very common way to separate proteins is just like as an elestrin blot to separate by molecular weight and then analyze different bands of molecular weight to reduce complexity of the sample. Almost universally proteins in a sample are digested to turn the proteins into peptides, a common protease you use to do that is trypsin but there are other proteases which are sometimes used. And so then you go from a usually fairly highly complex mixture of proteins to a simplified and a more simple mixture of peptides. These peptides are then typically separated even further. And then input into the mass spec via an ionization process. After they're anano, ana, ionized. They can actually be analyzed via mass spectrometry based on their mass-to-charge ratio and input into a first mass analyzer, which is used to, as a separation device to pick out which are the most abundant peptides that are being input to the mass spectrometer at this stage. And then select those ones for further fragmentation sent into a second mass analyzer which analyzes the fragments of those peptides, from which one can deduce protein sequence and also post-translational modifications and relate that to the, amount or quantity of the peptide that was coming in to the mass spectrometer. So, one of the most common forms of separation in mass spectrometry is liquid chromatography. in, in many cases a liquid chromatography column is the step which is, interfacing a mixture of peptides. And the mass spectrometer it, itself through a ionization process we'll describe a little bit later called electro-spray ionization. So liquid chromatography setup usually consists of many pumps which is taking your sample usually diluting it with some kind of solvent. This solvent composition can change over time, which changes its affinity for the most important part of the chromatography setup, which is the column. The column is packed with a resin. A common resin for proteomics is a hydrophobic C18 column but there are many types of columns. Which can separate your mixture of peptides based on various physical chemical properties. Such as hydrophobicity or charge for example. And the affinity of peptides for this column can be changed by changing the solvent composition. So typically you would, you inject your sample onto this chromatography setup, it starts to be pumped through the column, and as time goes on you can change the solvent composition on a so-called gradient which then changes how your peptides elude from this column. And then by analyzing what comes out of the end of this column over time we simplify a mixture of peptides into an even, a simpler and purer, composition of peptides coming out and being input to the mass spec at any given moment. When we input our sample of peptides to the liquid chromatography column we have to have a way of inputting that into the first mass analyzer. And to ionize the peptides that are coming off of this column. The method that's used to do this is called electrospray ionization. There are, of course, many types of ionization that some of which are listed here. But the two of which which are commonly used in proteomics are the electrospray ionization and so called MALDI or matrix-assisted laser desorption ionization which I'll explain in the next two slides. So electrospray ionization was one of the key breakthroughs which allowed proteomics to really take off in, in mass spectrometry applications where one is able to interface the output of a liquid chromatography column to a mass spectrometer. And the idea is, actually remarkably simple, although technically very challenging, is to take that flow output from the liquid chromatography column and put it out of a very fine spray needle, which turns the the liquid coming out into droplets, and those droplets are, are subsequently dried, and then, with a high voltage being applied between the liquid chromatography column and the input to the mass spectrometer these, these peptides can become ionized and then can be input into the mass spectrometer. And this invention, the Electrospray Ionization technique, was actually awarded the 2002 Nobel Prize, because it allowed not only mass spectrometry based proteomics to take off but many other forms of mass spectrometry, which depends on separation by a liquid chromatography column. Another commonly used form of ionization in mass spectrometry based proteomics is called MALDI. Where you simply embed your peptides in a so called matrix. Which is able to transfer charge to your peptides when you shine a laser on the sample. So you put your sample on a so called target, and shine the laser on it, and there's a potential then between the target and the mass analyzer. So a, a laser is shined onto your sample some charges are transferred over to your peptides they become ions and because of the applied voltage they then travel into the mass analyzer where they can be further analyzed. So after your peptides are ionized, they can be analyzed by a mass spectrometer. And the, the functional unit of the mass spectrometer that analyzes things based on a mass to charge ratio is called a mass analyzer. So there's, of course, several types of mass analyzers. Three commonly used ones in proteomics are a quadrupole, a time of flight analyzer, and an ion trap, or an orbitrap analyzer. So a quadrupole analyzer works in the following way according to this schematic, where the peptides, or ions, are input into a source from the electrospray ionization source, for example. And then they travel through a column where you have four rods two of which are positively charged and two of which are negatively charged. So that there's voltages across these rods and then the voltages are changed in a time dependent manner. So that you can imagine if you're an ion traveling through this series of columns, that you oscillate then either towards the positive or towards the negative nodes in this, in this quadrupole. And based on the time-dependent behavior that's, that's induced on these voltages, which can be tuned, you only allow certain ions with, with a particular window of mass to charge ratios through the other end. To the detector so a quadrupole can act both as a, a filter and also as a detector for certain mass to charge based on our ability to change the time dependent behavior of the voltages applied to the quadrupole. So another type of mass analyzer which is typically interfaced with multi ionization is the time of flight mass analyzer, where as described before. You shine a laser onto the, onto your sample on the MALDI target, which then induces a, a pulse of ions which are then transferred into the mass analyzer. So. If you can imagine that you have a fixed voltage at one end of the time of flight mass analyzer the ones with higher mass to charge ratio travel at different speeds as the ones with smaller mass to charge ratio. So because we can have a, a very defined start point for when the ionization occurs, and then we can measure the times at which different mass-to-charge ratios arrive at the detector. On the other end of the time-of-flight mass analyzer, we have a way of determining how much of each mass-to-charge is in our sample by analyzing the times of arrival of each time of flight of, of each ion through the time of flight mass analyzer. The last and most sophisticated but is becoming much more common in proteomics in the past few years is a so called Orbitrap which is a type of ion trap. As you can see here on the left, it's not a very large device, as big as perhaps a common coin. but, the way that the Orbitrap works is, a packet of ions is injected into the Orbitrap, with, with a linear velocity. And then, there's a voltage that, that's applied to the inner core of this orbitrap, which then gets the ions to start spinning around the orbitrap. And after a short burn in period, the ion, the ions reach a stable circular orbit around this orbitrap, and The unique thing about the Orbitrap is that, because of it's shape the ions then, although they're spinning around the cylinder, they also oscillate radially across the axis, of the cylinder. And the period of that oscillation radially is uniquely relatable to the mass-to-charge ratio. So by detecting how these ions are oscillating radially across the Orbitrap in time. And then applying some advanced mathematical processing to those signals, namely Fourier transform-based mathematics. One can determine what is the mass to charge spectra and intensities of these mass to charges of the ions that have been injected into this orbitrap. So I can say there's, there's a lot of good information and videos about this orbitrap system on the web link I've put here on the right. So I've talked a little bit about separation, ionization, the different types of mass analyzers but I haven't talked how that do you actually couple those mass analyzers together through fragmentation. And what are some typical combinations of mass analyzers that one uses in a tandem aspect setup. So a very common method of fragmentation is so called fast atom bombardment or collision induced dissociation. Where essentially you, you have your ions in a chamber and you simply bombard them with, with atoms. Which induces their their fragmentation and so in many cases ions are collided with an inert gas such as helium which then just causes them to randomly fragment causing the types of fragmented spectra that we, that I showed you a sample of in the first lecture. And this is a very common interface between two mass analyzers. One very common combination is a quadrupole with an orbitrap. So the quadrupole allows you to filter certain whole peptides through whichever are the most commonly and most abundant ones that happen to be coming off of the liquid chromatography column. And being amp, ionized by the electric spray ionization at the current time. Which you can then select for fragmentation via, for example, fast atom bombardment. And then take those fragmentation products put them into an orbitrap and then analyze what are the different fragments that I have here that were a result of fragmentation of this particular peptide that was selected by my quadrupole. So there's, there's a very nice and beautiful animation of this from Thermo Fissure who makes a quadrupole orbitrap tandem mass spect system called a Q Exactive. a, I can highly recommend you to go to the website here. I've put on the left and, and check this video out to really see you know, what happens to these ions as they go through the tandem mass spec process. And in a few lectures we actually take you into a lab to show you how does one actually use such a Q Exactive machine and how does one have to actually prepare a sample so that you can get data. And, and input your sample into such a Q Exactive machine. So in the next lecture I'm going to be talking about how you can take this mass spectrometry data. And how you can design your experiments in such a way such that you can quantify the resulting data.
