 Thank you guys for hanging in there and sorry for, you know, being a little bit late I had to find parking which was a little challenging. So today we're going to be discussing and it's rotation that your university acquired two three years ago maybe. So this is the megatop, MOLDI top mass spectrometer that is really focused on doing high mass analysis. Is everybody here doing MOLDI or just kind of interested in the capabilities of MOLDI? Yeah, great, great. So I'm gonna start off the talk just doing a brief kind of MOLDI recap just in terms of how the sample preparation works and a little just about the technology. After that I'm going to just review some of the Shimatsu offerings before going into some of the high mass applications and the megatop. And then after that I'm just going to touch on some really cool new developments that are going on at Shimatsu Corporation in the Mass Effect Research Laboratory as it relates to MOLDI mass spectrometry. So MOLDI is a technique that's been around for quite a number of years now and what's really nice about it is simplicity in the method especially as it relates to sample preparation. With LCMS you typically have to separate out your compounds or do direct infusions as well. But here you just spot your sample that co-crystallizes with the matrix and and then you are going to ionize the dry sample. This is just a video. So typically in sample preparation you have a mixture of your sample that's typically in the peak of mold that's a lower range and then on top of your sample you then spot the matrix. It's just called the dry droplet method. Some people use sandwich method where they'll pre-spot matrix, let that dry and then spot the sample that can help to concentrate the sample around the matrix crystals. So very straightforward sample preparation. Once you spotted all of your samples you let them dry. You're then going to load them into the instrument and this is basically the door where you would load your plate. It's like that side. I rotate it all of a sudden. And so then following loading the plate into the instrument you're then going to need to form the ion. So in MOLDI you're using a laser that is kind of wavelength that is close to the maximum absorbance of the matrix. The laser energy then is transferred by the matrix to the sample to induce ionization after which the analytes are going to separate based on their M over C through the time of flight. In the example of the megatop in linear mode instrumentation you have a direct path from the ion source to the detector. This is very beneficial for measuring very high molecular weight species but for lower molecular weight species you can then send them through a reflectron to help focus some of the dispersion of the ions so that you get very high resolution. So we would typically look at mass range say 1 to 5,000 to get that basic topic resolution and accurate mass using reflectron mode. You can still look at higher molecular weight species in reflectron but you would typically be then still using average molecular weight. So Shimatsu has a pretty long history in MOLDI mass spectrometry. The method was kind of developed simultaneously by Kuiichi Tanaka and the Hill and Camp School but in the end it was the work that was done by Kuiichi Tanaka that was noticed and he received the double prize in 2002. So his method was a little bit different from traditional MOLDI in that he was looking at a soft ionization technique where he mixed blister in with the matrix in a sample to induce the ionization. So that's a soft laser distortion method. Well anyway the work that Kuiichi Tanaka did then went to form the first commercially available MOLDI the LAMS 50K. Back in the day it was quite an accomplishment because it was the first time that people could look at high molecular weight emulates without any sort of fragmentation and this year we are celebrating 30 years of MOLDI mass spectrometry at Shimatsu so we're super excited about that. And then over the years Shimatsu has continued to evolve their product lines so we had the after the LAMS 50K they went to a much smaller format with the compact and then they started introducing the CFR CFR pluses which started to introduce Reflectron the top squared which then led to the Axima series which the megatopsis is built off of and then about five years ago we launched the MOLDI 79U which is our high performance MOLDI and then just last December we released the new batch top MOLDI linear linear MOLDI system that's been quite popular so this is our current offering ranging from the small bench top through to the high performance MOLDI system. So in terms of applications for MOLDI the the demands have kind of changed over the years so looking at back in the early days in the late 90s early 2000s people were really focused on looking at proteins typically using Joe electrophoresis and doing peptide mass fingerprinting because then there was no commercially available PSD post-horsed decay or CID and then from there people were able to use the high mass capabilities start looking at intact proteins but maybe a little bit limited in terms of the molecular weight range then in the 2000s people started using this the method of MOLDI imaging I just came from a MOLDI imaging conference and it's amazing to see kind of how instrumentation has developed around that application and then really though the resurgence of MOLDI came in the form of microbial identification using protein fingerprints to be able to quickly identify microorganisms by comparing them to a database and this is popularly commercialized by a Bionary unit group or a Bionary unit partnering with Shimansu to provide the Vitech MS system so you know there's a whole diverse range of MOLDI applications which oftentimes doesn't matter which instrument that you have as long as you have that MOLDI source so as it relates to high molecular weight proteins there's or just in terms of characterizing a protein there's many types of experiments that be done using MOLDI to investigate like the N-terminal sequence so people are using a technique called in-source decay where you're basically using the laser energy to destabilize the protein and allow to fragment along the flight path to get the N and C-terminal amino acid sequence of the protein you can do peptide mapping using MSMS you can do glycosylation analysis deamination post translational modifications basically a lot of this will require a collision cell the ability to isolate precursor molecules what the megatop will really focus on is some of the more high mass applications so looking at intact masses of very high molecular weight species such as antibodies you can also use it for looking at protein aggregation protein complexes peggulated proteins so peggulation is a very popular method for stabilizing drug compounds recently it's half-life and biological matrices ABC anybody drug conjugation and protein complex analysis we kind of covered a little bit of the benefits of MOLDI very simple straightforward sample preparation has a high tolerance for salt so if you may have a some salts in your buffer for your sample you don't necessarily have to clean it up sometimes you do very it leans towards the high mass detection so you can easily detect your proteins of interest typically generating singly charged molecules so the spectra are very simple to interpret and get good specificity as well so this is an example of looking at high mass discrimination due to the low velocity of ions so when you're looking at high molecular weight species as they're traveling down the flight path the velocity the impact on the detector is it's it's not so I don't know another word to say except for that so good and and as a result you get very low sense it low sensitivity as you increase in molecular weight so that's going to affect the amount of sample that you need and also the mass range that you can use to look at your at your sample so this is just using a simple electron detector now a lot of times the electron will be sufficient again assuming that you have enough sample to analyze and so in my experience on just a standard detector you can easily get up to 150,000 200,000 but once you get beyond there your sensitivity drastically will drop off and it also you have done with the shoots regarding some resolution as well this is just kind of laying out for you some of the types of molecules that you could analyze using a standard detector versus using a high mass detector now there's going to be a lot of overlap with regard to a lot of these proteins like pegalated drug anybody contributes because what you're going to gain with the high mass detectors and great deal of sensitivity as well as improved resolution so even though you can detect them with the standard detector you're going to see improved results with the high mass detector so when you're talking about your standard Mali I kind of like to think of the the megatop that is like the super hero Mali and that it unlocks some of the powers that a standard instrument wouldn't have so just as Clark Kent changes costume and Superman you've got to do a little costume change for the for the for the megatop and so we've exchanged the panel but we also have the high mass detector sitting on top and really the what you're able to do is to easily exchange between the standard mode of operation using a typical electron multiplier detector and the colex detector just using it's not an iPhone it's the eye touch or I forgot what they call them these days but it's just a little tablet device that you can click a button it'll switch in and out of position so the way that the colex detector works is basically a conversion dyno and so as the as the antibodies or the high molecular weight species hit the detector the the conversion dyno is going to then kick off secondary ions but these are going to accelerate through the through the dyno to a secondary detector where these with cascade effects so you're going to kind of be amplifying the signal to increase the sensitivity and so this is just kind of to lay out for you where the linear detector specification for Shimazu ends and how much farther you can go with the colex detector and so this is just kind of showcasing what the difference is in terms of the sensitivity levels that can be achieved so these are scaled to the same value where on the top you have the electron multiplier and the very top detector on the bottom you can see you get much higher sensitivity of detection with the colex detector alright so here with with sample preparation for these high mass applications you're typically going to do a serial dilution of your samples to try to optimize the for analysis you're then going to take these dilutions mix them with matrix you can either pre-mix them in a tube or you can mix them on the plate and just as illustrated before you then load the plate into the instrument for analysis so I wanted to talk a little bit about just kind of the issues in analyzing protein complexes by volume tough mass of trometry so what ends up happening when you ionize a protein complex the animation got a little bit ahead of me you are going to dissociate the the on covalent interactions so what you end up seeing is not the protein intact protein complex but you see the individual subunits of the of the protein complex and so what typically is done is to do some sort of chemical stabilization so this can be traditionally this is using NHS ester stratification to kind of cross-link the the amino groups of the two different proteins and it's a very non-specific interaction so it's basically going to attach to all the lysines that you have there all right so once you've done your chemical stabilization then you end up detecting mostly the protein complex so here's an example that it's just kind of illustrating some different types of cross-linkers these are all NHS esters that you can then look at this glutathione as transferase and looking at the comparing the the efficiency of the cross-linking with the two-minute reaction and so GST forms a dimer and so you would expect that with the cross-linking you should see the the double mass the 2M plus as opposed to the as opposed to the single mass so you can see with these the purpose of this publication was basically to showcase some cross-linking some new cross-linking reagents and showing kind of a lack of efficiency of some of the traditional cross-linking reagents and cobalex who builds the detectors they have created these homo-bifunctional amine reactive cross-linkers so you've got the reactive the reactive groups on both sides and they created kind of a a recipe of different lengths of cross-linkers to help not only improve the efficiency of cross-linking but also the completeness of cross-linking now one of the questions that often will come up is when you're doing these types of cross-linking experiments is to make sure that you have some specificity and that you're not cross-linking just random molecules together and so oftentimes what people will do is they will during them at the development is they'll spike in some protein that they know will not bind to the protein of interest so they'll have their their protein complex and then additionally some protein like albumin or in this case ubiquitin to ensure that they're not getting those non-specific complexes now in this case there is a small amount of non-specific complex formation so what you want to do is to also have some sort of control experiment where you do not cross-link and just verify that the levels are are very similar so this so these are going to be related to plume aggregates so the which we'll talk about a little bit so you can get some complex formation in the gas phase as well so this is just another example of complex analysis looking at looking at antibody antigen by binding interactions so here they're using their their mixture cross-linking reagents and just showing that you're seeing what you should be seeing with cross-linking and without cross-linking so in the no cross-linking you see exclusively the individual subunits with cross-linking you're seeing the the complex formation and this is two examples you have the antiprion protein antibody and then you're also for a of course with an antibody you would expect to have two groups attached to an antibody so what you would expect to see is maybe a certain degree of a single a single unit bound to the antibody and then and then sometimes you'll see two two antigens binding so although it's maybe not necessarily complex formation protein aggregation analysis is very similar in terms of how you would do this type of analysis and so you can see with antibody cross sorry antibody aggregation the molecular weights can really kind of get out and control very quickly so even with six antibodies you're already at the list of a million adults and so just like with the protein complexes what you're going to do is to do the cross-linking of your antibodies in solution it takes maybe a couple of hours for that reaction to and then you're going to analyze your sample without cross-linking and then compare it to what it would look like with cross-linking and what you want to do is of course control for these plume aggregates so this is with no cross-linking but if you crank up the laser power you're going to see a lot of these diners, trimers, and tetramers even though they're not really existing in this in solution and so you want to decrease your laser power to the point where you don't see any plume aggregates to try to control for that so it's just a question of kind of controlling that laser power so once you've done your cross-linking and you verify that you've got the right conditions to analyze for these aggregates then you can see you get very you get the stabilization and you can start to analyze for these for these submissible protein aggregates so these are the types of molecules that you wouldn't necessarily be able to see by traditional light scattering techniques you can take it a step further and you can do offline separation so this can be a orthogonal to GPC analysis so you can do the GPC fractionation and collect the fractions and then analyze for these aggregates and that might give you a little bit more sensitivity towards the higher so I'm not really an expert in the other areas but this is just to kind of show that that there's other orthogonal techniques to to moldy and that moldy is kind of right along there with these other methods in terms of its reliability and ability to detect some of these some of these high molecular weight species one of the things to kind of keep in mind is that the some of the methods like size exclusion chromatography is you mean to kind of target specific mass ranges and if you go outside of those mass ranges then you might get an inaccurate measurement so specifically like some of the dimers might be underestimated with size exclusion chromatography so they might not have the diversity of mass range that all you can offer and additionally in some cases again I don't I can't even read these figures but you know what you need to know is that the assumptions sometimes need to be made with using light scattering techniques and ultracentrification methods so when you're comparing to some of these orthogonal methods moldy has several advantages including speed resolution mass accuracy I think there could be you could gain more information especially when you're comparing moldy to size exclusion techniques you get all that mass information and then the sample prep is very straightforward and pretty quick and now the detector goes up to 2 million Dalton so it gives you even more capabilities so another area of interest has been the protein drug conjugates so in this case what people are doing is they're cross linking like drug compounds to antibodies or other proteins to try and get more specific targeting of their biomarkers so it's a way to specifically deliver a drug target to just the cancer or disease disease cells so this is typically the workflow so you are going to it's very similar to cross linking where you would have some sort of derivitized drug compound that would then bind to the antibody so you have a linker that's NHS that's typically NHS is terrified and then you're going to incubate it for a couple of hours and then once you've got your linker on there then you bind your drug compound to the linker and this is an example that was done by our collaborators at Walter Reed and they were looking at albumin bound to haptic and so basically what you can do is you are going to acquire your mass spectrum with no cross linking and then you can do different time points to determine kind of what is the optimal condition for the cross linking reaction and what you'll do is you can see the linker ratio increasing over time sorry I keep looking here but it's in there so you have the linker ratio and the number of sorry they're not optimizing me sometimes they're increasing the linker ratio to to try to increase the number of happenings that are binding to albumin and this was all about trying to find a vaccine against opioid addiction and generally speaking samples are run in triplet kit and the reason to do that is basically to kind of average out any discrepancy in a molecular way that might be related to just the shape of the peak or any sort of decreased resolution from laser power this figure is just basically showing how the moldy system compares to some of the traditional methods so in terms of analyzing these drug conjugates people will oftentimes use like a derivation method followed by just some UV detection and so the TMES or trinitribenzene sulfonic acid derivation basically you can see is going to underestimate the number of happenings that are attaching to the drug molecule the other point to make is that these derivation methods are both indirect whereas moldy is giving you a direct measurement of what you actually have okay so protein pegalation is still very similar you're pegalating your protein using a linker and the molecule of a certain length that this is done to basically stabilize drug compounds in biological solutions and so we were looking at a goat anti-rabbit antibodies and looking at a dilution curve so here we have the goat anti-rabbit standard so this is without any cross linking and then we have goat anti-rabbit 10k so this is just basically the 10,000 Dalton peg linker and then we have the 25k as well and all you need to do at that point is to just compare the different conditions and look at the mass differences over multiple acquisitions and you can see why it's somewhat important to do multiple acquisitions from this figure because you can see the peak shape has some variability here and even though the apex of the peak is the same it could be it could be shifting around a little bit and so we typically do our measures three to three to five typically of off of the same sample and so from here you can calculate with the GR 10, GR 25 the number of linkers on it's a pretty straightforward measurement of just doing a division of the mass shift by the molecular weight so from this presentation you know I've given a lot of the positives but there are some drawbacks to baldy as a method it's considered more to be a qualitative method people are not typically using it for absolute quantitation but it could be very complementary to more quantitative methods such as like q-top analysis you might require some cleanup a lot of times you can get away with no sample cleanup so this could be a very simple you know putting a drop of water on your sample and then removing it 10 seconds later to try to remove wash off some salts there's a lot of tips that you can buy that would allow you to just do it quite clean up like it's basically like a little hplc column on a tip and you can do that quite straightforward dialysis is another technique that I've used in the past there's lots of different methods for that and then for looking at drug conjugates looking at mass shifts and stuff like that the resolution of the instrumentation might not be sufficient for looking at small changes because you can see in some of these figures like this one peaks can be quite broad so looking for a small shift in molecular weight might just not be possible are there before we move on to some of the future future directions are there any questions yeah so what's the maximum mass that you can see and might be talking using your like you say that you think it does and it does a little bit but what actually you can see no I've never seen yeah so I've we've seen the most that I've personally seen is in that figure on the aggregates I think it was like 1.2 million dolphins for the nine bird I haven't looked at anything beyond that but I'd be I'd be curious to know like if you could see a diameter of IGM at like almost 2 million adults that would be like 1.8 million adults I haven't tried it though so I don't know where the specification comes from or how they even verify that spec but it's a good question great so so let's see so Shaman's you I mentioned a while ago that Koichi Tanako won the Nobel Prize he still works at Shaman's you and has a mass spec research laboratory that's dedicated towards promoting place sciences and particularly his preferred method is Mali top mass spectrometry and there's been a couple developments that recently come out of his lab one of which we've commercialized and this is the eight that multi matrix the eight that matrix is a special multi matrix that focuses on ionization of hydrophobic species so this could be hydrophobic proteins like membrane proteins or the digestive peptides from these proteins we're looking for new ways to use this matrix so if you have any ideas about what you might want to try with this we'd be happy to send you some some sample matrix to just try it out on your own and this is an example just kind of showing let's see the transmembrane the transmembrane domains and being able to selectively ionize the hydrophobic peptides so you can see the stars are representing the hydrophobic peptides and the circle is more the hydrophilic it's a very few hydrophilic peptides are being detected there excuse me and then finally we're one of the areas that Shaman's who's really been focused on has been Alzheimer's disease there's a big epidemic in Japan is the population's continuing to age in dementia and so Shaman's who's really taken on this this whole project of improving detection and yeah basically trying to come up with easier and earlier ways to detect dementia and so the collaboration with two universities whose names I can't think of off the top of my head they've developed this test that basically circumvents the ability the need for PET scans or the need to do a spinal tap to get the CSF those are currently the only methods for detecting these amyloid beta proteins and so what they do is they take a drop of blood and they basically are going to do immunoprecipitation of the amyloid beta protein fragments and they're going to be looking at the ratios of these of these protein fragments to correlate the data to a disease state or a non-disease state and they've been able to show with this data much earlier detection than the current methods and it's also much less invasive much less expensive and a lot faster as well so so we're very excited about that it's a little bit tricky because you have a test for a disease that has no treatment so what do you do with that you know so you know the idea would be that this could really help facilitate drug discovery and looking at clinical trials to see the effectiveness of drug treatments so we've been very excited about that and we're starting to offer that as a service so and that's going to be kind of based on this whole quality 80-20 platform which is the benchtop quality system and I don't know that I need to I don't know how we're kind of over time but we started you know pretty far into the presentation anyway but just wanted to kind of showcase some of the nice features of our new instrument it's pretty inexpensive relative to a lot of the systems out there very fast so this is working with the 200 hertz laser and the pump down time is really quick as well very the whole idea of it is to make it a very simple to use instrument and just kind of to tell you a little bit about future development you know we are investigating the possibility of making a top platform over to a benchtop model you know there's some complications because it's a much smaller plate path so there could be some limits there but we're we're just getting started on that project so hopefully you know next time I come out I can share some more information about that so I just wanted to acknowledge some of the work that's been done so Professor Almeyer, Almeyer out of the in Austria, Ryan Wentzl from Coblex, you know Professor Almeyer is always very willing to help us and share his data he's been a pretty early adopter of the Coblex detector and the detector course comes from Coblex where Ryan Wentzl does all the kind of business development and then my team back at SSI and Ryan Wash so that concludes my presentation I don't know if you have any questions you can discuss any if you're working on some samples and have any questions I can try to answer the wrap up Ryan we'll be over in a bit you're interested in why I'm here staying around you might come over and talk to it about problems or anything like that or any ideas you know it doesn't have to be problems no one will problem yeah Delaney did you know anything you wanted to cover no sorry you're good okay great well thank you so much for your time and for your patience it was a little bit of a a mess this morning but thank you for hanging in there it's all good