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hello we're glad you've joined us for this webinar clinical uses of mass spectrometry i am michelle ashton of laberd's and i'll be moderating this session today's educational web seminar is presented by laberd's the leading scientific social networking website and provider of virtual events and webinars advancing scientific collaboration it is made possible by an open educational grant from Agilent Agilent has had no input in the selection of speakers content or mode of presentation let's get started you can pose questions to the speakers during the presentation while they're fresh in your mind to do so simply type them into the drop down box located on the far left of your screen labeled ask a question and click on the send button to enlarge the slide window click on the arrows at the top right hand corner of the presentation window if you experience technical problems seeing or hearing the presentation just click on the support tab found at the top right of the presentation window will report your problem by typing it into the ask a question box I now present today's speakers Neena Audrey and Phillip Fatima's both are from the laboratory prism at the University of Lille to learn more about our speakers please visit the speaker tab at the top of the presentation window Nina in Phillips you may now begin your presentation so Thank You Michelle for the very kind introduction thank you so today we will talk about clinical uses of mass spectrometry specifically focusing on the new advances of the past years which we believe will make a huge impact in the clinical world so just to be more specific we will be talking about the methods which are not yet routinely used but that have a great potential of being used in oncology either during surgery or during a pathological exams so first just a bit of introduction of about mass spectrometry in clinics in these days it is routinely used to diagnose metabolism deficiencies to determine biomarkers and enzymes then for biological toxicological testing and there are many advantages of mass spectrometry over other techniques because they can be used to analyze fluid and solid tissue and not really a lot of amount of sample is necessary there is no labeling required we can multiplex analysis we can obtain detail molecular identity we can do qualitative quantitative analysis and we can analyze a large variety of biomolecules such as metabolize proteins and pharmaceutical compounds and also what is nice about these methods that we can use them for targeted or untargeted analysis and the last part is really more relevant for discovery based research there are several groups of techniques which have been involved in clinical research such as classical mass spectrometry techniques used in clinical laboratory and for example GT and LPNs mass spectrometry imaging has been used as well as ambient techniques and the recently is the development of intra operative techniques also called IOM s used for real-time and in vivo analysis during surgeries so let's just briefly go through the classical techniques here we outline a short timeline of the important events of the techniques that have been routinely used in clinical labs so historically the major impacts have been made by confirmation of immunoassays with positive drug screens by GCMs the identification of inborn errors of metabolism and analysis of steroid hormones more recently ms has dramatically improved the time required for micro by micro microbial identification and this is the part that we will also speak a little bit more about once direct screening became mandatory for employees in us several toxicology as a lab started using this technology and several studies have demonstrated that urine testing was very cost effective and they also started using this methodology for four other four other analysis such as your therapeutic drug monitoring the major limitation of GC NS is that the analyte needs to be volatile and the foremost clinical stage requires the multiple extraction and purification steps along with the chemical derivatives Asian to randomly analyze sufficiently volatile analysis atmospheric pressure ionization techniques such as the electrospray ionisation combined with AI performance and TMS while the next major analytical improvement that enabled ms for clinical laboratory uses so indeed this technology is very high throughput and cost-effective especially the developer of multiplex analysis and in the microbiology lab lab the development of MALDI combined with top mass analyzers allowed for rapid identification of microbes for implementation of mallet of microbiology laboratory depended on gram stain's culture biochemical tests and susceptibility testing but with the development of the Brooker bio typer it became very simple and very fast so basically what happens is that after culturing this mirrors are deposited on to metallic sample plate and mixed with multi matrices these are just regular organic matrices the sample plate is inserted in the instrument and a laser is shot onto the sample for analysis the molecules are then desorbed and ionized and separated based on their mass to charge ratio in the top analyzer you can see a very nice schematic in the middle of the slide the specific Peaks are then generated are generated and compared to the spectral libraries that is as as estimated that using these techniques has got the total cost of reagents and Labor's by 50 percent which is a great advance the spectral library can be custom ly modified so users can build their own database so this is just one of the great example of ms being used in everyday clinical analysis so now we have reached now we have reached a part which we want to emphasize more about which is a recap of technological advancements that we believe will have a really important part into the clinical world we will present these techniques and a lot of use nice examples performed either in the clinical or biomedical lab doll aids we will start with explaining about MALDI this was developed in 1985 by car essential intent that uses solid-state samples mixed with specific molecular matrix to generate gas phase ions to be analyzed in the late 90s MLG msi was introduced and demonstrated its best potential for biomarker discovery and has played a central role in clinical research linking classical histology and molecular analysis so here are a few break points such as the identifications of first diagnostic markers but also integration with astrology peptide MSI analysis on AFP tissue and lad the multicenter studies in a way to standardize these techniques so here we have the MALDI imaging ms this technique allows us to directly map molecular species such as proteins step-size lipids and metabolites and is considered a cutting-edge approach to better understand underlying processes in cells and viola so on top you can see a slide with the tissue section with apply g matrix we shoot the laser on top of the section and the molecules are dissolved and ionized and then furthermore analyzed in the mass spectrometer on the upper right side you can see the mass spectrum that is created and if we select a specific mass we can actually generate a two dimensional map of this specific mass MALDI MSI is used in discovery-based research for biomarker discovery as I mentioned it can be used to analyze metabolites lipids and proteins we can analyze fresh frozen tissue FFP tissue but we have to be aware that a specific sample preparation is required depending on what kind of molecules we would like to detect so in cancer research and miss MSI has been used for tumor typing and especially the identification of tumor origin in metastatic tissues as well as grading and may provide prognostic information while looking at the tumor microenvironment we can study for example a hypoxia derived prognostic biomarkers such as glycans which can be used to predict cancer progression and patient outcome for personalized medicine so here is a an example on the bottom right corner of the analysis of a tumor in Hodgenville at about unity and on the bottom-left corner an example of analysis of glycans on tissue mccoy's where we can distinguish some specific signals in tumor and non tumors tissues so one of the main breakthroughs in this field as we mentioned already previously is the possibility to image not only fresh frozen but also FFPE tissue which gives us access to large clinical tissues FFP stands for formalin-fixed paraffin-embedded in which the fresh tissues are fixed in formalin and then transfer for conservation so here I will just show you briefly how the sample preparation of these tissues work so first we have an FFA slide which is cut to appropriate thicknesses we wash the tissue and specific solvent to remove lipids and policin we do the antigen retrieval step to break the disulfide bonds of proteins which were caused by the fixation we spray trypsin or for other enzymes in order to break the proteins into smaller peptides and then the final step is to apply the matrix on top of the tissue here is just two images which show the distribution of specific triptych ly digested peptides and the mass spectra that we usually acquire so here we can see an example of the film reanalysis of patients with the high-grade serous casino MO and a round robin study of FFP DNA analysis I don't go further in details of this Kimbra example as we will dig deeper into it later but not only can we see proteins but also we can see metabolites in these 15 so these strategies MSI allow us to obtain the spatial distribution of whole of adjusted proteins but the idealistic identification is still very difficult directly from the tissues in fact it really relies on ms/ms fragmentation spectral quality so for a long time top-down FST analysis was difficult and for bottom up strategy the enzymatic digestion is complicated to read so how could we breach this localization aspect to the classical large-scale proteomics don't I teach through extraction and a CMS well in prism lab we aimed at filling this gap and correlate the protein localization with their identification by using specially resolved micro proteomics so what happens is that we use Pacific techniques call them micro sampling by the liquid micro Junction micro extraction with liquid extraction surface analysis Russa and in this particular example what we have done is that we use the segmentation analysis on a small d image of a red brain we were able to select specific regions on which we did two specifics micro extraction on approximately 1900 South extracted thousand and 350 identified protein proteins were found specifically a major majority of them were common between the zones but we also sign between 200 and 300 of protein specific to each position so we tested them the Republic the reproducibility of the extraction by processing several following slides of the brain at exactly the same location which showed us less than 5% variation in the protein content between each run one of the main application in the technique of the technique in the lab is to study the Cimbri are presenting high-grade serous carcinoma this specific a GSC series represents 85 days of epithelial cancer of the reproductive organs and it's partition in 70% of serous type and 30% of Nokomis state when the lesions usually diagnosed deletions are usually diagnosed in the advanced stages so the prognosis is usually bad even with heavy surgery and chemotherapy by analysis by an icing digestant proteins we were first able to analyze this tumor and detect specific molecular signatures that were that we extensively studied by using a chemical inkjet printer to deposit trypsin directly onto discrete regions of less than one millimeter scope so after protein digestion the liquid we conjunction is performed to retrieve all the peptides that were digested and that are coming from the identification of proteins in both cancer and benign regions our campaigns it has been possible by this approach to obtain different important signaling pathways involved in cancer processes and some proteins already known as biomarkers in the geography the comprehension of the pathology has really evolved these past 10 years and clinicians now think that the majority of the ovarian cancer arising not from the ovarian tissue but from the embryo part of the fallopian tube the communication between these secretory cells of the embryo and the ovarian epithelium by especially reactive oxygen species or rows of believed to induce a molecular differentiation of the secretory population into click on cells feelings and these regions are often detected from brca1 mutated patients who undergone or selected over ectomy and can be traced from p53 lesions to different intermediate grade rates leading to the migration of cancerous cells on the ovarian causing addresses a GSC so in order to evaluate the progression of the lesions it to HD SC we decided to look for those small numbers of cells which are bearing specific phenotypes of precancerous lesions the originality of our approach was to use the p53 and ki-67 immunohistochemistry staining obtained during routine analysis of pathologists so after they analyzed these slides and detect the second source lesions they gave them to us to be analyzed so when the cells were responding a delta p53 ki-67 or both then we use that entering to the universal chemistry to direct our analysis so we first unmount our cover slips from the slides and then we use them for imaging and micro extraction extraction to lc/ms analysis we were able to significantly the different lesions based on their proteins we could distinguish the pathways between the early stages of normal p53 and still samples which showed an overexpression of the silicon for mediating signaling pathways that affect cellular proliferation migration and endocytosis but also integrins family cell surface interactions which are involved in metastasis so the widely used techniques in a math analysis was invented in parallel to non G and is the electrospray ionization or easy it was developed by Fanning collaborators in 1984 in this technique the the analytes are present in a liquid phase and this is solvent is created in front of a mass spectrometer and on we apply an electrical current in order to promote the ionization of the analytes by definition the ionization sources is working under atmospheric pressure or aims so the samples that we analyzed can be in the native space so just to go quickly through some of the points in the timeline for the creation of nano is a to improve protein identification by lc/ms then the creation of daddy to perform imaging mass spectrometry lacey for laser ablation electrospray analysis and previously mentioned some in here we just want to mention that desi has been the most widely spread among them today to do 2d and 3d imaging either for tumor lipid profiles tumor subtyping or prediction of metastatic status in this variable technique of atmospheric pressure mass spectrometry will focus on some of them and begin with in panel a will be desi an aside so in this schematic diagram we can see how an electrospray is directed onto the sample surface to desorb molecules that are then analyzed and its application to brain tumor so the top spectrum shows the low-to-mid cell concentration region infiltrating into the gray matter and the bottom spectrum shows the CMS of dense tumor regions with high tumor cell concentration just under we can see desi ms iron image which shows the distribution of specific glycerophospholipids depending on the area so the second technique depress the MSO probe electrospray mass spectrometry shows the principle of the operation so the probe is held in contact with the tissue to be analyzed and then it's placed inside of the as a mobilization plume here we show the application to colon cancer and on the top and the bottom spectrum with the presence of specific glycerophospholipids in either normal or cancerous colon tissue so in panels see the diagram explain us how the TSN eyes works for touch prey ms so similar to the busy needle adsorbed molecules of interest by touching solids or solutions then this needle is covered with a small drops of solvent and we apply a current on it to directly gene ate a spray at its tip so in the application here the prostate cancer analysis is showed with its mass spectrum which is revealing the presence of specific molecules in the region outlined below which correspond on the biopsy to prostate malignant tissue so as it was determined by Esther palace' logical evaluation so on the right hand side you can see a table which summarizes the potentially relevant aims techniques you can be used in the ultra operative circumstances and the principle molecules or biomolecules that can be seen with these commitments we will dig a little bit further in the into the following part right after this recap slide so to summarize how does how do molecular information obtained by ms can be related to health and disease well you know in fact any small modification in overpasses leads to changes in biological pathways and when pathology brings imbalance the molecular profiles are dysregulated and all biomolecules are equally affected so basically by following proteins and lipids can therefore get insight into health and disease states so now we have reached in part of our presentation corresponding to the emergence of the more or less real-time and in vivo techniques which have been created since 2010 the first one was developed was developed in in UK intelligence a knife based on Reims rapid evaporative a ionization mass spectrometry and so although techniques are used with ranges as the sampling method like the pearl for people ii infrared laser mass spectrometry developed in 2015 and the spider masks developed by the Prisma laboratory in 2014 then in 2017 the math back pain was introduced the ions techniques are developed to help with the cancer diagnosis and margin evaluation principally which are difficult to observe during the pathological exam and that can take between 30 to 40 minutes during surgery in classical operation there are two main approaches one which is off line and watch one with which is on line the first one is taking the sample out of the patient and perform effect on potenuse analysis in the room next to the operating theatre it can be a biological or pathology lab the second approach is the use of a surgical Henty temple directly by the serger and performed in vivo and in real-time so here are the several techniques that have been developed over the years in this field such as the ionized technology developed by Zoltan caucus at the Imperial College of London the spider mass system developed here in prism labs the mass spec pen developed by a Berlin lab in University of Texas in Austin Austin LD AMS develop isotopes in 2009 in Germany the Perl developed by Doreen SR in University of Toronto and stereotactic TMS developed by a girl lab at Harvard Medical School in Boston but in this final example they used as the extra SM for example Arius analysis but all of these techniques are in common the same goal to help cancer diagnosis and to ultimately help the patient survival in outcome so we will mainly focus and explain the uses of the knife the mass spec then in the spider mass technology to give you a taste of the different methodology used so first we will start off with the ionized technology here is the first system so the intelligent I my based on rate the ionized produces gas phase ions of evaporated tissue after the contact with the surgical tool because phase ions are then subsequently transmitted to the mass spectrometer through the Teflon to the Teflon tubing or tygon tubing which is biocompatible which can be more than 1 meter length using a venturi jet jet pump so this technique is compatible with the commonly used surgical devices such as the electro cylindrical electrodes but also surgical diathermy people are four sets of curvature an ultrasonic logical operator and utilizes the surgical area I also saw smokes form doing normal surgical interventions the analyse detection detected our metabolites and lipids in the negative ion mode polarity so the analysed molecular signatures are then subjected to classification using multivariate classifiers so over the years it has been used in different applications such as the oncologist surgery breast cancer diagnostic and margin assessment brain tumor analysis industrial internist and gastroenterology such as implementation of an endoscopic system and the atmospheric pressure bio typing so the other techniques we mentioned is the mass segments so it consists of a syringe pump delivering a defined water volume to the sampling probe which we can see here on the right side of the screen it's connected to PTFE tubing which are integrated with valve to transfer the water to and from the tissue this device is the pen size for probing of biological so during the experiments as we can see on the left side of the screen a water droplet is deposited onto the tissue for three second period allowing the attraction extraction of the analytes so this droplet is vacuum extracted to the mass spectrometer and analyze the system is based on non-destructive liquid solid destruction and can be applied in vivo for in vivo tissue analysis so here's just a couple of examples of how the mass effect pen has been used in different applications while subjected to moly various statistical analysis and here we can see a different example such as breast cancer acai riod cancer ovary cancer and lung cancer with the to classify between normal and cancer tissues with a large number of patients so now let's go to the spider math system which we are developing in the in the lab for in vivo and real-time analysis using a laser in the ephra red wavelength and this system was introduced in 2014 so the spider base is tuned to excite the most intense vibrational band of water molecules at 2.94 micro minute micrometer you know process described as water assisted laser desorption and ionization so the spider master system is composed of the mini invasive laser microprobe to generate the ions which is the first part of the system then we have a transfer line which is transferring in real time the I analyzed from the probe to the MS systems and then the acquired molecular spectra are then subjected to multivariate analysis and classification models are built based on the different tissue type so basically what happens is that we can teach we can teach the muscle to recognize for example cancer or normal tissue once the model is created we can interrogate the model in vivo this has already been performed in one of our previous studies and ianka vet clinic indeed since the data is very complex to analyze and we are often very limited with the amount of tissue we have been currently developing software based on DNA and in this current state these spider medicine provides the imaging such so the spider mass prototypes can be connected to the mass spectrometer we have the biocompatible tygon tubing and two different variety of mass spectrometers here are a few here are the few examples um it can be linked to a queue top instruments such as the synapse and Devo from waters or high mass resolution spectrometers such as the Orbitrap that really shows the versatility of the technique and the adaptation depending on the user's needs we also did quite a lot of different applications with a spider med system in the first upper top left corner you can see the example how non-invasive each spider my system is because we were able to perform analysis in vivo on skin we can see the different metabolites and lipids that are coming off from the tissue the technique is also able to analyze protein standards deposited on the sample holder here is an example on the bottom left on the bottom left part of how we analyze protein standards such as the pigmenting and gives us the electrospray like spectra with a multiplied chart state the last example on the top right corner you can see the bacterial bio typing where we were able to separate different strands of bacteria based on their molecular profiles but one of the major studies was conducted on dog sarcoma samples Takuma is the type of highly heterogeneous tumor which are difficult from time to time to distinguish by apology so you know in Moore there is a large number of pathological type of Tacoma so we were we were up to the challenge to see whether we can distinguish between normal and cancerous tissues and different pathological types so in this study we used so ex-vivo biopsies obtained from surgery of dogs we had to cut these samples in half to have a morphological control and the spyder math analysis on its mirror please so using these techniques to generate the spectra corresponding to each situation we were able to to to classify the samples according to their biological content so we can see on the top on the bottom corner that the PCA is showing really a nice separation between the normal and the cancerous tissues and we can also see that the cancerous tissues are split into subgroups which we will extend your region just right after so in fact this separation is coming from the presence of necrotic tissues inside the cancerous subtype and we have been able to create a classification model taking into account this necrosis signals so the using this system we were able to see that the necrosis part is really distinguishing from the cancerous one and the normal tissues on the top right side of the slide you can see that we were able to classify the sarcoma according to the grade which is great for grading during surgery so we did some across validation of the markets detected with the Spyro mask using the MAL DMS a technique which has a father proven to be robust so here we can see that we have some specific markers from the normal on top and cancerous on the bottom part regions and dues the signals are I were identified in real-time and some markers share their specific localization between the spider mass and then all the MSI so in order to do to interrogate the classification model in real time and to do the actual assessment of one of the tissues we selected a tissue which has a mixed composition of normal cancer and necrosis we we started shooting the tissue and based on the Emmis spectra acquired we interrogated the classification model and give us back the real-time classification feedback and as you can see it was able to specify between normal cancer and necrosis part of the tissue so then we went to the vet surgery room to to see if the system is able to work during surgeries so we first installed the system and set decided the surgeon while doing these surgeries we were getting the ex vivo biopsy and analyzing them in real time then we gave the handful directly to the to the surgeons which used it during these surgeries and the generated mass spectra showed their ability to be linked to tissue specific molecular profiles and for the last example we want to show you that we are able to do direct protein analysis of human saliva here we use the 5 microliters of human saliva with five microliters of si and water and a CN directly onto a glass slide and analyzed the mess that's were produced as you can see we have a lot of specifically Peaks and then we also have this match range where we can see mainly the proteins and here one of these proteins came started really popping up which these as a mess 875 0.8 we perform some ms/ms analysis and we were able to see that we detect celebrity as acidic protein reach forceful protein 1/2 which is present in the human saliva DPR h1 that we detect provided a protective and reparative environment for the dental enamel so as you can see we really can use the spider mask technology in a variety of different application in the clinical world so to recap how the spider-man's development prism can help clinicians in their daily routine please well the pyramid can help providing any type of material as which showed and giving its molecular signature like a barcode so these can then be used to differentiate between various classes of samples and classify them so on during surgery it can be used for grading modeling detection and even extent of rejection assessment without damaging the surrounding tissues and let's not forget that in the lab it can be used for drug screaming bacterial bio typing a most other non clinical related application like pesticide analysis or food safety so let the our last learning objectives was to treat how the Alma development has advanced in the clinical labs and Hospital so it has developed from vacuum based ms to AMS and from process to direct analysis of samples the MS technology has made a lot of huge step forward in these advocates bringing us into the era of in vivo and real-time analysis plus the bioinformatics tools that have been developed have part of the ability to analyze huge data sets and make classification or discover new biomarkers so we wanted to finish this talk by thanking all of the prizm members which are doing a great state of the art work obtaining these deep analysis using imaging and multi-omics techniques for diagnosis and prognosis biomarker venting our aims are to better understand the physio pathology in oncology and in Neurology and discover new mechanism to treat them by their article or technological innovations and we want to specifically thank the partners involved in the spider mass project and last but not least all of you for attending this webinar and we will be more than happy to answer any of your questions and thank you for your attention thank you Thank You Nina and Philip for your presentation a quick reminder for our audience on how to submit questions simply type them into the drop down box located on the far left of your presentation window labeled ask a question and click on the send button our speakers will answer as many questions as time permits okay let's get started our first question is when do we expect the first experiment performed during human surgery so we expect our first experiments to be performed in 2026 yeah which is the quite objectives because we have several steps to to achieve before going into the surgery room because it's really the last and the more important goal of the project great thank you okay next question when do we expect to sell the first commercial spider mass prototype at the moment we have a lab prototype but we plan soon to have something to be available so we think that maybe next year in the next two years we will be able to commercially provide prototypes in order to run at least research in vitro and outreach for now yes exactly thank you okay it looks like we have time for one more question is is there any link between all the omics database for example genomic transcriptomic proteomic and genomic data or lipid omec data well that's a huge question in fact there is no direct link between the genomics transcriptomics and proteomics data banks and omics results as we know today more and more that not every gene which is expressed is transferred into a protein what we can say still is that maybe we can do the link between the lipid omics data we are generating and the Palio mcdhh at a-- we have when there is a direct link between the protein and biosynthesis of the lipid in questions I would like to once again thank Nina and Philip for their presentations I'd like to thank the audience for joining us today and further interesting questions any questions we did not have time for today will be addressed by the speaker via the contact information you provided at the time of registration we would like to thank lab roots and our educational sponsor Adeline for today's educational webcast mavar's will alert you via email when it's available for replay we encourage you to share that email with your colleagues who may have missed today's event until next time goodbye