Mass spectrometry for proteomics - part one

in modern proteomics the mass spectrometer is the key piece of instrumentation that allows the global analysis of complex proteome to happen in a reasonable timeframe this presentation is all about how mass spectrometers actually work because you need to understand how an instrument generates data before you can properly interpret the data the first question is why is math so important for identifying proteins each element in the periodic table has a unique mass in this table listing the masses of the main elements making up organic compounds such as proteins it can be seen that each element and the isotopes of those elements have a unique mass the difference can be averaged to one Dalton or the mass of a proton or neutron carbon has three isotopes which vary by the number of neutrons in their nuclei but otherwise they are chemically identical the carbon 13 isotope is only a very small proportion of the total carbon on the planet but it is extremely important for mass spectrometry as we will see because elements have unique masses compounds made of those elements can also have unique masses for ethanol when the masses of the elements making up ethanol are summed the compound molecular mass can be calculated this can be repeated for carbohydrates or other organic molecules such as this toxin from potatoes and more importantly proteins and peptides the mass of a peptide can be calculated from its sequence by summing together the masses of each amino acid but what if we don't know the sequence of the peptide this is where the mass spectrometer becomes useful mass spectrometers measure the mass of ions which are charged particles so unless you can either add a hydrogen atom to an amine or remove a hydrogen from the carboxyl group of a peptide you cannot measure its mass mass spectrometers consist of three basic parts that can be put together in different combinations to make specific types of mass spectrometers that are useful for specific types of analyses first you need something to Jenner the ions called the iron sauce then you need something to measure the mass of the ions sometimes called a mass filter combinations of mass filters can be put together for different purposes lastly you need something to detect and count the ions the mass filter and detector are always under vacuum so that random molecules floating around in the air don't get measured once all of this happens a spectrum is generated in this case for solanum the spectrum shows a number of populations of molecules with each peak being the accumulated signal of hundreds to thousands of molecules of exactly the same thing the major Peaks show the molecules of salonen whose carbon atoms are all the c12 isotope the next peak appearing at one dot and larger other molecules of solanum that have one carbon atom with an extra Neutron the c-13 isotope in different molecules the c-13 atom can be at a different place in the structure but the mass of the intact compound will be the same thus we can see the accuracy of the mass spectrometer that can measure the difference in mass 2 less than the mass of a hydrogen atom the difference in mass between the c12 and c-13 isotopes of ions is important when measuring peptides mass spectrometers don't measure mass but mass divided by charge ratios or M over Z so for salonen which takes up a single proton to become an iron the mass over charge ratio for the c12 iron is 868 and 1/2 divided by 1 charge and the c-13 isotope with that extra Neutron is 869 and 1/2 divided by the one charge thus the difference in mass between these isotopes is 1 Dalton reflecting the mass of the neutron peptides such as this one because they have two primary amines can take up 2 hydrogen atoms and thus have 2 charges the calculated mass of this peptide sequence is fifteen hundred and seventy Dalton's but the mass spectrometer measures the mass / charge ratio for the c12 isotope of 785 point 8 this is because it is measuring the mass of the peptide plus two hydrogen atoms divided by the two charges of those hydrogen atoms the c-13 isotope containing the extra Neutron of the peptide is measured as 786 point 3 or 1/2 adult and larger then rather than one Dalton because the mass spectrometer is measuring the mass of the peptide plus a neutron plus two hydrogen atoms and then dividing it by two charges the mass spectrometer can now be programmed to use this as a selection device looking for 1/2 Dalton or less differences between adjacent mass Peaks to select ions for further analysis now that we've got that sorted out it's time to look at the ways of getting peptides into the mass spectrometer or looking at the ion sources the first we will look at is matrix-assisted laser desorption ionization or maui invented in the mid-1980s by Franz Helen camp and Michael Karras in this technique the peptides are spotted onto a metal target plate with an ultraviolet light absorbing compound called a matrix and allowed to dry the two main matrices use our alpha cyano cinematic acid or CHCA and cinah Pennock acid the plate is then put into the mass spectrometer and a high voltage is applied to the plate about fifteen kilovolts a UV laser is then fired at the sample and energy absorbed absorbed by the matrix which transfers this energy to the peptide molecule this ionizes the peptide molecules off the target plate and they are accelerated past a counter electrode at twelve kilovolts into the mass filter which is normally a time-of-flight detector it is important to note that now be predominantly generates singly charged ions not multiplied charged time-of-flight is the simplest form of mass filter measuring the time it takes for an iron to travel from one end of a tube to the other end and strike a detector the laser pulse starts the clock and an iron strike in the detector is the measure of time the larger the iron the slower it travels the mass can then be calculated by inserting the flight time into this equation essentially it's like measuring how long it takes for something to get from slingshot to target as with everything else we've discussed we are not measuring a single iron of a particular mass but hundreds to thousands of molecules are the same identical peptide when the plume of ions is first produced they are spread out a little in space and thus time at the detector the number of irons hitting the detector is measured in bins of half a nanosecond after a specified time range has been scanned we can connect the intensities together to draw a peak the apex of which is the mass of an iron all of these intensity lines are the result of the same mass iron hitting the detector but it's slightly different times over a few nanoseconds thus to increase the accuracy of the mass measurement and increase sensitivity we want all of the ions to hit the detector at the exact same time making the peak as narrow as possible the first place to compress the ions into a smaller space is at the iron source using a technique called delayed extraction where electrical pulses are used to allow lagging ions to catch up before entering the time of flight tube another way of compressing ions is to use an electrical reflector this uses an electrical mirror to reflect the ions back to a detector this does two things firstly it makes the flight path longer so that ions of slightly different masses are better separated in time secondly it compresses the ions of the same mass together in space and thus time resulting in a sharp peak for a mass the difference between a linear Tov and a reflector Tov can be seen here these are both the same set of peptides the lower trace being linear Tov and the upper trace being the reflector Tov the in sets are a zoom on the same specific peak in the linear Tov none of the isotopic forms of the peptide can be resolved whereas the reflector Tov is able to resolve the c12 isotope from the c13 isotope with ease the alternate form of iron source is electrospray again invented in the 1980s by John Fenn in electrospray the peptidyl protein is in a liquid that flows out of a capillary that is under high voltage usually between 1,500 and 5,000 volts depending on the flow rate of a liquid with a lower flow requiring lower voltages for peptides and proteins the liquid is normally an acidified mixture of water and organic solvents such as a seed a nitrile the acidic conditions means that there is an excess of hydrogen ions in solution and the peptide molecules become protonated on their primary amines as previously described as the liquid is sprayed it forms a plume of droplets that rapidly evaporate making the droplets smaller and smaller and pushing the positively charged peptides together closer the molecules of the same charge don't like being too close together so there is a point where the charge density becomes too high the Rayleigh limit and the droplet explodes leaving the ions to accelerate into the mass analyzer often a quadrupole that can be placed before a time of flight detector electrospray generates multiple charged ions as seen in this example of lysozyme which has an intact mass of about fourteen thousand Dalton's as mass spectrometers measure mass divided by charge ratios the iron masses here show a population of lysozyme ions with either 7 charges 8 charges 9 charges ten charges and so on and so forth to determine the mass of the lysozyme iron with one charge and thus it's true mass we can use simultaneous equations computer algorithms exist to perform this task much faster thus electrospray can be used to very accurately measure the mass of a protein using a mass analyzer with a relatively small mass range which makes the mass spectrometer more sensitive to illustrate the main difference between Maori and electrospray we can observe these two spectra of myoglobin a protein with a mass of sixteen thousand nine hundred and fifty-two Dalton's in the upper spectra from the from Mao D we see the singly charged iron at sixteen thousand nine hundred and fifty-two and an iron of myoglobin with two charges at half of that mass in the electrospray spectra we can see the multiple charge states of different populations of myoglobin Enza ions the main advantage of using electrospray is that it can be interfaced with liquid chromatography more easily than Maori but we'll discuss this further when we have discussed chromatography in more detail electrospray is most often interface with quadrupole mass filters although iron traps were very popular for a long time we will discuss those briefly later a quadrupole consists of four rods arranged in a square that can have different amounts of voltage or radiofrequency applied to them at a certain voltage and radio frequency a certain mass to charge ratio iron will be passed through the quadrupole to a detector while other ions are lost to the vacuum system to scan a mass range the voltage and radio frequency must be varied and so sensitivity is reduced so why would you use a quadrupole Quadra files are fantastic at selecting specific mass to charge ratio ions and transmitting them to other types of mass analyzers one configuration is the triple quadrupole shown in this diagram the first quadrupole is used for iron selection for selection of a specific iron with all others being excluded from the quadrupole the selected iron is then transmitted to the second quadrupole which is a collision cell this cell is filled with nitrogen gas molecules and the ions are accelerated into the collision cell where they collide with the nitrogen molecules and get smashed into fragments the fragments then get transmitted to the third quadrupole which is set to transmit only one fragment to the detector while the rest are lost the result of this is an extremely sensitive and specific assay called selected reaction monitoring which can be thought of as doing a Western blot with a mass spectrometer it requires that you know the identity of the peptides that you want to measure to program the mass spectrometer in other words you can target specific peptides in a complex mixture these spectra represent full scans of the intact peptides selected by quadrupole one and the fragment peptides selected by quadrupole three however the output is a time-based chromatogram showing when the iron selected by Q three is Trent is detected and this can only happen when the intact peptide is present and selected by Q one is transmitted to the collision cell for fragmentation and a specific fragment is transmitted by Q three to the detector these combinations of Q one mass and Q three mass are referred to as a transition and the mass spectrometer can be set to scan transition combinations for about 50 milliseconds before moving on to a different combination for another 50 milliseconds thus a list of a few hundred transitions and thus peptides can be scanned through over a few seconds allowing numerous proteins to be assayed in the one experiment rather than one at a time for a Western blot but in the majority of proteomics experiments we do not know the identity of the peptides or proteins so we need to operate the mass spectrometer in a discovery mode using scans of a mass range as mentioned Quadra holls do this poorly with low sensitivity and resolution so hybrid instruments are often used one of the most popular is the quadrupole time-of-flight hybrid which is shown in this diagram it consists of the front two quadrupole zuv a triple quadrupole instrument the q1 for iron selection and the collision cell for measuring for fragmenting peptides the third quadrupole is replaced with the time-of-flight mass analyzer as mentioned earlier the time of flight requires a start time to measure the ions flight time but using chromatography to produce ions at the source means that ions are continuously flowing through the quadrupole we create a start time by making the ions turn 90 degrees into the toff every 250 milliseconds the pusher pulses a packet of ions into the toff the ions fly down to a reflector and are reflected back to the detector thus we can scan a mass range and get high-resolution data this animation shows things in more detail all of these balls represent ions the grey ones represent uncharged ions that will get sucked out by the vacuum system the positively charged ions will continue through from the sampling capillary through a series of electrical lenses into the quadrupole in the quadrupole here the only iron being selected is the green one all of the rest of the excluded by the vacuum system the green iron has been selected and then transmitted to the collision cell where it will collide with nitrogen gas molecules to produce fragments you can see the nitrogen gases the hazy things here and you can see the fragmentation of these green ions occurring the fragments then get transmitted out the other end of the collision cell to it to the time of flight tube the fragment ions are compressed into a nice flat packet before they reach the top tube so that all of the ions have the same starting time before they are pulsed into the top as you can see them rising straight up they all have the same start time and ions of the same mass stay together but ions a different mass have a different flight time they get reflected back from the reflector on and down to the detector they strike the detector and cause a cascade of ions which is then measured by a photomultiplier tube thus different masses are detected in bins you there are not many uses of cyclotrons nowadays because the problem with them is that they're very expensive to run as the magnets require cooling with liquid helium in proteomics applications they have been replaced with an alternative type of cyclotron called an orbit wrap which does not have a magnet in an orbit wrap the iron circle or orbit around a central spindle again inducing current in collection plates which is then interpreted by Fourier transform to produce a mass spectrum more detail on how these works can be found in this video from thermo as with quadrupole tofs orbit wraps are most often used with other mass analyzers as a hybrid the original instruments produced were paired with ion traps which we are not going to discuss any further except to say that they are able to trap specific ions and fragment them in the same space unlike a quadrupole which transmits ions to another quadrupole for fragmentation recent Orbitrap based instruments use the Orbitrap for scanning a mass range to measure intact peptide mass before using a quadrupole to select a specific iron and fragmenting that iron in a separate collision cell before transmitting the fragment ions back to the Orbitrap for measurement for proteomics purposes either a quadrupole toff based or an orbit rock trap based mass spectrometer would give good data but specific differences can make one type more useful for certain types of analyses while the Orbitrap incorporates a detector by inducing a current in detector plates triple quads quad tofs and iron traps need detectors after the mass analyzer triple quads and iron traps used inodes where a charged iron hits an electrode creating a secondary electric electron that increases or amplifies the signal the mass is determined by the software knowing what the voltage and the frequency of the quadrupole was and those values determine a certain mass time of Fey analyzes time-of-flight analyzers are coupled with microchannel plates a charged iron hits the front of the plate and causes a cascade of electrons to hit an electrode resulting in signal amplification the mass is determined by measuring the time from the start signal coinciding with the ions being pulsed by the pusher and the ions of a single mass hitting the detector we revisit this diagram from earlier where ions arriving are counted in bins of half a nanosecond after enough time has passed to measure the desired mass range typically 350 to $1,500 the intensity bars are joined to produce a spectra this concludes our very basic introduction of how mass spectrometers are used in proteomics work in the next presentation we will discuss how the instruments can be used to solve questions in proteomics

Channel: Matthew Padula

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Length: 22min 59sec (1379 seconds)

Published: Tue Jul 25 2017