panorama0000ProteomicsEric KuhnHomo SapiensRattus NorvegicusMus MusculusMacaca Fascicularishttps://panoramaweb.org/yVRdi1.urlBroad Institute of MIT and Harvardekuhn@broadinstitute.orgPanoramaPublicTherapies currently in preclinical development for prion disease seek to lower prion protein (PrP) expression in the brain. Trials of such therapies are likely to rely on quantification of PrP in cerebrospinal fluid (CSF) as a pharmacodynamic biomarker and possibly as a trial endpoint. Studies using PrP ELISA kits have shown that CSF PrP is lowered in the symptomatic phase of disease, a potential confounder for reading out the effect of PrP-lowering drugs in symptomatic patients. Because misfolding or proteolytic cleavage could potentially render PrP invisible to ELISA even if its concentration were constant or increasing in disease, we sought to establish an orthogonal method for CSF PrP quantification. We developed a multi-species targeted mass spectrometry method based on multiple reaction monitoring (MRM) of nine PrP tryptic peptides quantified relative to an isotopically labeled recombinant protein standard for human samples, or isotopically labeled synthetic peptides for nonhuman species. Analytical validation experiments showed process replicate coefficients of variation below 15%, good dilution linearity and recovery, and suitable performance for both CSF and brain homogenate and across humans as well as preclinical species of interest. In <i>n</i> = 55 CSF samples from individuals referred to prion surveillance centers with rapidly progressive dementia, all six human PrP peptides, spanning the N- and C-terminal domains of PrP, were uniformly reduced in prion disease cases compared with individuals with nonprion diagnoses. Thus, lowered CSF PrP concentration in prion disease is a genuine result of the disease process and not an artifact of ELISA-based measurement. As a result, dose-finding studies for PrP lowering drugs may need to be conducted in presymptomatic at-risk individuals rather than in symptomatic patients. We provide a targeted mass spectrometry-based method suitable for preclinical quantification of CSF PrP as a tool for drug development.Domain-specific Quantification of Prion Protein in Cerebrospinal Fluid by Targeted Mass Spectrometry.Minikel Eric Vallabh EV, Kuhn Eric E, Cocco Alexandra R AR, Vallabh Sonia M SM, Hartigan Christina R CR, Reidenbach Andrew G AG, Safar Jiri G JG, Raymond Gregory J GJ, McCarthy Michael D MD, O'Keefe Rhonda R, Llorens Franc F, Zerr Inga I, Capellari Sabina S, Parchi Piero P, Schreiber Stuart L SL, Carr Steven A SAdmBest1, Disorders, NKEF-B, Surrogate Endpoints, Product, PhrB photolyase activity, Laboratory, Amentias, Natural killer cell-enhancing factor B, growth and development, pityriasis rubra pilaris, Pharmaceutical Development, DmelCG6264, Relative, Thiol-specific antioxidant protein, Polypeptides, Inherited, cerebral spinal fluid, Biological, Method, responsivity, 3, NUP96, Analysis, dementia, epithelium, ASCR, C4BP, C4bp, ABC, Analyses, Torin, Assays, Amentia, Proline-rich protein, Familial, suprasegmental levels of nervous system, Mink, SUPPRESSOR OF AUXIN RESISTANCE 3, PrP-lt-C-gt-, medicine, scientific observation, Homo sapiens disease, PRX2, "prion induced disorder" EXACT [CSP2005:2042-5006], Diagnostic Findings, suprasegmental structures, Viral, anatomical protrusion, Immunosorbent Assays, NKEFB, Inherited Human Transmissible Spongiform Encephalopathies, Human Transmissible Spongiform Encephalopathies, prion, Procedure, Spectrum Analysis, PrP33-35C, Enzyme-Linked Immunosorbent, Progressive dementia, Prediction, Marker, Relative Risks, dipyrimidine photolyase (photosensitive), Prion Protein Disease, NkefB, TU15B, CJD, Target Prediction, Dementia, LAMB2T, Transmissible, End Points, dbest1, Spectrometry, Immunologic, Methodological, Laboratory Marker, Prion-Associated Disorders, Cerebro, cjd, human, GSS, Band-8, TPx-B, progressive, Clinical Finding, umat, "Prion protein disease" EXACT [SNOMEDCT_2005_07_31:193172009], Drug Target Predictions., Prion-Induced, Encephalopathies, Clinical Marker, KURU, GM-CSF, AI325101, protein-containing complex, DUH3, Human, Gene Products, Cerebro Spinal, disease or disorder, prion disease pathway, Technique, Man, Senile Paranoid Dementias, Clinical, VMD2, F23A5.3, AltPrP, BMD, AL022839, Study, drugs, TDX1, prP27-30, Transmissible Dementia, peptidos, PrP27-30, species, prp(c), LAN, TPx, Biologic, Pharmaceuticals, RP50, Transmissible Spongiform, Products, measuring, p27-30, Proteins, Serum Markers, Assay, disorders, Fluids, spinal fluid, encephalon, Spinal Fluid, Immune Marker, Spongiform Encephalopathy, TSA, Spinal Fluids, MS, Fluid, native protein, Surrogate End Point, Transmissible Spongiform Encephalopathy, chemical analysis, condition, Enzyme-Linked Immunosorbent Assays, Mass Spectrum Analyses, Relative Risk, Biologic Markers, ELISA, CD230, PrPSc, RPE, Spongiform, Pharmaceutic Preparations, best, Paranoid Dementias, underdeveloped, "Spongiform Encephalopathy" EXACT [NCI2004_11_17:C27585], Surrogate, Endpoints, postnatal growth, CSF, p. pigmentosa retinae, plan specification, Gene Proteins, Senile Paranoid, LAMNB2, PLF-RP, Prn-p, Prn-i, deoxyribonucleate pyrimidine dimer lyase (photosensitive), Pharmaceutical Products, Prion Disease, BEST, Immune Markers, Biological Markers, Viral Marker, TDPX1, single-organism developmental process, determination, postnatal development, Thioredoxin peroxidase 1, Biochemical, cleavage, Endpoint, "Prion disease pathway" EXACT [NCI2004_11_17:C38853], protein, Serum, pigmented epithelium, Prion-Induced Disorders, B2T, Laboratory Markers, Techniques, peptido, diseases, "prion induced disorder" EXACT [CSP2005:2042-5040], Pharmaceutical Product, ARB, liquor cerebrospinalis, diseases and disorders, synganglion, Encephalopathy Virus, dementia (disease), protein aggregate, Prion, Paranoid Dementia, Mass Spectrum Analysis, human disease, Man (Taxonomy), peptides, hypoplasia, pigmented retina, Tdpx1, ABC14, DNA cyclobutane dipyrimidine photolyase activity, Immune, Markers, Methodological Studies, Pharmaceutical, Viral Markers, PrP, Mink Encephalopathy Virus, TPX1, MCOPCB7, Familial Dementias, MTABC3, TR, Cerebro Spinal Fluid, SIGNS SYMPTOMS, PRE, Senile, PrP<C>, CG6264, Surrogate Endpoint, BIMP2, Sinc, PRIP, Prn, DESC, PRP, 1.11.1.15, Modern, deoxyribonucleic cyclobutane dipyrimidine photolyase activity, Biochemical Markers, Biologic Marker, Multiple Reaction Monitoring, Cerebro Spinal Fluids, Spectroscopy, retinal pigment, PrxII, prp, Diseases, Pharmaceutic, PRXII, retinal pigment layer, dBest1, Senile Paranoid Dementia, Risk, Encephalon, Dementias, Encephalopathy, Colony-stimulating factor, Csfgm, Methodological Study, PSORS2, disease, phr A photolyase activity, CARMA2, DNA-photoreactivating enzyme, Patient, Biochemical Marker, spine, Polypeptide, AA960666, Prion Induced Disorder, the brain, other disease, human being, Procedures, Clinical Markers, "Spongiform encephalopathy (disorder)" EXACT [SNOMEDCT_2005_07_31:230284004], Gene, Spectrum Analyses, Development, Medication, photoreactivating enzyme activity, anon-WO0118547.380, Symptoms and Signs, Cerebrospinal, Surrogate End Points, protrusion, Surrogate Markers, method, Homo sapiens, reduced, method used in an experiment, Studies, Mass, Enzyme-Linked, stratum pigmentosa retinae, tiny, Finding, Immunosorbent Assay, Drug Target Prediction, GMCSF, Mass Spectroscopy, Drugs, Biomarker, reactivity, MOS3, deoxyribocyclobutadipyrimidine pyrimidine-lyase activity, MRM, PRECOCIOUS, BM600, Biological Marker, Transmissible Dementias, Enzyme Linked Immunosorbent Assay, Medication Development, prP33-35C, EBR2, Thioredoxin-dependent peroxide reductase 1, non-neoplastic, prion disease, spongiform encephalopathy, Molgramostin, Immunologic Markers, Clients, Disorder, Cerebral Spinal Fluid, disorder, prion protein disease, biological marker, Preparation, Immunologic Marker, F23A5_3, small, ng/ml, Prion-Induced Disorder, protein complex, EBR2A, PrPc, End Point, Cerebrospinal Fluids, medical condition, Spongiform Encephalopathies, Medications, Client, Peptide, development, Isotope, PSS1, Signs and Symptoms, Transmissible Spongiform Encephalopathies, MODIFIER OF SNC1, prion induced disorder, Sargramostim, Protein, deoxyribonucleic photolyase activity, PrPC, Mass Spectrum, Dbest, Serum Marker, photolyase activity, Plfr, Prion Protein Diseases, "Prion disease (disorder)" EXACT [SNOMEDCT_2005_07_31:20484008], Risks, Surrogate Marker, Protein Gene Products, Drug, Familial Dementia, Computational Prediction of Drug-Target Interactions, Preparations, concentration, Modern Man, Drug Target, Peptid, HEL-S-2a, assay, response, PTX1, growth, Pharmaceutical PreparationMass Spectrum Analysis, Cerebro Spinal Fluid, Mass Spectrum, Analyses, protein complex, Proteins, Fluids, Spectrometry, Gene, Cerebrospinal Fluids, protein, spinal fluid, CSF, Spectrum Analyses, prion, protein-containing complex, Cerebro, cjd, Spectrum Analysis, Mink, Spinal Fluid, Protein Gene Products, Cerebrospinal, Cerebro Spinal Fluids, Spectroscopy, Gene Proteins, Spinal Fluids, MS, Fluid, cerebral spinal fluid, native protein, Protein, prp, Gene Products, Mass, Mass., Mink Encephalopathy Virus, Cerebro Spinal, liquor cerebrospinalis, Analysis, Encephalopathy Virus, prp(c), protein aggregate, Prion, Mass Spectrum Analyses, Mass SpectroscopyMultiple Reaction Monitoring, measuring, development, postnatal development., MRM, single-organism developmental process, determination, scientific observation, chemical analysis, prp, Mink Encephalopathy Virus, postnatal growth, assay, growth and development, Encephalopathy Virus, prp(c), prion, Prion, growth, cjd, MinkBiological Markers, Viral Marker, TDPX1, Disorders, artificial sequence, NKEF-B, Surrogate Endpoints, single-organism developmental process, Product, Laboratory, Amentias, postnatal development, Thioredoxin peroxidase 1, Biochemical, cleavage, Endpoint, Natural killer cell-enhancing factor B, growth and development, "Prion disease pathway" EXACT [NCI2004_11_17:C38853], protein, Serum, pityriasis rubra pilaris, Pharmaceutical Development, Relative, Prion-Induced Disorders, Thiol-specific antioxidant protein, Polypeptides, Inherited, B2T, Laboratory Markers, Techniques, cerebral spinal fluid, peptido, diseases, Biological, Method, "prion induced disorder" EXACT [CSP2005:2042-5040], Pharmaceutical Product, 10.5, liquor cerebrospinalis, diseases and disorders, synganglion, Analysis, Encephalopathy Virus, dementia (disease), protein aggregate, dementia, Prion, Paranoid Dementia, 10.9, ASCR, C4BP, C4bp, Mass Spectrum Analysis, ABC, human disease, F, Man (Taxonomy), peptides, DmelCG2328, Analyses, Torin, Assays, Amentia, hypoplasia, Proline-rich protein, V, Familial, Tdpx1, ABC14, suprasegmental levels of nervous system, Mink, Immune, Markers, PrP-lt-C-gt-, Methodological Studies, medicine, Pharmaceutical, Viral Markers, PrP, Mink Encephalopathy Virus, TPX1, MCOPCB7, Homo sapiens disease, Familial Dementias, PRX2, "prion induced disorder" EXACT [CSP2005:2042-5006], MTABC3, TR, Diagnostic Findings, Cerebro Spinal Fluid, SIGNS SYMPTOMS, Senile, PrP<C>, suprasegmental structures, Viral, anatomical protrusion, Surrogate Endpoint, Immunosorbent Assays, NKEFB, BIMP2, Sinc, PRIP, Prn, DESC, PRP, 1.11.1.15, Modern, Inherited Human Transmissible Spongiform Encephalopathies, Biochemical Markers, Human Transmissible Spongiform Encephalopathies, prion, Procedure, Biologic Marker, Spectrum Analysis, Multiple Reaction Monitoring, Cerebro Spinal Fluids, Spectroscopy, PrP33-35C, Enzyme-Linked Immunosorbent, Progressive dementia, Prediction, PrxII, Marker, Relative Risks, prp, Diseases, VI, Pharmaceutic, Prion Protein Disease, NkefB, PRXII, CJD, Target Prediction, Dementia, Senile Paranoid Dementia, LAMB2T, 20.35, Transmissible, End Points, Risk, Encephalon, Spectrometry, Dementias, Encephalopathy, Immunologic, Methodological, Colony-stimulating factor, Csfgm, Laboratory Marker, Prion-Associated Disorders, Cerebro, Methodological Study, cjd, human, PSORS2, disease, SYNTHETIC CONSTRUCT sequences, CARMA2, GSS, Patient, Band-8, Biochemical Marker, spine, eve2, artificial, TPx-B, Polypeptide, AA960666, progressive, Clinical Finding, umat, humans, "Prion protein disease" EXACT [SNOMEDCT_2005_07_31:193172009], Prion Induced Disorder, CG2328, the brain, 14.10, Drug Target Predictions., other disease, Prion-Induced, human being, Encephalopathies, Procedures, Clinical Markers, "Spongiform encephalopathy (disorder)" EXACT [SNOMEDCT_2005_07_31:230284004], Clinical Marker, KURU, Gene, GM-CSF, AI325101, Spectrum Analyses, Development, Medication, protein-containing complex, DUH3, Symptoms and Signs, Cerebrospinal, Surrogate End Points, Human, protrusion, Surrogate Markers, method, Homo sapiens, reduced, method used in an experiment, Gene Products, Studies, Mass, Cerebro Spinal, Enzyme-Linked, disease or disorder, even, tiny, synthetic genetic interaction (sensu inequality), Finding, Immunosorbent Assay, Drug Target Prediction, prion disease pathway, Technique, Man, GMCSF, Mass Spectroscopy, Senile Paranoid Dementias, Drugs, Biomarker, Clinical, MRM, BM600, Biological Marker, Transmissible Dementias, Enzyme Linked Immunosorbent Assay, Medication Development, synthetic genetic interaction defined by inequality, prP33-35C, AltPrP, EBR2, Thioredoxin-dependent peroxide reductase 1, AL022839, non-neoplastic, Study, prion disease, spongiform encephalopathy, Molgramostin, Immunologic Markers, drugs, Clients, Disorder, TDX1, Cerebral Spinal Fluid, prP27-30, Transmissible Dementia, disorder, peptidos, prion protein disease, biological marker, PrP27-30, species, Preparation, prp(c), LAN, Immunologic Marker, TPx, Biologic, Pharmaceuticals, Transmissible Spongiform, small, Products, Prion-Induced Disorder, protein complex, p27-30, EBR2A, Proteins, Serum Markers, Assay, disorders, Fluids, PrPc, End Point, Cerebrospinal Fluids, artificial gene, spinal fluid, medical condition, Spongiform Encephalopathies, Medications, synthetic DNA, encephalon, Client, Peptide, Spinal Fluid, Immune Marker, Spongiform Encephalopathy, development, TSA, Spinal Fluids, PSS1, Signs and Symptoms, Transmissible Spongiform Encephalopathies, MS, prion induced disorder, Fluid, native protein, Sargramostim, Surrogate End Point, Transmissible Spongiform Encephalopathy, Protein, synthetic, condition, PrPC, Eve, EVE, Enzyme-Linked Immunosorbent Assays, Mass Spectrum Analyses, Relative Risk, Biologic Markers, ELISA, CD230, PrPSc, Mass Spectrum, Serum Marker, Spongiform, Pharmaceutic Preparations, Paranoid Dementias, underdeveloped, "Spongiform Encephalopathy" EXACT [NCI2004_11_17:C27585], Surrogate, Plfr, Prion Protein Diseases, "Prion disease (disorder)" EXACT [SNOMEDCT_2005_07_31:20484008], Endpoints, Risks, postnatal growth, l(2)46Ce, CSF, synthetic constructs, Surrogate Marker, l(2)46Cg, Protein Gene Products, Drug, l(2)46CFj, plan specification, Familial Dementia, Gene Proteins, l(2)46CFh, Computational Prediction of Drug-Target Interactions, Preparations, Senile Paranoid, l(2)46CFp, concentration, Modern Man, LAMNB2, Drug Target, PLF-RP, Prn-p, Peptid, HEL-S-2a, Prn-i, PTX1, Pharmaceutical Products, E(eve), growth, Prion Disease, l(2)46CFg, Pharmaceutical Preparation, Immune Markers0falsePrion MRM assay development and clinical studyTherapies currently in preclinical development for prion disease seek to lower prion protein (PrP) expression in the brain. Trials of such therapies are likely to rely on quantification of PrP in cerebrospinal fluid (CSF) as a pharmacodynamic biomarker and possibly as a trial endpoint. Studies using PrP ELISA kits have shown that CSF PrP is lowered in the symptomatic phase of disease, a potential confounder for reading out the effect of PrP-lowering drugs in symptomatic patients. Because misfolding or proteolytic cleavage could potentially render PrP undetectable by ELISA, we sought to establish an orthogonal method for CSF PrP quantification. We developed a multi-species targeted mass spectrometry method based on multiple reaction monitoring (MRM) of nine PrP tryptic peptides quantified by stable isotope dilution analysis. Analytical validation experiments showed intra-day and inter-day assay reproducibility coefficients of variation less than 15% (below the best practices threshold), 3 orders of dynamic linear range that encompass the entire expected range of PrP concentration in CSF, lower limits of detection near 10 ng/mL and a similar recovery response from both CSF and brain homogenate matrices. Critical for preclinical assay development, comparable assay performance was found for peptides unique to 4 species. In N=55 CSF samples from individuals referred to prion surveillance centers with rapidly progressive dementia, all six human PrP peptides, spanning the N- and C-terminal domains of PrP, were uniformly reduced in prion disease cases compared to individuals with non-prion diagnoses. Thus, lowered CSF PrP concentration in prion disease is a genuine result of the disease process and not merely an artifact of ELISA-based measurement. As a result, dose-finding studies for PrP lowering drugs may need to be conducted in pre-symptomatic at-risk individuals rather than in symptomatic patients. We provide a targeted mass spectrometry-based method suitable for preclinical and clinical quantification of CSF PrP as a tool for drug development.Tue Nov 19 00:00:00 GMT 2019PXD01478110090954196061011631558565