BroadE: Fundamentals of peptide and protein mass spectrometry

all right so this is a section on fundamentals try to introduce you to the concepts that are probably moat that first off the words that you'll hear repeatedly as we go through the presentation and what's really kind of the important stuff about how mass spectrometry and mass spectrometers used in proteomics work see if this is it's this one okay so every mass spectrometer system has these components in it we have some sort of a means of introducing samples to the instrument called the inlet and most of the work that you're going to be hearing about over the course of today and tomorrow involves introduction of samples using HPLC the samples go in to the ion source whose job in life is to take molecules that are neutral uncharged analytes in solution and create gas phase ions out of them that's the job of the ion source and the reason why this this line does not extend all the way to the end of the ion source is for a specific reason many mass spectrometers in fact most in the past the entire region here was in the high vacuum region this is very high vacuum nowadays as I'll get to when we introduce electrospray mass spectrometry which is our go-to technique for proteomics you'll find out that we actually create the ions at atmospheric pressure and then they go into the high vacuum region so post generation of the ions we separate the ions by mass and charge and that's really kind of critical it's not this thing doesn't measure molecular weight it measures mass and charging you have to derive the molecular weight from that measurement the ions are separated by the mass analyzer based on their mass and charge the principles of this I thought we all thought were kind of you know unnecessary for the biology and chemistry communities here at the burrowed if you're interested there are references at the end of each of our SEC and so you can look up the more technical details of this but the the ions are separated and then detected in a mass spectra mass spectrum is recorded nowadays by a combination of onboard microprocessors on the mass spectrometer and an external data processing environment so this is mass spectrometry it you know just you know I refer to as the Dark Ages is sort of where where I began my career in mass spectrometry across the street at MIT this is what they they looked like and I'll tell you that underneath the shiny plastic covers that all mass spectrometers have now there's a lot of this stuff still there so there's an enormous amount of electronics and other gizmos that have all been compacted now and made to look much prettier but this is what mass spectrometers used to look like they had lots of knobs and dials on them this is what a computer system originally looked like this was an IBM 1800 mainframe and it had literally massive 10 megabyte storage capability these were disks about this big that you had to physically remove of course all that's now well in the past this is what was the yellow hand crank oh this yeah it's a hand crank that was for winding film yeah we actually had to record this some of the stuff on on on film at one point okay this is a and please this is meant to be informal if you have questions raise your hands or speak up so I can know that you've got a question and will will will try to take them so nowadays these are the types of instruments that we have in the proteomics facility here at the brode we have this is an orbit wrap instrument q exact 'iv these are what's important about this is that these are very high performance instruments meaning very high sensitivity but also as I'll come to explain the terminology high mass precision and high resolution instruments and we these are used for our or global proteomics experiments this type of an instrument is a triple quadrupole mass spectrometer these are also very sensitive instruments they don't have quite the resolution of mass accuracy as these machines have but they have other positive attributes that you'll hear about when the when Hasmukh and Su present on the targeted approaches so I've already mentioned these tasks that the mass spectrometer has to accomplish one that I didn't bring up in that original introduction is this one to select and fragment ions of interest to provide structural information in the case of peptide sequence information using ms/ms this is referred to as tandem mass spectrometry hence the ms/ms part and I'll come back and explain what I mean by this in a in a moment yeah said that already so I'm gonna at least introduce something called matrix assisted laser desorption we actually do not have one of these instruments in the proteomics group there are baldie instruments here at the brode they're used in fact in the genome sequencing platform but and there's also one recently acquired in the chemistry group here here at the brode so let me walk you through this it's not that it's an irrelevant technology it's actually very relevant it's just that our go-to technique always involves coupling to liquid chromatography and one of the drawbacks of this method is it's not so straightforward to interface to liquid chromatography so the way this works is again inside the high vacuum region of the instrument there we apply on a generally a metal plate a thin layer of a liquid matrix um I'll come back by what I mean by that in a moment it's basically a small organic acid molecule that's used then your sample is dissolved in that matrix and it's allowed to crystallize on the surface it's put into the vacuum envelope and a laser beam is introduced through an optical port into the instrument and it the surface and what comes off the surface before what that that ionization method sorry that that laser blast does is to cause an ablation of the an explosion if you will of the matrix that is on the surface there is a large amount of energy that is that is put in as a result of the laser blast and what happens is that protons and other small charged molecules atoms are transferred from the matrix to the analyte molecules that are dissolved in that matrix so energy transfer protonation of the analyte molecule and once it's protonated it's sitting in a very high electrical field and it can't hang out any longer it says I see this field I'm going to get ripped out and projected down the pipe so mix in matrix flashed the laser and ionized generally by proton transfer so these are the types of molecules that one uses in MALDI and they're all small acids no more detail is needed their laser time-of-flight instruments are amaldi time-of-flight instruments come in two basic flavors one is the linear system so after the blast you form ions which can in fact because of the energy put in fragment in the process of transmittal down the pipe but because they they have the same velocity as the ion from which they originate it they're really not discriminated so they all arrive at the detector pretty much at the same time in reflecting instrument you can these ions get bent in another electric field which then does take into account the mass the difference in mass of the fragment ions that may have formed from this parent and they then arrive at the final detector with differing times and you can measure their masses that way MALDI is any questions about that no MALDI is particularly good for measuring intact proteins and this is really where it's made its mark and i think it's one of the areas where it continues to to really really outperform other types of instruments this is a typical spectrum this is an immunoglobulin you'll see a protonated molecular species and in many cases you will get multiple charging so this is a protein which has picked up two protons and is therefore doubly charged and that species that has three proton as therefore triply charged we'll come back to this multiple charging phenomenon electrospray because it is the one of the characteristics of that technique okay the rest of this is going to be on electrospray methodologies and the reason as i alluded to earlier is that ease of coupling to liquid introduction techniques has made this approach the go-to technique for all biological mass spectrometry which proteomics is a subset so liquid is introduced and now this is what this is the expansion of the critical region so that liquid is going through a needle which is one one instance the needles held at high voltage in other instances this is at ground and the interface plate here is at high voltage so the important point is that there again is a charge difference between the liquid coming out of the tip and the entrance plate to the mass spectrometer and what happens is the the needle is actually very fine and pointed the charge difference here creates a what's referred to as a Taylor cone there's a a plume of very small droplets that emerges from the from the tip and as the droplets travel the distance between the end of the needle and the entrance port to the mass spectrometer which is a distance of a few millimeters typically the ions begin to dissolve eight they begin to dry and as they dry they collapse on themselves and if you think about these these particles as having not just single charge in this droplet but many molecules with lots of charges on them at some point the droplet can't sustain the charge density any longer and there's something that's referred to as a columbic explosion that happens in these particles this is all of course physics hand waving but that's what what the wood is believed to happen these particles blow apart and charged analyte molecules are then brought in to the to the mass spectrometer and on the next any questions about that step so this next slide just illustrates this is the same thing you saw on the previous slide just showing you where where we're talking again just to emphasize this ions are actually generated at atmospheric pressure not inside the vacuum chamber those ions already formed get dragged into the into the mass spectrometer and in this particular instance this is a tandem mass spectrometer if you will where there is a mass separation device first mass separation device here there is a collision cell and we'll come back in terms of what that does in a moment and a second mass analyzer and then a detector which feeds the collected information to the data system so here is this is not in the handouts but it will be up on the website as a slide I added last night this is a typical mass spectrum of a very simple peptide mixture it happens to be for a mixture of various forms of this bioactive peptide no susceptor this is a piece of it that is goes from residue 1 to residue 11 and other piece that goes from residue 1 to residue 6 and another piece that represents the full length so one of the things that you strike immediately is huh the bigger piece has a lower apparent mass than the longer piece than the shorter piece here and that's again because the multiple charging phenomena and we'll come back to that in a little bit but you can clearly see the three precursor species for the three peptides present here and when I say precursor what I mean is the intact peptide you see these primarily as nated forms but you also see occasionally species in which the proton has been exchanged for a sodium or potassium in some cases ammonia these are typical processes that happen electrospray as I mentioned in Trudeau let me just go back here it's one of the things I want to point out why these particular residues are in red is because those are the places in the peptide we're charging occurs so if you think about this this is looking at gas phase protonation the most obvious places for a proton to get transferred are two basic residues on the peptide so there is a primary amine at this end of the peptide that gets charged the side chains of arginine and lysine are obvious places to be charged so the peptide that goes from residues 1 to 6 which is this and ends right here has only one basic charge on it which is the amino terminus hence this is ends up only being observed primarily in a singly charged form as the M plus h this peptide is goes from 1 to 11 right here breaking between the arginine and the lysine it has two charged sites the sidechain of the arginine and the n-terminus it's observed primarily in the doubly charged States and this guy which is 1 through 17 has 1 2 3 4 5 potential charges now interestingly we only see it in the triple e charge State or primarily in the triple e charge state and the reason is when you have adjacent charged residues you tend not to get charged on both it's too much too high a charge density ok that's how peptides behave and of course this extrapolates up to larger molecules and you can do small proteins pretty easily by electrospray this is an example for beta casein which is a phospho protein in which there in fact are known amino acid substitutions in the backbone in this mixture and that with these different precursors represent so you'll see there's a big charge envelope here that stretches probably up to 30 charges and down to about eight over this mass range that's being analyzed if you blow up one of these charge states this 14 plus charge state and scale expand it you'll see these three components now I'm putting these molecular weights on here and the question is well how did I get to that okay how do you know what the charge is for any one of these species I'm not going to spend time going through this in detail now because it's on the slide but basically the assumption is that peaks in a lectin a multiple charge spectrum from electrospray differ in mass by the charge a unit of a proton so any gap between here here here here here here is just adding one more proton on to the molecule so the in here is 1 and you can measure the mass the charge of each of these components accurately that's x2 and x1 and so you can determine the charge by the difference in met in mass to charge x1 minus x2 x2 over that is the charge state it's not generally come out to be an exact integer but you round it to the integer and then you test that integer again by plugging it in as the as the charge state for each higher charge and you actually get multiple measurements of the molecular weight that way and therefore a determination of precision of the molecular mass you can look through this and if it's confusing you can look at this reference or you can come talk to anyone of us afterwards and we'll explain to some greater detail to you or more clearly ok talked about molecular generating molecular species from electrospray now how do we get sequence information so this is done by a process referred to as collision induced dissociation and it really is just that precursor ions or parent ions go into this collision cell region let me just back up here it's this thing that I talked about before it's a discrete region of the mass spectrometer they and this region is filled with collision gas not high pressure but much higher pressure than the regions that surround it so this might be at 0.1 Torr and the regions outside of it might be at ten to the minus six to ten to the minus eight tour so this is a high pressure region by comparison to the mass analyzer regions to either side of it the ions are moving at some velocity and they basically are striking or coming in too near near collision if not real collision with the gas molecules that are in here it's typically nitrogen or argon sometimes helium is used as the collision gas and you generate generate fragment ions from these charged species they literally break apart I so you also get loss of small neutral molecules typically water carbon dioxide ammonia is readily lost and then the both the fragment ions as well as any residual parent ion go traveling out the end of the collision cell so here's that process in a little bit more detail so this is an MS scan at the top let's just focus on that so in an MS scan we scan the first mass spectrometer over whatever the desired mass range is here it's 350 to 1200 here the collision cell is effectively off there's no gas in it or we're doing something else that causes no collisional excitation to occur therefore all of the parent ions all the precursors that were formed back here in the ionization event passed through and we scan those out in the second mass spectrometer and that's what's shown here this is the mass spectrum and each one of these species here is a intact peptide ion either in its singly charged State doubly charged state or triply charged State and here is an ms/ms spectrum okay in an ms/ms spectrum again we have all ions going into the first mass spectrometer but now we are telling it only pass a certain mass region so here we're passing the region 834 to 838 so that's what this arrow represents now we have gas in this cell so all of the ions that have mass to charge 834 to 838 at that particular point in time coming off the HPLC column fragment break apart and those fragment ions then go into the mass but into the second mass spectrometer and get scanned out so effectively we've taken this ion because that's the ion that happens to be between 834 and 838 in that region we've passed it into the collision cell broken it apart and scanned out all the ions and this without the labels on it is the ms/ms spectrum that that you obtain the labels get added on later by data system and or manual interpretation and Carl clauser is going to tell you more about that in a in a few minutes so I'm not going to spend much time on this because Carl has a detailed presentation on on this but here is just very simply this is a representation of the peptide backbone and protonation as we said was the first step this may not be in your handout it will be up on the website so here's the n-terminus that's a sight of protonation basic side chain residues like lysine our protonation sites but interestingly in the gas phase and you have to remember this is a gas phase process certain things that you wouldn't think of as a chemist as being basic are so a mid-back bones are actually basic in the gas phase as are the mi carbonyls these are sites where proton transfer can readily occur but the order is generally amino terminus and the side chains tend to be more basic than the than the backbone with with some and histidine for example is another residue that readily charged again Carl's going to go into a lot more detail on this but this is what we mean by breaking the backbone so in general the primary cleavage that we observe in electrospray is cleavage at between of the backbone amide with charge being retained on either the amino terminal end generating these so-called B ions or on the c-terminal end generating the so-called Y I in series and you basically can fragment all the way along this backbone and Carl is going to tell you a lot more about that that's redundant spectrum okay I want to tell you in a few minutes about three important parameters in the mass spectrometer that we pay a lot of attention to one is mass accuracy this has to do with how accurate is the measurement of the mass that we're making second parameter is resolution this is really about you have species which have nearly the same molecular mass therefore they're coming out close to one another on the mass-to-charge scale how well are these Peaks going to be separated from one another that's what resolution is all about sensitivity not going to talk too much about that but clearly it's the it's a really critical parameter it is really more a function of how we prepare the samples and how and what instrumentation we're using for their analysis and I'm not going to go into in much more detail about this because sensitivity will come up in the course of the following talks quite a bit okay but how do we define mass so going back to your college chemistry course our definition of mass is based on carbon-12 with this being the reference point and by definition a the international unit of mass or the Dalton is defined as 1/12 the mass of a single carbon atom where carbon is defined as 12.000 at infinitum okay so that's the starting point and most elements including carbon have more than one stable isotope carbons particularly important because most of the molecules we analyzed have a lot of carbon in them and the natural abundance of carbons most abundant isotope c 13 is 1.1 percent so 1.1 percent of the carbon atoms on this planet have one additional Neutron in them yeah so why do we care about this you care about it because if your resolution is high enough you will in fact see the isotopes of these of these molecules in the mass spectrum and it's actually important to have high enough resolution because the higher this is a little bit of a maybe an oversimplification but more resolution gives you a better measurement of the mass it's not absolutely true but in it certainly in a complex mixture it is true and most of the time we're analyzing pretty complex mixtures so here's the here are the elements that you're probably most concerned about missing from this or phosphorus and sulfur you can look those up in tables but here's the one point 1 percent of carbon 13 hydrogen has deuterium as its abundant element most abundant isotope you can see it's actually tenfold less abundant than carbon 13 is nitrogen 15 has some significant abundance and oxygen 18 has some significant abundance and these are all stable isotopes of these of these atoms when you look at a spectrum of a typical peptide and this one is got you know on the order I think I don't actually have the composition down here but it's probably got about 6070 carbons in it this first peak so the mono isotopic mass what is that the mono isotopic mass is effectively the accurate mass including the decimal component so including you know this carbon of course it's decimal component is zero zero zero but for nitrogen we take into account the zero zero three one for oxygen it's not quite 16 it's 15 nine nine four nine we do take that into account so the month the mono isotropic mass is the sum of those accurate masses for the most abundant isotope of each of the elements present now in most cases those are the lightest isotopes but there are exceptions okay if you have iron for example in that in the case of iron the most abundant isotope is not the lightest isotope but four peptides the lightest isotopes are the most abundant and therefore this first peak is generally the mono isotopic this next peak is the one that has one carbon atom well why does this why is this not at 1.1% well because this thing's got 70 carbon atoms in it so it's 70 times 1.1 and therefore when you look at an ensemble of molecules that's how many are likely to have one carbon 13 represented in a large ensemble of molecules and this is how many the percentage a little bit close to 30% that have two carbon 13 s in them now what's missing from this is the fact that this species also has nitrogen 15 under it and oxygen and this guy's got oxygen 18 underneath it I haven't shown you that but they are making contributions and as these molecules get bigger those contributions get more and more significant and we do take those things into account in our measurements so back to this spectrum I showed you earlier to this point that we measure mass the charge of not molecular mass directly so in looking at this peak the first thing we would look at is what is the spacing in mass between the isotope Peaks in this spectrum so this guy's a 549 point one this guy is at five forty nine point six there's half a mass in half a Dalton difference between these Peaks okay what does that mean well you can't have half a charge you can't have yeah you can't have half a charge on this so therefore this thing has to be doubly charged so it's this species represents a 2m with protons to H so it's molecular mass is fundamentally twice this and then take away two which is ten ninety six point two similarly this guy if you look at its isotope spacing is five eighty five point two two five eighty six point two that's one Dalton spacing therefore this is singly charged if you look at the this guy the spacing here is 603 point two two six zero three point five that's point three of a mass unit so that means that guy is triply charged and therefore this represents a three M plus 3 H 3 plus so to get to the molecular weight you take the observed mass you multiply it by three and you take off the three protons from it to get to 1806 point six that's how this works now in the bad old days we did all this interpretation by hand now the mass spectra the data analysis software that Carl will describe pretty much does all of this for you without even thinking about it and that's the problem okay I want you to think about this I want you to think about what's going on behind the curtain because sometimes they it's it's not done right and you have to realize when it's not being done right okay so that's that's mass accuracy and isotopes this is a little bit about resolution so again resolution is the ability to resolve closely spaced Peaks it has a definition that we typically use now in the old days we use the 10% valley definition but now we we use it what's known as a 50% full width half maximum so that is the width of this peak in Delta M at the half height of the peak and we do this because a lot of times we have a peak in isolation and there aren't two peaks next to each other like this that we can readily determine the resolution from so why is why is this important resolution is also a function of mass so I drew this up on the blackboard here to illustrate this so here are two peaks differing by one mass unit at mass 500 and 501 and we have a static resolution of 1000 the peak widths here at 0.25 of a Dalton as a result of that and they're very cleanly resolved you can easily tell that you have two peaks here now as you go up in mass still retaining maintaining a resolution of a thousand we're now at mass a thousand and a thousand one for these two peaks they're still separated by one mass unit now the peak with half a mass unit and the peaks are still separated but just barely from one another because the width of the peak has increased now once we're up to mass 1500 to 1503 this is this region again now we have a peak at mass 1500 and a peak that has a peak top at 1502 now the width is 0.75 and you're seeing that we're just barely able to tell that there are two peaks and of course as you go higher and mass this gets worse and worse so here's a slide illustrating the effect of this showing you the change in resolution at 4a a peptide at mass roughly 1271 and another peptide with a mono isotopic mass of 25 36 or roughly double you notice that if we have a resolution of 500 we really do not resolve any of the isotope peaks at all the peak top here which is the thing that is most easily measured doesn't represent the mono isotopic mass nor does that represent the average mass if we used the average masses that combine all the isotopes together into the weight of the of the atom but once we get up to a resolution of a thousand at this mass we were beginning to separate the isotope Peaks and we can cleanly determine the mono isotopic mass of the the accurate mass of this peptide and a resolution of five thousand you've got basically sticks which are even easier to determine now of course is you this is just an illustration of this again as now doubled the mass you can see that at resolution of a thousand we no longer resolve these Peaks whereas it now requires a resolution of five thousand in order to get decent resolution of the peaks for a peptide of this mass so that's two different ways of illustrating the effect of resolution any questions about that No okay so maybe the most important thing for you to take away from this is that in a complex mixture you don't get a measurement you don't get accurate measurement of your masses unless you've got decent resolution so here's an example where this is a low resolution spectrum in this case these are the peptides these are just small molecules there are four low small molecules in a mixture and on a an instrument like an ion trap with a resolution of you know around a thousand this is what that envelope looks like it's unresolved and the best thing you can do is to take a centroid of it which doesn't give you the molecular mass of any of these species and obviously doesn't tell you that you've got a mixture present but if you go to an instrument like our high-resolution orbitrap machines or cube executives you now cleanly baseline reads more than baseline resolve these species and can determine the accurate masses of each one of these components so in complex mixtures like you know cell lysate biofluids etc you want to use instrumentation that has both high mass accuracy as well as high resolution and this is just the Surgeon General from years ago warning you that insufficient mass accuracy is dangerous to your health I remember anybody remember his name somebody say coop that's the coop that's right C everett Koop the guy that was responsible I think for the warnings on cigarette packages so Carl I don't know Carl you don't have the slide near deck do you okay so this just shows you that that accurate mass really is a is a as it says a powerful against when you're searching peptide mass information against a database so this is a peptide of mass 1005 this shows you increasing mass accuracy in part per million on the measurement of that peptide and this shows you the number of possible amino acid compositions independent of what actually exists in the database this is just the number of possibilities and you'll notice that G you know going from what is already pretty high resolution sub 10 part per million to one part per million yeah that that constrains the number of amino acid compositions but G not by a whole lot you're going from you know four thousand down to three hundred it's a constraint but you still have three hundred things you'd have to figure out now but when you're talking about searching a database so let's say this is a mammalian cell lysate now that represents two levels of constraint the accurate mass and secondly what is known to exist in nature in the mammalian genome and so now at one part per million you have only 18 potential tryptic peptides so while you have three hundred potential amino acid compositions there's only 18 actual tryptic peptides that have that composition and you can then begin to work out well which one of these is it likely to be and of course how you do that is using your ms/ms data that we've generated as part of that same experiment okay how are we doing for time I don't have a watch one you may have a quick read quarter to one okay so I'm almost almost done most analyses in proteomics are done by digestion of peptides to proteins and this is referred to as bottom-up proteomics it has and this is what you're going to be hearing about throughout the course of the day it has a number of advantages listed here the data acquisition is very highly automated now and Carl will be talking about that importantly the reason why this is done is that the fragmentation of tryptic peptides ie taking a protein digesting it with trypsin has been studied for several decades and it's really very well understood and the rules of fragmentation are what underlie the software tools that are used for automated data analysis forth but also very importantly is that it's much easier to get high-resolution separations of peptides than it is to separate proteins okay a lot of proteins are very sticky you never get them off the column it's much easier after you've digested them to be able to separate these complex mixtures of peptides now that said there's a lot of disadvantages as well the biggest one being that you've taken what was already a very complex mixture now you've magnified its complexity by however many tryptic peptides each individual protein can generate so using roughly estimating twenty to a hundred times more complex in this it's a very high analytical demand on the instrument to be able to make measurements of lots and lots of peptides that are in your mixture fortunately those instruments that I showed you have gotten increasingly fast over time maintaining their sensitivity and that's just over the last couple of years so we see really a continual arithmetic improvement in the performance of mass spectrometers over the next three to five years and every three to five years by history in mass spectrometry there's been a leap in terms of a new analyzer that's been introduced so I you know the one the one thing I would never do is discount that there could be factors of 10 improvement in what we do okay moving on you can also just to make sure you're aware of this you can in fact do something called top-down proteomics and what this means is taking an intact protein and analyzing it directly not just for molecular weight information but to try to derive information about the sequence of that protein and this is most useful for relatively simple mixtures or single proteins and it has the advantage that if you have variants that are isoforms or which have multiple modifications on them representing some kind of a code like in the case of histones for example you can read out in one go on a sec tably one population the molecules what the what the arrangement or the combinatorial modifications are that are present on that population you can't do that once you've digested the protein two peptides now and there's a couple of references here if you're interested in that but while it's useful it requires really quite specialized instrumentation you can't easily apply this to complex mixtures the data interpretation remains its spite advances that have been made relatively recently quite complex and the breadth and depth of coverage is nowhere near what you can do with bottom-up proteomics you get maybe a few hundred proteins able to be detected in these studies not the thousands even tens of thousands that you can do now by modern proteomics so we have to do some sample handling so this is really a preamble to what you're going to be hearing from Monica later the first thing we do is to reduce and alkylate the proteins and the reason for doing this is to break disulfide bonds and make them make the protein more amenable to digestion then we use highly specific proteases I've talked a lot about trypsin but we also use things like lysine staff v8 nos pan and their specificities are shown here but occasionally we'll because there's a need to I to look at a certain part of a specific protein will resort to less specific protease is like chymotrypsin proteinase k or thermal Ison in order to get to that part of the molecule but it's not our first recourse we tend to stick to the more highly specific proteases lycée and trypsin in particular there are some problematic amino acids so you should just be aware of this the finding no matter what you do is pretty easy to oxidize so we observe it +16 in not all but many many of the peptides that contain the fining if in the process of cutting up a protein you generate a gluten or a carboxyl amino methyl cysteine at the n-terminus of your peptide those will cyclize these are just thing and these are factors that now are actually incorporated into the automated interpretation algorithms and taken into account but you should be aware of them one thing that we we try to avoid although we do use urea as a denature --nt extended exposure to urea causes carbonylation of the n-terminal of proteins and peptides so we try to avoid this okay so this is the experiment you're going to be hearing about repeatedly biological samples down to taking out mixtures of proteins which are digested the peptides separated and analyzed by LC ms/ms on modern mass spectrometers to generate rich patterns of mass to charge and intensity into peptides and fragment information for sequence that data feeds into a data analysis package which car we'll be talking about in a moment which leads to peptide identity protein identity and information about relative abundance and I'm just gonna skip over these because they're redundant and I'll finish with this slide just to point out for those people who do a lot more genomics on microarray work than they've done proteomics there are some pretty distinct differences between what you do in the microarray or genomics area in proteomics in micro arrays you know the features that you've put down on the chip in mass spectrometry we don't know all the features that were likely detect because we observe for example the modifications and those are not things that are sometimes predictable as to whether we'll see them or not your sample in a transcriptional profiling experiment is static during the analysis where in our case the sample is dynamic it's coming off that liquid chromatography column in time and we have to be fast enough to be able to sample whatever is coming off at any given moment in time from that from that chromatographic system in transcript profiling space you measure all the features but in mass spectrometry interestingly enough we only get to something like today 25 maybe 50 percent of all the peptides actually attempted to be sequenced and of those a smaller percentage are than sequence by the automated analysis software so there's a drop-off in information here and this is one of the places where there's gonna you will see substantial improvements as the instruments get faster and resolution and so forth improve you guys have a way of genomics guys have a way of amplifying their low blow signals we have no such amplification method in proteomics and in any of our systems whether it's a mammalian cell line or bio fluid there's a tremendous dynamic range in the protein abundance we have to be able to deal with that dynamic range directly so it's a complication analytical challenge and here in in transcript profiling space if you don't see a signal it means that thing isn't there okay whereas in mass spectrometry particularly in the case of discovery experiments if you don't detect a species that you're interested in it means either that it's not present or you're sampling wasn't efficient enough to pick it up in that experiment and you can't tell which this is one of the reasons we have worked on developing and now implementing something called targeted mass spectrometry you're going to be hearing about that today so here are some suggested readings at the end of your present end up the presentation I'm going to stop there take any questions and then hand it over to Carl anybody you're all awake that's good yes yes so it's a very good point it's an important point not only our pet are some proteins ionized more efficiently because they have more chargeable sites on them which leads to which relates directly to their detect ability but peptides also have large differences and it's something which now that you've mentioned it I need to actually fold back into the presentation when you even if you've got equivalent digestion of all peptides from a protein they don't all respond the same they don't have all the same signal response even if you've got stoichiometric yield of all the peptides now what contributes to this is in in some cases yes the degree of chargeability but it's not just that some of it relates to hydrophobicity more hydrophobic peptides in some cases tend to actually give you a stronger response part of this relates to how easy is it to dissolve eight to take the water molecules away from this from from the peptide so there's a lot of factors not all of which are well understood but the the the practical observation is that you can have ten and sometimes even up to a hundredfold difference in the abundance of a peptide the observed height intensity of a peptide from the same protein even with equivalent release efficiency so again this represents a challenge so direct quantification from a mass spectrometry doesn't work unless you have some kind of normalization or internal standard and we're going to come back to that because it relates directly to talks you're gonna be hearing about from Jake Jaffe and others this morning I'm sorry is it intact yep yes so that it's exactly what I'm referring to not just fragmentation I'm talking about the peptide precursor intensity is 1/10 to 1/100 of another peptide from the same protein full proteins you know I don't you know there I could wave my hands and give you a solid answer but I don't have the data to support this but it it's clear that with a even a simple mixture of proteins they give quite different ratios with what you think is equivalent amounts so because we don't measure Prout a lot of mixtures of proteins any longer most of what I'm telling you relates to peptides and it's very true for peptides yes yeah so the the resolutions on the instruments that we're typically running now just to give you a sense is not a thousand but more like 15,000 to even a hundred thousand that's the kind of resolution and in terms of the I'm not sure that I think on a quadrupole correct me if I'm wrong guys on a triple quad I think the resolution actually is constant across the the mass range whereas on you know instruments like these ion cyclotron resonance or Orbitrap instruments it's not the resolution has to actually be specified at a given mass typically it's like a mass 400 you get a hundred thousand resolution but if you're at mass a thousand the resolution drops down you

Channel: Broad Institute

Views: 93,660

Keywords: Broad Institute, Proteomics, Steve Carr, peptide, protein mass spectrometry

Id: PFOodSbH9IY

Length: 49min 56sec (2996 seconds)

Published: Mon Sep 30 2013