>> GOOD MORNING, WELCOME TO THE PROTEOMICS INTEREST GROUP LECTURE SERIES. IT'S MY PLEASURE TO INTRODUCE DR. MICHAEL STRADER FROM FDA. DR. STRAYEDER DID HIS Ph.D. AT THE UNIVERSITY OF TENNESSEE AND POST-DOC AT OLD CREEK LABORATORY AND RESEARCH FELLOW FOR QUITE A FEW YEARS AT NIH HERE AND IN 2011 HE MOVED TO FDA TO ATENURE TRACK POSITION. AND -- A TENURE TRACK POSITION. HE H TALK ABOUT RESEARCH ON PROTEOMICS OF BLOOD CELLS. >> FIRST I WOULD LIKE TO THANK EVERYBODY IN THE AUDIENCE AND ALL THE PEOPLE WATCHING THE VIDEOCAST SYSTEM. IT'S ALWAYS A PLEASURE TO COME TO THE NIH, I HAVE FOND MEMORIES OF THIS PLACE AND HAVE LOTS OF FLASH BACKS WALKING ON CAMPUS ONE OF THE MAJOR GOALS OF OUR LABORATORY IS STUDIES AIMED AT DESIGNING HEMOGLOBIN BASED OXYGEN CARRIERS THAT CAN BE POTENTIALLY UTILIZED AS BLOOD THERAPEUTICS AND WHAT TODAY WE'RE GOING TO -- I'M GOING TO TALK SOME ABOUT SOME PROTEOME RESEARCH THAT WE HAVE DONE LOOKING AT MICROPARTICLES AND WE ARE ACTUALLY INVOLVED IN LOOKING -- AT INDs THAT ARE INVOLVED MICROPARTICLES AS POTENTIAL PRODUCTS DOWN THE LINE. SO ANYWAY BEFORE I START I WOULD LIKE TO GET SOME BASIC INFORMATION. AS YOU GUYS PROBABLY KNOW HEMOGLOBIN IS A TETRAMER MADE OF TWO ALPHA TWO BETA SUBUNITS, EACH SUBUNIT CONTAIN PROSTHETIC IRON CONTAINING HEME GROUP AND IN THE RIGHT OXIDATIVE STATE THE SUBUNITS COMBINE OXYGEN. THERE ARE TWO PRIMARY OXIDATIVE STATES THAT EXIST PHYSIOLOGICALLY. THAT IS THE FERRIS FORM AND THE FERRET FORM. FERRIS FORM, WHEN OXYGEN IS BOUND IS A RED COLOR, AND THE DEOXYGENATED FORM HAS A BLUE COLOR. SO THE FERRET FORM OR MET HEMOGLOBIN DOESN'T BIND OXYGEN, LESS STABLE AND TURNS OVER RAPIDLY. I'M GOING TO DISCUSS MEDIATED TOXICITY IN THE NEXT SLIDE BUT THERE'S ALSO ANOTHER FORM THAT'S TRANSIENT, HIGHLY REACTIVE, THAT IS THE PLUS FOUR FERRELL FORM. IT IS VERY TOXIC AND I WILL DISCUSS THAT SHORTLY AS WELL. ONE OF THE BY-PRODUCTS OF OXIDATIVE -- YOU HAVE OXIDATIVE PROCESSES WHERE FERRIS IS OXIDIZED TO THE FERRET IN PEARL FORMS AND AT THE SAME TIME THERE'S REACTIVE OXYGEN SPECIES SUCH AS SUE OXIDES AND PEROXIDES PRODUCED THAT FACILITATE THE OXIDATION TO CREATE THESE SPECIES. THERE ARE ENZYMES TO MITIGATE THESE PROCESSES SUCH AS CATALASE, MET HEMOGLOBIN REDUCTASE AND SUPEROXIDE DISMUTASE TO ENSURE THAT THE HIGHEST AMOUNT OF HEMOGLOBIN PRESENT IS IN THE PRODUCTIVE OXYGEN BINDING FERRIS FORM BUT THESE PROCESSES STILL HAPPEN. WHAT WE HAVE LEARNED FROM PATIENTS THAT SUFFER FROM HEMOGLOBIN ON THINKS, THESE -- ON OPATHIES, THESE PROCESSES ARE EXACERBATED TO GET A HIGHER LEVEL OF OXIDATIVE STATES. AND THE FERRELL FORM WE HAVE SEEN IN PREVIOUS STUDIES TO BE MORE AMONEY CAN'T IN THESE STATES,LY WILL TALK BRIEFLY GIVING AN EXAMPLE, THAT IS SICKLE CELL HEMOGLOBIN IS HIGHLY REACTIVE. THE FERRELL HEME NOT ONLY ATTACKS MOLECULES WITHIN CLOSE PROXIMITY OF THE HEMOGLOBIN, IT ALSO ATTACKS ITSELF. SO THERE ARE LITERALLY RESIDUES WITHIN THE BETA SUBUNITS PREDOMINANTLY, THAT ARE OXIDIZED. THIS OXIDATION ACTUALLY FACILITATES INSTABILITY LEADING TO TURN OVER HEME MEDIATED TOXICITY. HEME RELEASE, HEME IS TOXIC, HEME CAN CAUSE SERIOUS CONDITIONS PHYSIOLOGICALLY CAN ATTACK ENDOTHELIUM THROUGH LIPID PEROXIDATION, IT CAN -- CAUSES DYSFUNCTION MITOCHONDRIAL DYSFUNCTION, ACTUALLY THE HIGH PRESENCE OF FERRELL HEMOGLOBIN OR HEME ITSELF CAN CAUSE BIOENERGETIC INCAPACITY OF TISSUES THAT ARE IN PROXIMITY TO FERRELL HEMOGLOBIN OR HEME. THIS IS TO RECAPITULATE WHAT I HAVE SAID. WE HAVE DONE LOTS OF STUDIES, WELL DOCUMENTED, THERE ARE RESIDUES KNOWN AS WE CALL THEM HOT SPOTS, THAT'S A VERY UBIQUITOUS TERM USED IN SCIENCE BUT IN -- WHEN WE REFER TO HOT SPOTS WE'RE DISCUSSING THOSE RESIDUES THAT BECOME OXIDIZED AND DAMAGED WHEN YOU HAVE THESE HIGHER OXIDATIVE STATES DURING OXIDATION. ONE OF THE KEY RESIDUES THAT WE HAVE LEARN, I'M REFER TO THIS RESIDUE THROUGHOUT THE TALK. THROUGH OUR STUDIES WE FIND THAT WHEN WE GET FERRIS OXIDIZED TO FERRELL, SISTINE 93 SEEMS TO BE THE END POINT FOR FREE RADICAL OXIDATION. I WOULD ALSO LIKE TO POINT OUT IF I DON'T SAY IT LATER, THAT WHEN IT COMES TO OXIDATION OR OXIDATIVE TOXICITY, OXIDATIVE INSTABILITY WITH REGARD TO THESE AUTOOXIDATIVE PROCESSES THE BETA SUBUNIT TAKES A GREATER DEGREE OF THE OXIDATION. THE ALPHA SUBUNIT IS MORE STABLE. THIS IS JUST A TABLE SHOWING SOME OF THE HEMOGLOBIN WE STUDIED IN THE PAST. OUR ULTIMATE GOAL IS TO LEARN WHY THAT THEY DYSFUNCTIONAL, WHAT WENT WRONG? WHAT IS THE MECHANISM OR CHANGE IN THE PROPERTIES OF THESE HEMOGLOBINS THAT MAKE THEM TOXIC AND HOW CAN WE USE THIS INFORMATION TOWARDS DESIGNING BETTER HEMOGLOBINS OR OXIDATIVELY MORE STABLE HEMOGLOBINS? TODAY I'M GOING TO DISCUSS ONE OF THE HEMOGLOBINS WE HAVE DONE WORK ON, SICKLE CELL HEMOGLOBIN. SO BRIEF INFORMATION ABOUT SICKLE CELL HEMOGLOBIN, THAT IS, AS A REVIEW, THE GENE ENCODING THE BETA SUBUNIT, THERE'S A CODON SUBSTITUTION WHICH RESULTS IN AMINO ACID SUBSTITUTION. WHERE GLUEMATIC ACID OCCURS ON BETA SUBUNIT YOU HAVE A VALINE. ONE OF THE OUTCOMES OF THIS IS THAT IN THE DEOXYGENATED STATE WHERE THE T CONFIRMATION, HBS, BETA SUBUNIT, THIS VALINE IS SURFACE EXPOSED OR CREATES A HYDROPHOBIC STICKY PATCH. SO WHAT HAPPENS IS THAT YOU GET POLYMERIZATION OF THESE HEMOGLOBINS TO FORM FIBRILS. BECAUSE RED BLOOD CELLS ARE PREDOMINANTLY MADE UP OF BLOOD, THESE FIBRILS CAUSE DISTORTION OF THE RED BLOOD CELLS IN A SICKLE CELL YOU'RE SICKLING. AND THE SICKLING LEADS TO VASO-OCCLUSIVE CRISIS. THIS RIGHT HERE IS VERY PAINFUL, AND THERE ARE VERY LIMITED ALTHOUGH WE UNDERSTAND THE DETAILS BEHIND THIS POLYMERIZATION MECHANISM THERE'S VERY LITTLE THERAPEUTICS ON THE MARKET TODAY OTHER THAN HYDROXY UREA, I'LL DISCUSS IN A SECOND, THAT CAN BE USED TO HELP A PATIENT GOING THROUGH VASO-OCCLUSIVE CRISIS. THERE IS ANOTHER DETAIL ABOUT SICKLE CELL HEMOGLOBIN. EVEN UNDER NORM OXIC CONDITIONS, NOT HYPOXIC WHERE YOU HAVE AN EXCESSIVE AMOUNT OF DEOXYGENATED HEMOGLOBIN BUT UNDER NORM OXIC CONDITIONS, SICKLE CELL HEMOGLOBIN IS OXIDATIVELY TOXIC. THERE'S MORE AND MORE EVIDENCE THAT INDICATES THAT PATIENTS EVEN UNDER NORMAL CONDITIONS THEY STILL ARE AT A DISADVANTAGE. OUR LAB, WE HAVE RECENTLY FOUND THAT SCD HEMOGLOBIN HBS AUTOOXIDIZES THREE TIMES MORE RAPIDLY THAN WILD TYPE NORMAL HEMOGLOBIN MOST PEOPLE HAVE. SO THERE'S AUTOOXIDATIVE PROCESSES ARE MUCH MORE RAPID, YOU'RE PRODUCING A LOT MORE OF THESE FERRELL SPECIES. THAT'S WHAT WE FOUND. WE HAVE DONE ASSAYS FERRELL ASSAYS AND THERE IS A GREATER DEGREE OF FERRELL, THAT AUTOREDUCTION OF THE FERRELL, REMEMBER THE FERRELL IS THE REACTIVE FORM, IT HAS A HIGH REDOX POTENTIAL AND IT WILL ATTACK EVERYTHING THAT'S PRESENT. YOU DON'T HAVE A LOT OF THAT AROUND. AUTOREDUCTION OF THAT BACK TO THE FERRET FORM WHICH IS STILL GOING TO UNFOLD IS MUCH SLOWER. PREVIOUS TALKS I DISCUSS I HAVE DEVELOPED A METHOD WHERE WE CAN QUANTIFY THE DEGREE OF OX DATION. I HAVE FOUND USING THIS METHOD WHICH I'M NOT GOING TO TALK ABOUT TODAY, BECAUSE WE'RE TALKING MORE PROTEOMICS IS THAT WHEN WE LOOK AT HBS VERSUS HBA SISTINE 93, THAT HOT SPOT RESIDUE WHICH I HAVE SORT OF COINED AS THE MARKER, WE USE THAT AS ONE OF THE DEFINING METRICS FOR AS CERTAINING OXIDATIVE INSTABILITY IN OUR HEMOGLOBIN VARIES THAT WE STUDY, IS MUCH MORE PROMINENT THAN HBA. AND IN A PAPER PUBLISHED IN JVC WE COLLATED ALL THIS TOGETHER, WE FOUND THAT YES, WHEN YOU LOOK AT THE MITOCHONDRIAL BIOENERGETICS, AND YOU INCUBATE MITOCHONDRIAL TISSUE IN PRESENCE OF HBS VERSUS HBA YOU GET GREATER DEGREE OF MITOCHONDRIAL DYSFUNCTION. (OFF MIC) >> THE OXIDATION PRODUCTS. SO FIRST OF ALL YOU WOULD HAVE -- >> THERE'S TYROSINE AND OTHER RESIDUES. YES, IT DOES. I'M SORRY, YES. SO TRIOXIDATION. OKAY I USED TO BETTER CHARACTERIZE, I THOUGHT THIS WOULD BE SOMETHING I LIKE TO BRING UP, TO BETTER CHARACTERIZE THE ROLE OF THE FERRELL MOIETY IN SISTINE 93 OXIDATION, WE DO ASSAYS WHERE WE MIMIC CONDITIONS OF OXIDATION, WE USE EXCESSIVE AMOUNTS OF PEROXIDES. THIS FACILITATE IT IS AUTOAX DAYTIVE PROCESSES. ONE THING I WANT TO DO IS SHOW A -- USING A LABELING METHOD THAT THE OXIDATIVE DIFFERENCES THAT WERE SEEN BETWEEN HBS AND HBA INVOLVE THE PEARL FERRELL MOIETY SO WE USE THE LABEL PROCESS. SO AS I WAS SAYING, WHEN YOU EXPOSE HEMOGLOBIN TO PEROXIDE YOU CREATE THE PORPHYRIN RADICAL CATION. IT WILL ATTACK THE BETA SUBUNIT AT THE SISTINE 93 TO CREATE AGLOBIN RADICAL. IN THE PRESENCE OF DMPO FIVE DIME THAT WILL IN OXIDE OXIDE, THE GLOBIN RADICAL WILL INTERACT WITH LABELING AGENT, PRODUCING A NIGH COURTHOUSE OXIDE FAIRLY UNSTABLE BUT THE FERRELL LINE REDUCES THAT TO A STABLE THAT WE CAN ANALYZE USING HAS SPECTROMETRY. SO THE NITRO WON'T BE UNLESS THERE'S A FERRELL ION. SO WE FOUND HBS HAS A HIGHER LABEL OF LABELING THAN DHBA SO THERE'S A RELATIONSHIP TO THE FERRELL MOIETY. THAT'S ADDITIONAL EXPERIMENTS WE USED IN THE JBC PAPER TO CONFIRM OR CHARACTERIZE THESE CONDITIONS. SO WE'RE BUILDING ON A PREVIOUS WORK. AND SO WHAT WE'RE INTERESTED IN DOING IS DETERMINE THE GLOBAL IMPACT OF OXIDATION T. WE UNDERSTAND POLYMERIZATION AND DEOXYGENATED CAUSES VASO-OCCLUSIVE CRISIS BUT PEOPLE WHO HAVE THIS DISEASE ARE -- THEY HAVE A HEMOGLOBINOPATHY, THEY HAVE CONDITIONS THAT ARE PRETTY SERIOUS REGARDLESS. SO WE WOULD LIKE TO KNOW MECHANISMS BEHIND OX DAYSIVE TOXICITY. SO I'M GOING TO DISCUSS WORK WE HAVE DONE WITH TRANSGENIC MICE, THAT ARE -- THAT HAVE HUMANIZED HEMOGLOBIN. SO THERE ARE MICE THAT EXPRESS EITHER WILD TYPE HBA FOR HUMANS, OR SICKLE CELL HEMOGLOBIN, WHAT DIFFERENCES DO WE SEE BETWEEN PROTEOMES EXPAND OUR UNDERSTANDING OF THE VASCULOPATHY ASSOCIATED WITH SCD. ANOTHER THING TOO IS THERE'S COMPANIES INTERESTED IN POTENTIALLY DESIGNING PRODUCTS AS POTENTIAL HEMOSTATIC AGENTS. THE RESEARCH ABOUT MICROPARTICLES IS LACKING. THERE'S A LOT WE DON'T KNOW. WITH THAT BEING SAID, CAN OUR RESEARCH HELP BENEFIT OUR REGULATORY MISSION REVIEWING THE INDs AND BASICALLY APPROVING THEM OR HELPING THEM GET TO THE POINT WHERE THEY CAN BE APPROVED. SO ONE OF THE SCIENTIFIC MISSIONS, I THINK IT'S IMPORTANT, IS THAT WE -- IN ADDITION TO TRANSGENIC MOUSE MODELS I'M GOING TO DISCUSS SOME HUMAN DATA THAT WE HAVE. WE FOUND SOME REALLY INTERESTING RESULTS AND HOW DO THESE RELATE TO WHAT WE SEE WITH LIMITED NUMBER, THAT'S THE THING, WHEN YOU DO HUMAN STUDIES YOU HAVE TO HAVE A LARGE NUMBER OF HUMANS POPULATIONS BECAUSE YOU GET A HIGHER DEGREE OF VARIATION THAN YOU WOULD IN SAY A MOUSE OR ANIMAL MODEL. FROM THAT IS THE REALITY OF IT BUT WE HAVE AN VERY SMALL NUMBER OF HUMAN SAMPLES AND I'M GOING TO SHOW SOME OF THE DATA AND HOW IT CORRELATES WHAT WE SEE IN THE MOUSE MODEL. ED FROM ALSO WE WANT TO KNOW MOW DOES HYDROXY UREA CHANGE THE RELATED OCD. HYDROXY UIA IS THE ONLY FDA APPROVED DRUG ON THE MARKET USED TO TREAT PEOPLE BUT IT HAS LIMITED SUCCESS, THE DIRTY SECRET ABOUT HYDROXY UREA IS NOT EVERYBODY RESPONDS TO IT UNIFORMLY. SOME PEOPLE OVER TIME, THEIR BODY REJECTS IT SO THAT MAKES HYDROXY UREA A CHALLENGE SO YOU SEE SOME THAT DO WELL WITH IT AND OTHERS THAT DON'T. WHAT WE KNOW ABOUT HYDROXY UREA MECHANISTICALLY U, IT DOES UPREGULATE FETAL HEMOGLOBIN LEVELS. THERE'S A REDUCTION IN HBS. BUT THERE'S MORE AND MORE EVIDENCE TO SUGGEST HYDROXY HYDROXY UREA HAS ADDITIONAL MECHANISTIC BENEFITS AND WE'RE TRYING TO EXPLORE WHAT THOSE MIGHT BE. I'LL ALSO DISCUSS THAT IN PART IN MY TALK AS WELL. BRIEF INFORMATION ABOUT MICROPARTICLES. FIRST OF ALL, THE FORMATION OF MICROPARTICLES IS A RESPONSE TO CELLULAR STRESS. SO WHAT WE UNDERSTAND ABOUT MICROPARTICLES IS THAT MICROPARTICLE FORMATION IS A IMPORTANT PROTEIN, THAT MEDIATED PROCESS, THAT PRODUCES MICROPARTICLES THAT ARE RECOGNIZED AND REMOVED BY THE IMMUNE SYSTEM. IN NORMAL RED BLOOD CELLS OR NORMAL CELLS, MICROPARTICLE FORMATION IS ACTIVATED DURING APOPTOSIS. THIS PROCESS OF REMOVING SENESCENT CELLS BY THE IMMUNE SYSTEM UNDER NORMAL PHYSIOLOGICAL CONDITIONS WOULD BE LOW. CELLS UNDER NORMAL PHYSIOLOGICAL CONDITIONS SHOULD HAVE A SMALL AMOUNT OF MICROPARTICLES. WITH SICKLE CELL HEMOGLOBIN RED BLOOD CELLS WITH ANEMIA, WE HAVE HEMOLYSIS OCCURRING, THE DEGREE OF MICROPARTICLE FORMATION IS MORE RAPID. WE HAVE LEARNED THAT. SO OUR INTEREST IS WHAT IS THE PROTEOME BEHIND THESE MICROPARTICLES? IS THERE SOMETHING ABOUT THE PROTEOME THAT ARE PROTEINS ENRICHED ARE THIS THINGS WE CAN LEARN ABOUT THE THE TYPES OF PROTEINS OR GROUPS OF PROTEINS IN THESE MICROPARTICLES AND HOW DO THEY COMPARE TO MICROPARTICLES FROM SAY A NORMAL PERSON? OR WILD TYPE? THIS IS JUST A SLIDE SHOWING A WORK FLOW MOW WE GET MICROPARTICLES. FIRST OF ALL, MOST DISCUSSION IS GOING TO BE FROM MICROPARTICLES DERIVED FROM RED BLOOD CELLS FROM MICE, THESE WERE PROVIDED FROM US FROM OUR COLLABORATORS, UNIVERSITY OF MINNESOTA. I WILL DISCUSS SOME HUMAN DATA VERY LIMITED, BASICALLY WE USE A WELL ESTABLISHED STRESS METHOD TO GENERATE MICROPARTICLES IN BOTH PROCESSES. AND THEN WE USE DIFFERENTIAL OR ZONAL SENTRY FUMIGATION TO SEPARATE THE MICROPARTICLES AND USE FLOW CYTOMETRY AND NANOTRACKING ANALYSIS TO CHARACTERIZE AND CONFIRM WE HAVE A MICROPARTICLE POPULATION. THEN WE DO QUANTITATIVE PROTEOMICS. THE QUANTIATIVE PROTEOMIC OVERVIEW I WILL DISCUSS IS WE ARE INTERESTED IN CHARACTERIZING THE PROTEOME, ALSO INTERESTED IN CHARACTERIZING POST TRANSLATIONAL MODIFICATIONS. I ALWAYS SPELL THIS OUT A LOT OF TIMES PEOPLE IN MY AUDIENCE DON'T THINK ABOUT PTM VERY OFTEN, THEY'RE LIKE WHAT IS A PTM. SURE EVERYBODY HERE WHO DOES MASS SPECTROMETRY IS USE THAT A LOT. HOW DO PROTEOMES DIFFER BETWEEN AA AND S S MICE? I'M SAYING SICKLE CELL DISEASE MICE EXPRESSING WILD TYPE HEMOGLOBIN. WHAT IS THE PROTEOME, THE PTM LANDSCAPE AND HOW DOES IT DIFFER? DO WE SEE DIFFERENCES IN POST TRANSLATIONAL MODIFICATIONS? CAN THIS GIVE INSIGHT INTO THE DEGREE OF OXIDATIVE STRESS? WHAT IMPACT DOES HYDROXY UREA, THIS WILL BE THE CHERRY ON TOP, IF YOU WILL, WHAT CORRELATION DO WE SEE BETWEEN MOUSE MODELD ON HUMAN PROTEOME DATA SO THAT IS WHAT I WILL SHOW AT THE END. FIRST STEP WE GET OUR RED BLOOD CELLS, MICROPARTICLES, THE FIRST THING THAT I DO I QUANTIFY THE MICROPARTICLE PROTEINS USING BC ASSAY THAN SOLUBLIZE THE MICROPARTICLES. WE LOAD EQUAL AMOUNTS OF MICROPARTICLE PROTEIN, THAT HAVE BEEN SOLUBILIZED BY GEL LOADING, AND WE RUN THE SAMPLE SAY 30 TO 5 MOCKER GRAMS AT MOST. THAT'S A LOT OF PROTEIN. WE RUN FOR FIVE MINUTES ON DENATURING GEL SO WE HAVE A TIGHT BAND THAT I THINK CUT OUT, WE CUBE IT VERY METHODICALLY, AT LEAST 30 PIECES, I TRY TO BE AS CLOSE TO DOING EVERYTHING THE SAME. SO WHAT CONTAINS IN THIS TIGHT PLUG IS THE PROTEOME OF THE MICROPARTICLE. NOW, THERE IS AN ADVANTAGE TO DOING THIS, BECAUSE A LOT OF THESE PROTEINS WITHIN THE MEMBRANE AGGREGATE SO IS IT'S WAY OF USING THE GEL METHOD TO KEEP AS MANY OF THE PROTEINS OR TO TRIPS RECOGNIZE AS MANY PROTEINS AS POSSIBLE. THEN WE DO IN GEL DIGESTIONS, EXTRACT THE PEPTIDE USING CLASSICAL MEANS. BEFORE LOADING ON TO THE INSTRUMENT I WILL THEN DO A BC ASSAY TO QUANTIFY PEPTIDE. SO WHAT WE'RE WANTING TO DO IS WANTING TO ENSURE THAT WE LOAD EQUIVALENT AMOUNTS AS POSSIBLE. SO I AM USING A NEW SOFTWARE DESIGNED IT'S LABEL FREE SOFTWARE. IT ALLOWS FULL CHANGE DIFFERENCES BETWEEN PROTEINS OF DIFFERENT SAMPLES. SO I'M GOING TO SHOW YOU HOW WE CAN STATISTICALLY CONFIRM THAT WE HAVE DATA COMPARABLE BUT I WANT TO GIVE YOU AN OVERFLOW HOW IT WORKS. FIRST PEPTIDES AFTER YOU HAVE RUN -- YOU HAVE DONE YOUR INSTRUMENT RUN, SO BEFORE I START THIS, I WOULD JUST LIKE TO SAY THAT AFTER WE GOTTEN PEPTIDE ANOLYTES AND KNOW EXACTLY HOW MUCH TO RUN, SAY I WILL RUN ONE MICROGRAM TOTAL PEPTIDE REPRESENTING THE PROTEOME, I WILL RUN IT OVER TWO HOUR GRADIANT, WITH A LOOP COUNT OF 20, MY TARGETS WILL BE SET. THE GOAL IS TO OPTIMIZE NUMBER OF MSMS SPECTRA, ALSO MAKE SURE I GET A NICE CHROMATOGRAPHIC SEPARATION WITH SHARP PEAK CAPACITIES. THAT IS TAKEN CARE OF BUT UNDERSTAND YOU HAVE THAT YOUR DATA ACQUISITION WILL CONTAIN 20, 30,000 SPECTRA. ALL THIS YOUR ENTIRE PROTEOME DONE IN SINGLE DATA ACQUISITION. NOW THE SOFTWARE. IT TAKES THE RAW FILES, IT FIRST -- IDENTIFIES THE PEPTIDE IDs USING EXTANDEM. A DATABASE SEARCH ALGORITHM, WORKS SYNONYMOUSLY WITH MASS SPOT OR SIMILAR METHOD. IT THEN GENERATES EXTRACTED MS 1 CHROMATOGRAMS FOR EACH PEPTIDE IDENTIFIED. THIS IS ONE FEATURE I LIKE BECAUSE I DO EXTRACT CHROMATOGRAM WHEN I WAS CHARACTERIZING OXIDATIVE TOXICITY SO THIS IS AUTOMATED WAY OF DOING THAT. IT IDENTIFIES ENDOGENOUS REFERENCE PEPTIDES, THAT ARE COMPARABLE FROM SAMPLES BUT SIMILAR RETENTION TIME TO PEPTIDES, IT DOES PEPTIDE. SO WHAT IT DOES IS THEN IT WILL MATCH THOSE AND NORMALIZE YOUR PEPTIDE ABUNDANCE BASED ON RETENTION TIME SO YOU CAN HAVE A COMPARISON BETWEEN SAMPLES SO IF I DID A DATA ACQUISITION FOR SS THEN SA THE SAME ENDOGENOUS PEPTIDES WERE USED TO NORMALIZE THE PROTEINS COMPARABLE. ONE OF THE FEATURES IS YOU HAVE TO HAVE STATISTICALLY SOUND DATA. YOU HAVE TO HAVE GOOD RETENTION TIME, WANT TO SHOW YOU HAVE LITTLE RETENTION TIME DRIP. YOU WANT TO MAKE SURE YOU'RE LOADING EQUAL AMOUNTS OF SAMPLE, SO THAT'S THE NAME OF THE GAME. SO ONE OF THE THINGS THAT I FEEL IS MOST CHALLENGING AND ONE OF THE THINGS I USED TO HATE DOING ABOUT PROTEOMICS BACK IN THE DAY WHEN I HAD TO DO SPECTRAL COUNTING WAS THAT YOU REALLY HAD LIMITED -- YOU WERE LIMITED HOW YOU CAN CONTROL LOADING ISSUES. BUT WHAT THIS SOFTWARE DOES IS AUTOMATICALLY CALCULATES A TOTAL ION CHROMATOGRAM VARIATION CO-EFFICIENT. THEN IT WILL COMPARE ALL OF THESE AND SHOW THE DIFFERENCE. SO IF YOU HAVE ANYTHING BELOW 20%, YOU ARE GOOD. THAT MEANS BETWEEN ALL YOUR RUNS, FOR THIS, THESE ARE SAY FOR THESE I HAD -- THESE ARE BIOLOGICAL REPLICATES. SO I DID AN N OF THREE FOR WILD TYPE TRANSGENIC MICE, THE SICKLE CELL DISEASE TRANSGENIC MICE AND THEN WE DID SOME HYDROXY UREA TREATMENT. MORE ON THAT LATER. SO THERE'S 8% TOTAL VARIATION BETWEEN ALL THESE DIFFERENT TOTAL ION CHROMATOGRAM CO-EFFICIENTS. IT ALSO LOOKS AT PEPTIDE QUANTIFICATION. YOU CAN SEE THAT WHEN YOU HAVE THESE DATA, ALL OF A SUDDEN THE NUMBERS OF PROTEINS THAT YOU GET, THE DIFFERENCES, AND FULL CHANGES ARE RELEVANT BECAUSE YOU CAN SEE YOUR LOAD EQUIVALENT AMOUNT OF ANOLYTE. THAT'S ONE THING I LIKE ABOUT THE SOFTWARE. I THOUGHT IT WAS WORTH MENTIONING. SO BEFORE I TALK ABOUT THE DATA, I HAVE I ALREADY KNOW IT'S STATISTICALLY COMPARABLE. WHEN WE STARTED RESEARCH WE USED THE BIOCHEMISTRY LAB, WE OWN THE BUSINESS OF HEMOGLOBIN. SO OUR BREAD AND BUTTER METHODOLOGIES INVOLVE CLASSIC SPECTRAL SPECTROMETRY, WE DO STOP FLOW. HIGH MOCKS -- I CAN'T SAY THE WORD. WE LOOK AT OXYGEN BINDING AND SO WITH THAT BEING SAID THE FIRST THING WE DID IS SYSTEMATICALLY LOOK AT MICROPARTICLES, WE EVEN HAD A MICROPARTICLE TRANSGENIC LINE HETEROZYGOUS, I'M MAINLY GOING THE FOCUS ON AA AND SS COMPARISONS. WHAT WE LEARNED WAS IS WHEN WE COMPARE TO A CONTROL HEMOGLOBIN BUFFER WE SEE THERE IS A GREATER DEGREE OF OXIDATION THAT OCCURS AT THE HEMOGLOBIN WITHIN THE MICROPARTICLES. AND OVER A 24 HOUR PERIOD OF TIME YOU GET HEMOGLOBIN, YOU GET SOME FERRELL YOU GET HEMOCHROME. ONE INTERESTING FACTOR, WE DID HPLC ANALYSIS USING REVERSE BASED CHROMATOGRAPHY, AND MONITORED THE ABOUTSOR WANTS AT 280 NANOMETER OVER TIME WE FIND THAT WHEN YOU COMPARE THE HEMOGLOBIN FROM MICROPARTICLES WITH WILD TYPE VERSUS THE SICKLE CELL, YOU GET DIFFERENCES OVER TIME AND SUBUNIT ALTERATION. THE HEME -- AMOUNT OF HEME IS IDENTICAL IN BOTH. SO WE ATTRIBUTE THIS SUBUNIT ALTERATION TO BE CHARACTERISTIC OF OXIDATION. WHICH WE HAVE ALREADY SAID IN OUR PREVIOUS STUDIES, WE KNOW THAT'S WHAT'S HAPPENING. SO THIS IS IN VIVO STUDY THAT SUPPORTS EARLIER IN VITRO STUDIES. WE THEN DID TIME COURSE SAY AS MONITORING AUTOOXIDATION AS FUNCTION OF TIME. WE FOUND THAT FOR MICE YOU GET HIGHER LEVEL MET HEMOGLOBIN AT THE BEGINNING BUT OVER TIME THIS MET HEMOGLOBIN EXCEL AUTOOXIDIZING TO THE HBA VERSUS HBS, YOU -- WE FOUND THAT IN MICROPARTICLES WITH SICKLE CELL DISEASE HEMOGLOBIN, THIS AUTOOXIDATION TO MET HEMOGLOBIN WAS MORE PROFOUND AND ACCELERATED IN MICROPARTICLES WITH SICKLE CELL DISEASE THAN HBA. SO WE FOUND THE TREND TO BE REPRODUCIBLE WHEN WE DID THE SAME THING WITH HUMAN STUDIES SO WE HAVE AS I SAID WE HAVE LOOKED AT HUMAN PATIENT SAMPLE AND TRY TO DO AS MANY AS WE CAN. SO WE KNOW AT THE HEMOGLOBIN LEVEL SOMETHING GOING ON WE SEE IN THE TOWN MICE WHEN YOU HAVE HBS EXPRESSED IN RED BLOOD CELLS OF THE TOWNY MICE THE HEMOGLOBIN IS BEHAVING IN WAY THAT INDICATES OXIDATIVELY LESS STABLE THAN HUMAN WILD TYPE HBA. SO I THEN LOOKED AT THE PROTEOME, AT 0 AND 30 HOURS. NOW, THIS PANEL RIGHT HERE, REPRESENTS THE ZERO HOUR DATA. I WILL GO MORE IN DETAIL ABOUT VOLCANO PLOTS. FOR YOU THAT DON'T LOOK AT VOLCANO PLOTS, IT'S BASICALLY A TWO DIMENSIONAL DISPLAY OF PROTEOME DATA TO GET IDEA HOW PROTEIN ABUNDANCES ARE DISTRIBUTED IN THIS CASE. THE X AXIS IS LOG BASE OF TWO FOR THE RATIO OF THE SICKLE CELL PROTEOME OVER THE AA. AND THE Y AXIS ARE THE CORRESPONDING P VALUE, THE NEGATIVE LOG OF TEN OF P VALUE, THE P VALUE THAT WAS ASSIGNED TO PROTEIN ABUNDANCE. SO WHEN WE LOOK AT THIS, WE SEE IN THIS QUADRANT HERE, I WILL SHOW MORE VOLCANO PLOT DATA WE SEE GREATER DEGREE OF PROTEINS IN THIS QUADRANT INDICATIVE OF GREATER DEGREE OF CHANGE UPREGULATION IN SICKLE CELL PROTEOME. SO SOMETHING GOING ON IN THE PROTEOME. FIRST THING WE NOTICE WHEN WE LOOK AT THE ANTI-OXIDATIVE ENZYMES AMONG OTHERS THIS IS THE FIRST TIME WE DO THIS, WE SAW THE ANTIOXIDANT ENZYMES WERE MORE UPREGULATED AND THE MICE THAT HAD SICKLE CELL DISEASE HEMOGLOBIN. THIS IS A HALLMARK OF OXIDATIVE STRESS. WHAT FURTHER ILLUSTRATES MICROPARTICLES ARE OXIDATIVELY STRESSED OUT, DESPITE THE FACT WE HAVE HIGHER LEVELS OF ANTI-ENZYMES WE STILL HAVE GETTING HIGHER AMOUNT OF MET HEMOGLOBIN. SO WE REPEATED THE SAME EXPERIMENTS USING A DIFFERENT SET OF MICE. WE HAD ORIGINALLY FIRST STUDY, WE DID -- WE LOOKED AT THE MICE. AND FOUND THE RESULTS SO WANTEDDED TO DO THE SAME PROTEOME STUDY AND WE WANTED TO KNOW WOULD WE BE -- SEE A REPRODUCIBLE TREND? IF WE DO, WHAT IS THE DISTRIBUTION OF THESE PROTEINS? THIS VOLCANO PLOT HERE, AS I SHOWED BEFORE INDICATES THAT WE'RE GETTING A LOT OF THE SAME THINGS. WE'RE SEEING UPREGULATION OF THE PROTEOME. PROTEINS THAT ARE RELATED TO THE SICKLE -- THE SICKLE CELL MICROPARTICLES. WHAT'S INTERESTING IS THAT WE SAW THE SAME INCREASE IN ANTIOXIDANT ENZYME SO NOW WE HAVE TWO SEPARATE STUDIES WHERE WE HAVE BIOLOGICAL REPLICATES FROM STATISTICALLY SOUND DATA THAT REPRODUCIBLY SHOW US SOMETHING WE HAVE NEVER SEEN BEFORE. THAT IS THAT WE HAVE THESE ANTIOXIDANT ENZYMES THAT SEEM TO BE IN THE MICROPARTICLE PROTEOME AND ENRICHED. FURTHERMORE WE HAVE HIGHER LEVEL RETAINED MITOCHONDRIAL PROTEIN. THERE'S A RECENT PAPER THAT KIM OUT JCI FROM A GROUP THAT LOOK AT RBCs THEY FOUND YOU GET GREATER DEGREE OF MITOCHONDRIAL RETAINED SICKLE CELL BLOOD CELLS. THIS IS THE RED BLOOD CELL PROTEOME. WE FOUND SOMETHING THAT IS PARTICULARLY INTERESTING, WE FOUND LARGE NUMBER OF PROTEINS, IN ADDITION TO DOING PROTEOME ANALYSIS, WE DID PTM ANALYSIS. WE DID GLOBAL PTM ANALYSIS WHICH IS ONE OF THE THINGS YOU CAN DO WITH PRO TOELYZERS YOU CAN LOOK AT SEVERAL SIMULTANEOUSLY BECAUSE WE'RE USING APPROXIMATE -- I FORGET TO MENTION, WE HAVE HIGH MASS ACCURACY, HIGH RESOLUTION, WE CAN NOW USE FEATURES PEPTIDES THAT WOULD HAVE PTM AND LOOK AT MULTIPLE PTM, WHAT WE FOUND WAS SEVERAL PROTEINS IN THIS GROUP, IN GREEN, WERE UBIQUITINATED. THAT REALLY SURPRISED US. DOESN'T SEEM LIKE A BIG DEAL BUT THERE WAS A GROUP IN THE 80s AND EARLY '90s THAT SPENT A LOT OF TIME DOING CLASSICAL METHODS AND THEY ACTUALLY COME TO THE CONCLUSION THAT UPS MACHINERY WASN'T ACTIVE IN RED BLOOD CELLS. THERE'S RECENT PAPERS THAT SHOWS YEAH ACTUALLY UPS MACHINERY IS. IT'S MY PHONE. MY BAD. THIS IS -- YEAH. ALL RIGHT. I CAN HANDLE THAT. SO ONE OF THE THING IS THAT WE NOTICED RIGHT AWAY SOMETHING THAT WAS SURPRISING. THAT WE LOOKED AT THE PTM -- HEMOGLOBIN LEVEL. WHAT WE NOTICED WAS IN ADDITION TO INCREASE IN SISTINE 93 OXIDATION WHICH WE HAVE ALREADY -- I HAVE ALREADY TOLD YOU IS ONE OF THE HOT SPOTS, YOU HAVE THE CYSTIC ACID, WE SAW IN THE SS MICE IT WAS HIGHER THAN IN THE AA MICE. WHAT WE ALSO FOUND WAS REPRODUCIBLE TREND WE FOUND UBIQUITINATION LYSINE 96 AND LICENSE 145 -- LYSINE 145. CIRCULATING PLASMA WE FOUND THE MICROPARTICLES THAT ARE IN THE PLASMA TO BEGIN WITH, WE FOUND THESE LEVELS WERE REPRODUCIBLE SO THE SHOE STRESS MICROPARTICLES WE STUDY, IN CIRCULATE MICROPARTICLES, SO THAT WAS FASCINATING TO US BECAUSE K-96 AND K-145, ARE LOCATED IN PROXIMITY TO SISTINE 93 STRUCTURE. ONE THING TO POINT OUT IS THAT IN OUR WORK, WE HAVE KNOWN THIS FOR SOME TIME REASONS BEYOND SCOPE OF THE TALK, EXPLAIN TO ANYBODY THAT WANTS TO ASK ME AFTER I'M DONE, THE BETA SUBUNIT TAKES THE BRUNT OF OXIDATIVE STRESS OVER THE ALPHA SUBUNIT. THE UPS MACHINERY, THESE MODIFICATIONS WERE LIMITED TO THE BETA SUBUNIT. AND THEY WERE MORE PROFOUND IN HBS THAN HPA. SO WE HAVE A CORRELATION WITH UBIQUITINATION AND SISTINE 93. SO YOU TAKE THESE TOGETHER IT IS TEMPTING TO SAY CORRELATION WITH OXIDATION. AND YOU BICTY NATION. WE KNOW UBIQUITINATION, ONE OF THE DEFINING REASONS FOR UBIQUITINATION IS IT'S A TARGET PTM FOR DEGRADATION. SO WE HAVE ADDITIONAL DATA, I WOULD LIKE TO SAY MOTHER NATURE HAS GIVEN US THE GREEN FLIGHT THAT END CASE SICKLE CELL HEMOGLOBIN IS OXIDATIVELY LESS STABLE BECAUSE IT'S UBIQUITINATED MORE THAN HBA. IT'S LIKELY TARGETED DEGRADATION. THIS IS THE FIRST WE ACTUAL HI PRESENTED THIS WORK SINCE SUBMITTED IT AND BEEN ACCEPTED UPON REVISIONS, BUT THIS IS THE FIRST -- WE WERE THE FIRST GROUP TO IDENTIFY THIS UBIQUITINATION ANDSIS TAKE ACID RELATIONSHIP, WHEN I FIRST IDENTIFIED IT I WAS NERVOUS BECAUSE IT WAS GOING AGAINST THE GRAIN, WHEN I FIRST TOLD PEOPLE YEAH HEMOGLOBIN IS UBIQUITINATED THEY'RE LIKE I DON'T KNOW IFIT IS. I DON'T THINK SO. I GOT FURTHER STUDIES COMING UP THAT SUPPORT THIS MOUSE MODEL DATA. AND WE HAVE DONE SRM DATA ORTHOGONAL METHOD TO CONFIRM THIS. SO A COLLEAGUE IN MY LAB SHE IS INTERESTED IN LOOKING FOR ANTIOXIDANTS. PARTICULARLY ONE OF HER PROJECTS IS ANTI-SICKLING AGENTS. SHE LOOKED AT HYDROXY UREA AS POTENTIAL ANTIOXIDANT. THESE ARE SAY SAYS SHE DID. SHE FOUND WHEN YOU INCUBATE HEMOGLOBIN S WITH HYDROXY UREA, EXCUSE ME, THEN YOU SUBJECT IT TO FERRELL CONDITION YOU CREATE FERRELL AND ASSAY FOR FERRELL USING SODIUM SULFIDE SHE FOUND YOU GET A LOWER AMOUNT OF FERRELL WHEN HYDROXY UREA IS PRESENT. SHE ATTRIBUTED TO THIS, HER HYPOTHESIS IS YOU'RE MODIFYING DOING SOMETHING PROTECTIVE. TO THE -- SO THIS IS IN VITRO DATA. YOU ARE DOING SOMETHING THAT PROTECTS THE HEMOGLOBIN FROM THESE OXIDATIVE PROCESSES. I DID SOME ANALYSIS FOR -- I WENT AND FOUND THAT WE GOT THE SPECTRA WHERE WE FOUND S NITROSYLATION AT SISTINE 93. THE REASON WHY I SHOW THIS IS I WAS SURPRISED BECAUSE WE ALSO HAD THE DIGLYCENE MOIETY SHOWS WITH UBIQUITIN. I DON'T ACCEPT PEPTIDES WITH MULTIPLE PTNs, IF I SEE ONE I WON'T THROW THE BABY OUT WITH THE BATH WART, I WILL MANUALLY EXAMINE IT, LOOK AT IT, EXAMINE THE IONS, MY COLLABORATOR MICHAEL, HE WENT AND LOOKED AT IT, THERE WERE TWO Ph.D.s INVOLVED TO CONFIRM THIS IS REAL BECAUSE -- IT DIDN'T SURPRISE US BECAUSE WE HAD SEEN UBIQUITINATION. BUT WE ACTUALLY SAW EVIDENCE THAT YES YOU CAN GET S NITROSYLATION AND HYDROXY UREA, THIS EXCITED US SO LET'S GO AND DO THE MOUSE MODELS WHERE WE CAN LOOK AT THE IMPACT OF HYDROXY UREA. IF THERE IS ANY. SO WE DID AS I SHOWED YOU PREVIOUSLY, WE DID A STUDY WHERE WE LOOKED AT THREE DIFFERENT N OF THREE OF MICE, AND BIOLOGICAL REPLICATES AND LOOKEDDED TO SEE IF THERE WAS A DIFFERENCE IN THE PROTEOME OF HYDROXY VERSUS UNTREATED AND TREATED SO YOU REMEMBER THAT BEAUTIFUL YOU HAD PROTEINS ON ONE QUADRANT INDICATIVE OF PROTEOME GLOBAL DIFFERENCE, OBVIOUSLY SOMETHING GOING ON, THIS IS WHAT IT LOOKS LIKE WHEN NOTHING IS HAPPENING. WHEN WE DID COMPARISON OF UNTREATED HYDROXY UREA, EVERYTHING IN THE MIDDLE. WE DID NOTICE THERE SEEMED TO BE SOME DIFFERENCE IN REFERENCE TO HYDROXY UREA TREATMENT AND OXIDATION AND UBIQUITINATION PTN SO WHILE THAT'S INTERESTING WE'RE CAUTIOUS AND REALIZE THAT'S POTENTIALLY VERY POSITIVE. I'LL SHIFT THE REMAINING PART OF MY TALK I'LL GO OVER SOME SLIDES SHOWING LIMITED HUMAN DATA. THAT IS IN THIS CASE COMPARE ETHNICALLY MATCHED CONTROL, A CONTROL THAT DOES NOT EXPRESS THE SICKLE CELL. THEN WE'RE LOOKING AT SAY AN UNTREATED VERSUS A TREATED. I'M DISCLAIMING RIGHT NOW THAT THIS -- WE DID TECHNICAL REPLICATES BUT THIS IS AN N OF ONE. WITH HUMAN SAMPLES YOU CAN GET A LOT OF VARIATION. SO WE'RE WORKING ON GETTING SAMPLES TO DO THIS RIGHT BUT THIS IS WHAT WE HAVE GOT FROM N OF ONE. WHAT WE WERE INTERESTED IN IS DO SOME ANALYSIS TO SEE IF THE HUMAN DATA WE GET CORRELATES WITH OUR MOUSE MODEL DATA. SOMETIMES THEY DON'T. FIRST THING WE DID IS LOOK AT THE OBVIOUS. IN HUMAN WE KNOW HBF LEVELS ARE DIFFERENTIATED. SO WHEN YOU ADD HYDROXY UREA, YOU SHOULD SEE HIGHER LEVEL OF GAMMA SUBUNITS THAT ARE INVOLVED IN FORMING FETAL HEMOGLOBIN, IT'S TWO ALPHAS, TWO GAMMAS, ADULT HEMOGLOBIN IS TWO ALPHASES AND TWO BETAS. WHAT WE FOUND IS THAT FOR THE CONTROL, WE HAD VERY LIFT OF THESE AT ALL. SO THESE FULL CHANGE DIFFERENCES ARE COMPARING, THESE ARE P VALUES TO SHOW THE TEST SIGNIFICANTLY SIGNIFICANT, WHEN WE COMPARE FULL CHANGE TO CONTROL WE SEE SUBSTANTIAL AMOUNT OF GAMMA ONE AND TWO SUBUNITS. WE EVEN SEE SOME IN THE SED UNTREATED WHICH IS KNOWN TO OCCUR. BUT WE USE ORTHOGONAL METHOD. I LIKE TO DO ORTHOGONAL EXPERIMENTS. MOST PEOPLE IN THE FIELD OF PROTEOMICS REALIZES WE HAVE ARROWS ON OUR BACK WHEN WE RELY TOO HEAVILY ON MASS SPECTROMETRY DATA. THE BEST WAY TO WIN OVER THE BIOLOGICAL COMMUNITY IS TO WORK WITH THEM AND USE THE CLASSIC METHODS THEY LIKE AND WELL ESTABLISHED. I SEW ELECTRIC FOCUS HPLC CHARACTERIZATIONS, OLD SCHOOL METHODS THAT WORKED FOR YEARS, WHEN WE LOOK AT THESE SAMPLES WE FOUND THAT WE INDEED GET INCREASESES OF HBF. WE'RE CONFIRMING THAT OUR MASS SPECTROMETRY METHOD GIVES THE KIND OF RESULTS WE EXPECT FOR HUMAN SAMPLES. THIS IS A TABLE SHOWING SOMETHING THAT I THOUGHT WAS INTERESTING. THAT IS? ADDITION TO GETS SUSTAINING ACID, THESE ARE INDIVIDUAL PEPTIDES WE SEE, WE'RE AT THE I'M SHOWING PEPTIDE DATA SINCE WE HAVE N OF ONE. WHAT WE SEE IS THAT THE SISTINE 93 OXIDATION FOR SICKLE CELLS HIGHER THAN IT IS FOR ETHNIC CONTROL. THEY ARE ABOUT THE SAME. WE'RE DEFINITELY SEEING INCREASE. THIS IS ONE THING THAT REALLY EXCITED US IS THAT THE K 296 AND K 145 WE SAW UBIQUITINATEDDED IN MOUSE MODEL IS NOW HUMAN DATA. SO WE HAVE A CONSERVED POST TRANSLATIONAL MODIFICATION AND THAT ONLY STRENGTHENS OUR ARGUMENT THAT UPS MACHINERY IS INVOLVED IN TARGETING ABERRANT OR DEFECTIVE HEMOGLOBIN, HBS WOULD BE DEFECTIVE, IT'S OXIDATIVELY LESS STABLE AND IT ALSO DEOXYGENATED STATE POLYMERIZES SO NOW WE HAVE TWO DIFFERENT SETS OF DATA SHOWING THE SAME PTM LANDSCAPE. THESE ARE MESSY DATA BUT THAT'S OKAY BECAUSE THESE ARE THE STUFF WE HAVEN'T PUBLISHED. THIS IS THE STUFF WE'RE JUST -- WE'RE INTERESTED IN SHOWING. SO WE DID SOME SRM STUDIES ON THESE HUMAN SAMPLES. THAT'S SELECTIVE REACTION MONITORING, TO SORT OF SHOW ORTHOGONALLY THAT THE UBIQUITINATIONS AND -- ARE REAL. ONE THING I WANT TO DO, THIS IS A HOT PTM. WHEN YOU START TALKING UPS MACHINERY THERE'S A LARGE COMMUNITY OUT THERE THAT DOES A LOT OF STUDIES ON THIS, SO YOU WANT TO GET IT RIGHT. SO WE USE SRM STUDIES. INSTEAD OF DOING MS 1 CHROMATOGRAMS, WHICH I THINK ARE VERY VALUABLE, THE PROTEOLYZER CAN DO SRM AS WELL. AND IT GENERATES EXTRACT DICHROMATOGRAMS BASED ON SM 2 DATA OR EXCUSE ME GAGMENT ION CHROMATOGRAMS GENERATED FROM FRAGMENT IONS. SO THE ADVANTAGES THAT IT'S HIGHLY MORE SENSITIVE AND MORE -- AND EVEN THOUGH MS 1 DATA IS HIGHLY LIKELY CORRECT IT'S BULLET PROOF FOR BEING INCORRECT. WHEN WE LOOK AT UNTREATED FOR HYDROXY UREA VERSUS TREATED WE DIDN'T DETECT IT AND THE UNTREATED BUT WE DID SEE A WHOPPING AMOUNT EXTRACT ION CHROMATOGRAM AND THE K-96 THIS WAS OVERLOADED WE WERE SEEING A PEEK COMING OFF. THE POINT IS WE WEREN'T SEEING HYDROXY UREA TREATED SO WHILE NEEDING TO OPTIMIZE STUDIES WE WANT TO GET ENOUGH HUMAN SAMPLES BEFORE WE START. BUT THE THING IS THE SRM RECAPITULATES WHAT WE WERE SEEING FROM OUR DATA DEPENDENT ACQUISITION, MS 1 DATA, WHICH CORRELATES WITH MOUSE MODELS DATA SO WE'RE LOOKING GOOD. THIS IS ANOTHER THING. SINCE HUMAN DATA SAND MITOCHONDRIAL DID SOME ANALYSIS INDEPENDENTLY, ANTI-PHOSPHOTYROSINE ANTIBODIES, PHOSPHOTYROSINE ANTIBODIES, AGAINST HUMAN SAMPLES AND HE FOUND THAT FOR THE SICKLE -- THE SS 1 AND SS 2 PATIENTS, WE WERE IN FACT SEEING A HIGHER AMOUNT OF PHOSPHORYLATION. WE ALSO IDENTIFY PHOSPHORYLATION FOR BAND 3 AND FOUND IT WAS HIGHER. IN UNTREATED VERSUS HYDROXY UREA TREATED. ANOTHER THING HE DID WAS THAT -- IS THAT IT'S KNOWN HEMOGLOBIN DENATURES FORMS HEMOCHROME AGGREGATES THAT THEN -- POTENTIALLY INTERACT WITH 3. THEY CLUSTER AND CALL BAND THREE TO GROUP TOGETHER, CLUSTER AND ACTIVATE THE MICROPARTICLE FORMATION PROCESS SLOUGHING OFF FORMATION OF MICROPARTICLES SO WHEN HE DID A CO-IMMUNOPRECIPITATION HE FOUND THAT THERE WAS A RELATIONSHIP WITH HYDROXY UREA TREATMENT VERSUS UNTREATMENT. SO THESE ARE ALL SORT OF THINGS WE'RE PUTTING TOGETHER OUR NEXT STORY. I'M WRAPPING IT UP. THIS IS A MODEL WE HAVE PROVEN BUT WE HAVE DATA AND THIS IS WHERE WE'RE HEADED. THEN I'LL GET MY SUMMARY SLIDE AND MOVE ON. IN SUMMARY WE KNOW THAT OXYGEN BINDS FOR SICKLE CELL, WE GET TRIOXIDATION,SIS STAKE ACID, WE GET UBIQUITINATION WHICH TARGETS IT FOR DEGRADATION BY THE 20S PROTEASOME. OR HEMOGLOBIN CAN BE PARTIALLY DENATURED, FORM AGGREGATES THAT CLUSTER, WITH BAND 3 IS TO ACTIVATE MICROPARTICLE FORMATION SO THE BIG QUESTION NOW IS WHEN WE GET ENOUGH HUMAN SAMPLE WE WILL WANT TO DO MORE INTENSIVE ANALYSIS OF THE HYDROXY UREA TREATMENT TO SEE IF WE GET GOOD DETECTION OF S NITROSYLATION. IF IT'S POSSIBLE, WHAT WE SAW IN VITRO IS OCCURRING IN VIVO. ARE WE GETTING PROTECTIVE MECHANISM THAT INHIBITSSIS TAKE ACID WHICH DOESN'T -- WHICH PLAY AS ROLE DESTABILIZING THE HEMOGLOBIN AND CAUSING DEMISE OR DEGRADATION. SO I SORT OF ALREADY SAID THESE, I WON'T READ THROUGH -- I HATE READING SLIDES BUT WE HAVE USED A BATTERY OF ASSAYS TO CHARACTERIZE MICROPARTICLES, SPECTRA PHOTO METRIC METHODS, HPLC ANALYSIS, ISOELECTRIC FOCUSING. AND THE TAKE HOME MESSAGE IS THAT WHEN YOU EXPRESS HUMAN HEMOGLOBIN SICKLE CELL HEMOGLOBIN IN MICE AND LOOK AT THE RED BLOOD CELLS IN MICROPARTICLES YOU CAN AUTOMATICALLY SEE THERE'S A DIFFERENCE IN THAT HEMOGLOBIN, IT'S OXIDATIVELY LESS STABLE, IT HAS HIGHER SISTAIC ACID, UBIQUITINATED, AND THIS CORRELATES WITH OUR EARLIER IN VITRO STUDIES THAT WE WERE DOING. THAT'S A GOOD THING. WHEN WE LOOK AT THE PROTEOME WE SEE THAT IN ADDITION TO PROTEIN LEVEL, WE SEE THAT THE SICKLE CELL RED BLOOD CELLS OR MICROPARTICLES THE PROTEOME INDICATES THERE'S OXIDATIVE STRESS. WE'RE GETTING CHANGES IN TYPES OF PROTEINS, WE'RE GETTING UPREGULATION OF MITOCHONDRIAL PROTEINS OR MITOCHONDRIAL RETENTION, WE GET HIGHER LEVEL PTMs, SO WE'RE ACTUALLY EXPLORING UBIQUITYNOME FOR FUTURE WORK. THIS PROTEOME TELLS US SOMETHING MECHANISTICALLY WE DIDN'T KNOW BEFORE. WE LEARNED UBIQUITINATION IS OCCURRING. AND THAT IT MIGHT BE PLAYING A ROLE IN THE OUTCOME OF CELLS THAT EXPRESS SICKLE CELL HEMOGLOBIN. FURTHERMORE WHEN WE LOOK AT A LIMITED NUMBER OF HUMAN SAMPLES WE SEE THAT THE PTM WE IDENTIFY IN THE MOUSE MODEL ARE PRESENT IN THE TOWNNY MICE. BEFORE I GIVE A LARGE TALK ABOUT PROTEOME I AT LEAST WANT TO HAVE MEANINGFUL NUMBER OF REPLICATES OF HUMAN SAMPLES SO THAT WE CAN HAVE A NICE VOLCANO PLOT, THAT'S WHY WE DIDN'T INCLUDE THAT DATA BECAUSE WE NEED TO HAVE STATISTICALLY SIGNIFICANT NUMBERS OF HUMAN SAMPLES. FINALLY DOES HYDROXY UREA ACT ADS ANTIOXIDANT? WE HAVE IN VITRO DATA THAT INTERACTS WITH SISTINE 93 PROVIDING ANTIOXIDANT EFFECT. HUMAN DATA DEFINITELY SHOWS SOMETHING BUT IT COULD BE THAT IT IS DOING THAT BY VIRTUE OF DIMINISHING HBS AND BRINGING IN HBS SO WE HAVE TO WORK THAT OUT. WITH THAT BEING SAID, I WOULD LIKE TO THANK Y'ALL FOR YOUR ATTENTION AND ACKNOWLEDGE ALL THE PEOPLE I WORK WITH. GREAT GROUP. WE DO A LOT OF WORK TOGETHER AND WE'RE TIGHT KNIT GROUP. SHE'S THE GUY WHO MAKES IT ALL HAPPEN. I WOULD BE GLAD TO TAKE ANY QUESTIONS YOU HAVE. I THANK EVERYBODY ON VIDEOCAST FR LISTENING. WITH THAT, I'M DONE. [APPLAUSE] >> ANY QUESTIONS? (OFF MIC) >> I KNOW. THAT'S -- SO HE WAS MAKING A COMMENT, HE'S MAKING A GOOD COMMENT, THAT IS THAT YOU KNOW IT'S UBIQUITINATED OR UBIQUITINLATED, TOMATO, TOMATO, BUT ANYWAY YOU DON'T KNOWN WHEN IT'S OCCURRING, COULD BE OCCURRING FORMATION OF RETICULOSITES DURING SAY PERIOD WHEN YOU HAVE RED BLOOD CELLS. THAT'S TRUE. (OFF MIC) >> THAT WOULD BE A GOOD PROJECT TO WORK ON TO UNDERSTAND THAT MORE CLEARLY THAT'S ONE OF THE CAVEATS IS THAT THAT IS SOMETHING WE -- WHAT WE DO KNOW IS HBS IS MORE UBIQUITINATED. WHEN IS IT OCCURRING WE DON'T KNOW. BUT THAT IS SOMETHING WE WANT TO LOOK INTO. (OFF MIC) >> WE LOOKED AT SISTAIC ACID, TYROSINE OXIDATION, PHOSPHORYLATION. AND WE LOOKED AT UBIQUITINATION. AND OF COURSE WE LOOKED AT ME THIONEINE OXIDATION, THE CLAY I CAN ISES THE CLASSICS THAT OCCUR. (OFF MIC) >> GLYCOSYLATION IS -- >> YEAH. THAT'S THAT'S WHY JOHN CHIP LA HAS JOB SECURITY. I MEAN THAT WITH RESPECT, I HAVE A LOT OF RESPECT FOR PEOPLE WHO WORK WITH GLYCOSYLATED PROTEIN, YOU HAVE THE DO A NEGATIVE ION MODE. I -- -- I THINK -- I DON'T THINK IT DOES. I THINK THAT IT'S SOMETHING I WOULD ASK HIM ABOUT DOING. I THINK IT COULD BE -- ABSOLUTELY. BECAUSE JERRY HART, HE SPENT TWO DECADES BEING IGNORED AND THEN THEY FOUND CHEMISTRY METHOD FOR BEING ABLE TO STABILIZE AND CONFIRM GLUTE NATION, BUT IT'S DEFINITELY -- AND ANOTHER ONE IS CARBONLATION. THAT RIGHT THERE IS A TRICKY MODIFICATION, I HAVE BEEN WANTING TO SINK MY TEETH INTO THAT. BECAUSE I KNOW THAT THAT IS ONE THING THAT GETS HEAVILY OXIDIZED IN HEMOGLOBIN. I DON'T WANT TOW THIS THINK I'M IGNORING THOSE. THERE'S MORE WORK TO BE DONE. THANKS, GUYS. >> THAT'S A GOOD COMMENT, THAT IS ONE THING, THE ONLY THING I WISH -- I WOULD LOVE TO HAVE AN ANSWER TO THAT.