panorama0000ProteomicsHelian VunkHomo Sapienshttps://panoramaweb.org/orthogonal_dmd.urlhelian.vunk@scilifelab.seScience for Life Laboratory, KTH - Royal Institute of Technology, Stockholm, SwedenPanoramaPublic<h4>Background</h4>Molecular components in blood, such as proteins, are used as biomarkers to detect or predict disease states, guide clinical interventions and aid in the development of therapies. While multiplexing proteomics methods promote discovery of such biomarkers, their translation to clinical use is difficult due to the lack of substantial evidence regarding their reliability as quantifiable indicators of disease state or outcome. To overcome this challenge, a novel orthogonal strategy was developed and used to assess the reliability of biomarkers and analytically corroborate already identified serum biomarkers for Duchenne muscular dystrophy (DMD). DMD is a monogenic incurable disease characterized by progressive muscle damage that currently lacks reliable and specific disease monitoring tools.<h4>Methods</h4>Two technological platforms are used to detect and quantify the biomarkers in 72 longitudinally collected serum samples from DMD patients at 3 to 5 timepoints. Quantification of the biomarkers is achieved by detection of the same biomarker fragment either through interaction with validated antibodies in immuno-assays or through quantification of peptides by Parallel Reaction Monitoring Mass Spectrometry assay (PRM-MS).<h4>Results</h4>Five, out of ten biomarkers previously identified by affinity-based proteomics methods, were confirmed to be associated with DMD using the mass spectrometry-based method. Two biomarkers, carbonic anhydrase III and lactate dehydrogenase B, were quantified with two independent methods, sandwich immunoassays and PRM-MS, with Pearson correlations of 0.92 and 0.946 respectively. The median concentrations of CA3 and LDHB in DMD patients was elevated in comparison to those in healthy individuals by 35- and 3-fold, respectively. Levels of CA3 vary between 10.26 and 0.36 ng/ml in DMD patients whereas those of LDHB vary between 15.1 and 0.8 ng/ml.<h4>Conclusions</h4>These results demonstrate that orthogonal assays can be used to assess the analytical reliability of biomarker quantification assays, providing a means to facilitate the translation of biomarkers to clinical practice. This strategy also warrants the development of the most relevant biomarkers, markers that can be reliably quantified with different proteomics methods.Orthogonal proteomics methods warrant the development of Duchenne muscular dystrophy biomarkers.Johansson Camilla C, Hunt Helian H, Signorelli Mirko M, Edfors Fredrik F, Hober Andreas A, Svensson Anne-Sophie AS, Tegel Hanna H, Forstström Björn B, Aartsma-Rus Annemieke A, Niks Erik E, Spitali Pietro P, Uhlén Mathias M, Szigyarto Cristina Al-Khalili CAprotein translation, FBgn0003149, Carbonic Anhydrase, CA3 field of the hippocampus, Carbonate dehydratase III, L-Lactic Acid, Biological Markers, Viral Marker, Surrogate Endpoints, single-organism developmental process, Laboratory, Ass-1, Blood, postnatal development, DXS206, Biochemical, muscular dystrophy, 2-Hydroxy-, Endpoint, D-Lactic Acid, growth and development, DMD - Duchenne muscular dystrophy, Childhood Muscular Dystrophy, Serum, Becker Muscular Dystrophy, Anhydrase, MAGE-E1 antigen, Duchenne and Becker Muscular Dystrophy, Pseudohypertrophic Muscular, Polypeptides, Laboratory Markers, Donor Artificial Insemination, Techniques, DSmurf, Para, Muscle-Specific Carbonic Anhydrase, peptido, diseases, Biological, Method, CDA2, AA408052, Muscle-Specific, fold, diseases and disorders, Analysis, TRG-5, CG5939, methodology., cornu Ammonis 3, Donor Artificial, field CA3, Mass Spectrum Analysis, human disease, Muscle Tissues, Ldh2, peptides, Analyses, 2-Hydroxypropionic Acid, Becker Type, Tissue, DXSmh7, Duchenne muscular dystrophy, procedures, DXSmh9, elevated, L Lactic Acid, LDHBD, Duchenne musc. dyst., ASS, reaction, Immune, Markers, Methodological Studies, HCA1, Smurf, Viral Markers, 3B, DXS239, Homo sapiens disease, DXS230, CG4943, D-smurf, dys, Sarcolactic Acid, protein anabolism, Cardiomyopathy, Viral, protein biosynthetic process, Surrogate Endpoint, Dilated, DmelCG5939, Biochemical Markers, Procedure, Lack, Blood Serum, antibodies, Biologic Marker, Spectrum Analysis, results, Spectroscopy, Dp71, prm, Marker, DAMAGE, Hepatocellular carcinoma-associated protein 1, Diseases, protein formation, Heterologous Insemination, l(3)S010605, median, Childhood Pseudohypertrophic Muscular Dystrophy, D Lactic Acid, Dp427, Pseudohypertrophic Muscular Dystrophy, Becker's, CA-III, X-Linked, H-Ldh, End Points, DmelCG4943, Severe dystrophinopathy, 0106/05, PM/mPM, 2-Hydroxypropanoic Acid, Spectrometry, b-lactate, Immunologic, Methodological, DXS142, Laboratory Marker, Pseudohypertrophic Childhood Muscular Dystrophy, DMD, Methodological Study, field CA3 of hippocampus, CMD3B, DXS268, DXS269, muscle element, Car3, disease, Artificial Insemination, DXS270, DXS272, protein synthesis, Patient, Biochemical Marker, Duchenne Type Progressive Muscular Dystrophy, regio hippocampi proprii III, Carbonic anhydrase III, Polypeptide, Duchenne-Becker, mMage-e1, Duchenne-Becker Muscular Dystrophy, Ammonium, other disease, Procedures, Clinical Markers, Peptidomics, Clinical Marker, dSmurf1, 2 Hydroxypropionic Acid, Gene, DXS164, Spectrum Analyses, beta-lactate, Duchenne-Type Progressive Muscular Dystrophy, AID, Duchenne Muscular Dystrophy, Aid, Propanoic Acid, Surrogate End Points, Surrogate Markers, method, (2S)-, Ldh-2, mass spectrometry assay, region CA3, mPM, method used in an experiment, AI790582, Gene Products, Studies, Mass, disease or disorder, Pseudohypertrophic, Technique, Becker's Muscular Dystrophy, aid, Mass Spectroscopy, Insemination, Biomarker, Clinical, MeCH(OH)CO2 anion, Tissues, Biological Marker, Muscular Dystrophy Pseudohypertrophic Progressive, Ammonium Lactate, CA3 field of the Ammon horn, Duchenne Becker Muscular Dystrophy, BMD, AI847422, Muscular Dystrophy, BB219044, non-neoplastic, Study, Duchenne type, Immunologic Markers, d-smurf, HEL-S-281, HEL-S-284, Clients, musculus, disorder, HIGM2, peptidos, Ca3, Muscle-Specific Carbonic, 2-hydroxypropionate, Immunologic Marker, high elevation, Duchenne Type, Biologic, Heterologous, regio III cornus ammonis, Lactate, 2 Hydroxypropanoic Acid, Car-3, ng/ml, whole blood, Progressive Muscular Dystrophy, Proteins, Serum Markers, Alpha-dystrobrevin-associated MAGE Protein, disorders, End Point, ion(1-), CA3, medical condition, Dsmurf, Muscle, Duchenne and Becker Types, Client, Peptide, Immune Marker, Human Donor, development, dSmurf, Becker, MS, Muscle Specific, 4.2.1.1, Surrogate End Point, Dystrophies, Smurf ubiquitin ligase, Pseudohypertrophic Progressive, Protein, condition, protein biosynthesis, Donor, background, techniques, Mass Spectrum Analyses, Biologic Markers, Artificial, mdx, Mass Spectrum, Serum Marker, LDH-H, pke, RGD1560259, LDH-B, Surrogate, Arp2, ARP2, Endpoints, postnatal growth, Childhood, muscle, pseudohypertrophic progressive, Surrogate Marker, introduction, Protein Gene Products, plan specification, Gene Proteins, (2R)-, l(3)10631, Duchenne muscular dystrophy (disorder), 2-hydroxypropanoic acid, MRX85, Peptid, growth, Muscle Tissue, Serums, pm, PM, methodology, Immune Markers, Ammon's horn (Lorente de Ns)Biological Markers, Viral Marker, Surrogate Endpoints, Procedures, single-organism developmental process, Peptidomics, Clinical Markers, Laboratory, Clinical Marker, postnatal development, muscular dystrophy, Biochemical, Endpoint, growth and development, DMD - Duchenne muscular dystrophy, Childhood Muscular Dystrophy, Serum, Duchenne-Type Progressive Muscular Dystrophy, Becker Muscular Dystrophy, Duchenne Muscular Dystrophy, Surrogate End Points, Duchenne and Becker Muscular Dystrophy, Pseudohypertrophic Muscular, Surrogate Markers, Techniques, Laboratory Markers, Method, Biological, Studies, Pseudohypertrophic, Technique, Becker's Muscular Dystrophy, Biomarker, Clinical, Becker Type, Biological Marker, Muscular Dystrophy Pseudohypertrophic Progressive, Duchenne muscular dystrophy, procedures, Duchenne Becker Muscular Dystrophy, Muscular Dystrophy, Study, Duchenne musc. dyst., Duchenne type, Immunologic Markers, Immune, Markers, Methodological Studies, Viral Markers, 3B, Immunologic Marker, Duchenne Type, Biologic, Cardiomyopathy, Viral, Surrogate Endpoint, Immune Markers., Dilated, Progressive Muscular Dystrophy, Serum Markers, End Point, Biochemical Markers, Procedure, Biologic Marker, Duchenne and Becker Types, Immune Marker, development, Becker, Dystrophies, Marker, Surrogate End Point, Pseudohypertrophic Progressive, techniques, Childhood Pseudohypertrophic Muscular Dystrophy, Pseudohypertrophic Muscular Dystrophy, Biologic Markers, Becker's, X-Linked, Serum Marker, End Points, Surrogate, Endpoints, postnatal growth, Methodological, Childhood, Immunologic, pseudohypertrophic progressive, Laboratory Marker, Pseudohypertrophic Childhood Muscular Dystrophy, DMD, Methodological Study, Surrogate Marker, Duchenne muscular dystrophy (disorder), Biochemical Marker, Duchenne Type Progressive Muscular Dystrophy, Duchenne-Becker, growth, Duchenne-Becker Muscular Dystrophy, methodologyBiological Markers, Viral Marker, Surrogate Endpoints, Procedures, single-organism developmental process, Peptidomics, Clinical Markers, Laboratory, Clinical Marker, postnatal development, muscular dystrophy, Biochemical, Endpoint, growth and development, DMD - Duchenne muscular dystrophy, Childhood Muscular Dystrophy, Serum, Duchenne-Type Progressive Muscular Dystrophy, Becker Muscular Dystrophy, Duchenne Muscular Dystrophy, Surrogate End Points, Duchenne and Becker Muscular Dystrophy, Pseudohypertrophic Muscular, Surrogate Markers, Techniques, Laboratory Markers, Method, Biological, Studies, Pseudohypertrophic, Technique, Becker's Muscular Dystrophy, Biomarker, Clinical, Becker Type, Biological Marker, Muscular Dystrophy Pseudohypertrophic Progressive, Duchenne muscular dystrophy, procedures, Duchenne Becker Muscular Dystrophy, Muscular Dystrophy, Study, Duchenne musc. dyst., Duchenne type, Immunologic Markers, Immune, Markers, Methodological Studies, Viral Markers, 3B, Immunologic Marker, Duchenne Type, Biologic, Cardiomyopathy, Viral, Surrogate Endpoint, Immune Markers., Dilated, Progressive Muscular Dystrophy, Serum Markers, End Point, Biochemical Markers, Procedure, Biologic Marker, Duchenne and Becker Types, Immune Marker, development, Becker, Dystrophies, Marker, Surrogate End Point, Pseudohypertrophic Progressive, techniques, Childhood Pseudohypertrophic Muscular Dystrophy, Pseudohypertrophic Muscular Dystrophy, Biologic Markers, Becker's, X-Linked, Serum Marker, End Points, Surrogate, Endpoints, postnatal growth, Methodological, Childhood, Immunologic, pseudohypertrophic progressive, Laboratory Marker, Pseudohypertrophic Childhood Muscular Dystrophy, DMD, Methodological Study, Surrogate Marker, Duchenne muscular dystrophy (disorder), Biochemical Marker, Duchenne Type Progressive Muscular Dystrophy, Duchenne-Becker, growth, Duchenne-Becker Muscular Dystrophy, methodologyprotein translation, FBgn0003149, Carbonic Anhydrase, CA3 field of the hippocampus, Carbonate dehydratase III, L-Lactic Acid, Biological Markers, Viral Marker, Surrogate Endpoints, single-organism developmental process, Laboratory, Ass-1, Blood, postnatal development, DXS206, Biochemical, muscular dystrophy, 2-Hydroxy-, Endpoint, D-Lactic Acid, growth and development, DMD - Duchenne muscular dystrophy, Childhood Muscular Dystrophy, Serum, Becker Muscular Dystrophy, Anhydrase, MAGE-E1 antigen, Duchenne and Becker Muscular Dystrophy, Pseudohypertrophic Muscular, Polypeptides, Laboratory Markers, Donor Artificial Insemination, Techniques, DSmurf, Para, Muscle-Specific Carbonic Anhydrase, peptido, diseases, Biological, Method, CDA2, AA408052, Muscle-Specific, fold, diseases and disorders, Analysis, TRG-5, CG5939, methodology., cornu Ammonis 3, Donor Artificial, field CA3, Mass Spectrum Analysis, human disease, Muscle Tissues, Ldh2, peptides, Analyses, 2-Hydroxypropionic Acid, Becker Type, Tissue, DXSmh7, Duchenne muscular dystrophy, procedures, DXSmh9, elevated, L Lactic Acid, LDHBD, Duchenne musc. dyst., ASS, reaction, Immune, Markers, Methodological Studies, HCA1, Smurf, Viral Markers, 3B, DXS239, Homo sapiens disease, DXS230, CG4943, D-smurf, dys, Sarcolactic Acid, protein anabolism, Cardiomyopathy, Viral, protein biosynthetic process, Surrogate Endpoint, Dilated, DmelCG5939, Biochemical Markers, Procedure, Lack, Blood Serum, antibodies, Biologic Marker, Spectrum Analysis, results, Spectroscopy, Dp71, prm, Marker, DAMAGE, Hepatocellular carcinoma-associated protein 1, Diseases, protein formation, Heterologous Insemination, l(3)S010605, median, Childhood Pseudohypertrophic Muscular Dystrophy, D Lactic Acid, Dp427, Pseudohypertrophic Muscular Dystrophy, Becker's, CA-III, X-Linked, H-Ldh, End Points, DmelCG4943, Severe dystrophinopathy, 0106/05, PM/mPM, 2-Hydroxypropanoic Acid, Spectrometry, b-lactate, Immunologic, Methodological, DXS142, Laboratory Marker, Pseudohypertrophic Childhood Muscular Dystrophy, DMD, Methodological Study, field CA3 of hippocampus, CMD3B, DXS268, DXS269, muscle element, Car3, disease, Artificial Insemination, DXS270, DXS272, protein synthesis, Patient, Biochemical Marker, Duchenne Type Progressive Muscular Dystrophy, regio hippocampi proprii III, Carbonic anhydrase III, Polypeptide, Duchenne-Becker, mMage-e1, Duchenne-Becker Muscular Dystrophy, Ammonium, other disease, Procedures, Clinical Markers, Peptidomics, Clinical Marker, dSmurf1, 2 Hydroxypropionic Acid, Gene, DXS164, Spectrum Analyses, beta-lactate, Duchenne-Type Progressive Muscular Dystrophy, AID, Duchenne Muscular Dystrophy, Aid, Propanoic Acid, Surrogate End Points, Surrogate Markers, method, (2S)-, Ldh-2, mass spectrometry assay, region CA3, mPM, method used in an experiment, AI790582, Gene Products, Studies, Mass, disease or disorder, Pseudohypertrophic, Technique, Becker's Muscular Dystrophy, aid, Mass Spectroscopy, Insemination, Biomarker, Clinical, MeCH(OH)CO2 anion, Tissues, Biological Marker, Muscular Dystrophy Pseudohypertrophic Progressive, Ammonium Lactate, CA3 field of the Ammon horn, Duchenne Becker Muscular Dystrophy, BMD, AI847422, Muscular Dystrophy, BB219044, non-neoplastic, Study, Duchenne type, Immunologic Markers, d-smurf, HEL-S-281, HEL-S-284, Clients, musculus, disorder, HIGM2, peptidos, Ca3, Muscle-Specific Carbonic, 2-hydroxypropionate, Immunologic Marker, high elevation, Duchenne Type, Biologic, Heterologous, regio III cornus ammonis, Lactate, 2 Hydroxypropanoic Acid, Car-3, ng/ml, whole blood, Progressive Muscular Dystrophy, Proteins, Serum Markers, Alpha-dystrobrevin-associated MAGE Protein, disorders, End Point, ion(1-), CA3, medical condition, Dsmurf, Muscle, Duchenne and Becker Types, Client, Peptide, Immune Marker, Human Donor, development, dSmurf, Becker, MS, Muscle Specific, 4.2.1.1, Surrogate End Point, Dystrophies, Smurf ubiquitin ligase, Pseudohypertrophic Progressive, Protein, condition, protein biosynthesis, Donor, background, techniques, Mass Spectrum Analyses, Biologic Markers, Artificial, mdx, Mass Spectrum, Serum Marker, LDH-H, pke, RGD1560259, LDH-B, Surrogate, Arp2, ARP2, Endpoints, postnatal growth, Childhood, muscle, pseudohypertrophic progressive, Surrogate Marker, introduction, Protein Gene Products, plan specification, Gene Proteins, (2R)-, l(3)10631, Duchenne muscular dystrophy (disorder), 2-hydroxypropanoic acid, MRX85, Peptid, growth, Muscle Tissue, Serums, pm, PM, methodology, Immune Markers, Ammon's horn (Lorente de Ns)0falseOrthogonal proteomics methods warrants development of Duchenne Muscular Dystrophy biomarkersBackground: Molecular components in blood, such as proteins, are used as biomarkers to detect or predict disease states, guide clinical interventions and aid in the development of therapies. While multiplexing proteomics methods promote discovery of such biomarkers, their translation to clinical use is difficult due to the lack of substantial evidence regarding their reliability as quantifiable indicators of disease state or outcome. To overcome this challenge, a novel orthogonal strategy was developed and used to assess the reliability of biomarkers and analytically corroborate already identified serum biomarkers for Duchenne muscular dystrophy (DMD). DMD is a monogenic incurable disease characterized by progressive muscle damage that currently lacks reliable and specific disease monitoring tools. Methods: Two technological platforms are used to detect and quantify the biomarkers in 72 longitudinally collected serum samples from DMD patients at 3 to 5 timepoints. Quantification of the biomarkers is achieved by detection of the same biomarker fragment either through interaction with validated antibodies in immuno-assays or through quantification of peptides by Parallel Reaction Monitoring Mass Spectrometry assay (PRM-MS). Results Five, out of ten biomarkers previously identified by affinity-based proteomics methods, were confirmed to be associated with DMD using the mass spectrometry-based method. Two biomarkers, carbonic anhydrase III and lactate dehydrogenase B, were quantified with two independent methods, sandwich immunoassays and PRM-MS, with Pearson correlations of 0.92 and 0.946 respectively. The median concentrations of CA3 and LDHB in DMD patients was elevated in comparison to those in healthy individuals by 35- and 3-fold, respectively. Levels of CA3 vary between 10.26 and 0.36 ng/ml in DMD patients whereas those of LDHB vary between 15.1 and 0.8 ng/ml. Conclusions: These results demonstrate that orthogonal assays can be used to assess the analytical reliability of biomarker quantification assays, providing a means to facilitate the translation of biomarkers to clinical practice. This strategy also warrants the development of the most relevant biomarkers, markers that can be reliably quantified with different proteomics methods.Tue Jun 13 00:00:00 GMT+01:00 2023PXD038089960637308827