panoramaProteomicsHuu Hien HuynhHomo Sapienshttps://panoramaweb.org/lcmsIIIprocollagenserum.urlhhuynh89@uw.eduUniversity of WashingtonPanoramaPublicThe amino-terminal propeptide of type III procollagen (P-III-NP) is used with IGF-I to detect the illicit use of growth hormone and to monitor growth hormone therapy. However, the only currently available assays for P-III-NP are immunoassays, which are not well harmonized. In addition, other fragments of type III procollagen may better evaluate collagen turnover. We aimed to develop a high-throughput assay using immunoaffinity enrichment coupled to ultra-high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify peptides belonging to three different regions of type III procollagen in human serum simultaneously. To facilitate higher throughput, we transferred the assay from microcentrifuge tubes to a 96-well plate format with partially automated pipetting. The method was linear (Pearson's R ≥ 0.994) over an estimated concentration range of 1.35-13.3 nM, 0.04-2.28 nM, and 0.26-5.1 nM for each surrogate peptide of P-III-NP, collagen degradation products, and the carboxyl-terminal propeptide, respectively. Intra-day and inter-day imprecision were both < 13.6%, and the results of robustness testing were also encouraging. The method was successfully applied to capillary blood samples obtained using Tasso+ microsampling devices. Modest correlation of P-III-NP concentration was observed between our new method and a WADA-approved immunoassay (N = 40, Pearson's R = 0.789) with a significant bias of -87.8%. Our method simultaneously quantifies four peptides belonging to three regions of type III procollagen in human serum. High bias between assays highlights the need for common higher-order calibrators or reference materials to help improve the comparability of results across laboratories.A Novel High-Throughput Immunoaffinity LC-MS/MS Assay for P-III-NP and Other Fragments of Type III Procollagen in Human Serum.Huynh Huu-Hien HH, Barahona-Carrillo Lili L, Moncrieffe Danielle D, Cowan David A DA, Forrest Katrina K, Becker Jessica O JO, Emrick Michelle A MA, Thomas Andreas A, Thevis Mario M, Eichner Daniel D, Byers Peter H PH, Miller Geoffrey D GD, Hoofnagle Andrew N ANliquid chromatography tandem mass spectroscopy, GH05739, Collagens, human being, determination, Pituitary growth hormone, procollagen aminopeptidase activity, Type III, Somatomedin-C, alpha-Collagen, Procollagen Type III, Somatomedin, Serum, LC-MS-MS, type I/II procollagen N-proteinase activity, Human, Insulin Like Growth Factor I, Insulin-Like Somatomedin Peptide I, Polypeptides, Microfibril, III, peptido, GH14582, Homo sapiens, utilization, Dermodress, Collastat, LC-MSMS, 3, Collagen Type III, Zyderm, Man, IGF1A, Immunoassays, DmelCG2043, treatment, Somatotropins, IGFI, GH-N, Growth Hormone, Insulin-like growth factor I, LCMSMS, lcp3, Man (Taxonomy), peptides, alpha1 Chain, Assays, Microfibril Collagen Hemostat, LCP-3, Blood., Avitene, igf-1, procollagen aminoterminal protease activity, alpha Collagen, aminoterminal procollagen peptidase activity, Type III Collagen, Immunochromatographic Assay, Growth Hormones, CP3, collagen, scientific observation, Collagen Fleece, Collagen alpha1(III), xigf1, Mechano growth factor, disease management, Therapies, C730016P09Rik, peptidos, CG7776, Collagen, growth hormone receptor ligand, Collagenfleece, LC-MS2, Therapy, IGF-I, measuring, Somatomedin C, Immunochromatographic Assays, use, LcpIII, statistics and numerical data, Pituitary Growth Hormone, Modern, Assay, e(Pc), Igf-I, LC-MS/MS, Insulin Like Somatomedin Peptide I, type III procollagen, IBP1, Blood Serum, igf1-A, Recombinant Somatotropin, L[[3]]CP3, Peptide, LC/MS/MS, aminoprocollagen peptidase activity, IGF-1, Growth hormone 1, chemical analysis, IGF-I-SmC, Collagen Hemostat, Somatotropin, numerical data, Recombinant Growth Hormones, LCP3, Igf-1, Growth hormone, Pangen, DmelCG7776, Type III Procollagen, DMLCP3, l(2)28-28-12, other collagen, procollagen N-terminal peptidase activity, anon-35Fa, Avicon, Immunochromatographic, growth hormone, Treatments, Wachstumshormon, human, Recombinant, procollagen N-proteinase activity, DmelCG5861, GH, Recombinant Growth Hormone, Therapeutic, anon-35Fc, liquid chromatography-tandem mass spectroscopy, DmelLcp3, E(PC), BG:DS02740.11, anon-48Ac, MGF, Modern Man, Recombinant Somatotropins, Growth Hormones Pituitary, Collagen Felt, liquid chromatography tandem mass spectrometry, Treatment, Peptid, CG2043, assay, Polypeptide, procollagen N-terminal proteinase activity, Pituitary, growth hormones, Pituitary Growth Hormones, Recombinant Pituitary Growth Hormones, Serumsliquid chromatography tandem mass spectroscopy, LC-MS2, measuring, GH05739, LcpIII, human being, determination, Modern, LC-MS/MS, e(Pc), procollagen aminopeptidase activity, Type III, Procollagen Type III, type III procollagen, Blood Serum, Serum, L[[3]]CP3, LC-MS-MS, type I/II procollagen N-proteinase activity, Human, LC/MS/MS, III, aminoprocollagen peptidase activity, GH14582, Homo sapiens, LC-MSMS, chemical analysis, 3, Collagen Type III, LCP3, Man, DmelCG2043, LCMSMS, DmelCG7776, lcp3, Man (Taxonomy), DMLCP3, Type III Procollagen, l(2)28-28-12, alpha1 Chain, procollagen N-terminal peptidase activity, LCP-3, Blood., anon-35Fa, procollagen aminoterminal protease activity, human, DmelCG5861, aminoterminal procollagen peptidase activity, procollagen N-proteinase activity, Type III Collagen, CP3, anon-35Fc, liquid chromatography-tandem mass spectroscopy, scientific observation, DmelLcp3, E(PC), BG:DS02740.11, anon-48Ac, Collagen alpha1(III), Modern Man, liquid chromatography tandem mass spectrometry, CG7776, assay, CG2043, procollagen N-terminal proteinase activity, Collagen, Serumsliquid chromatography tandem mass spectroscopy, LC-MS2, measuring, GH05739, LcpIII, human being, determination, Modern, LC-MS/MS, e(Pc), procollagen aminopeptidase activity, Type III, Procollagen Type III, type III procollagen, Blood Serum, Serum, L[[3]]CP3, LC-MS-MS, type I/II procollagen N-proteinase activity, Human, LC/MS/MS, III, aminoprocollagen peptidase activity, GH14582, Homo sapiens, LC-MSMS, chemical analysis, 3, Collagen Type III, LCP3, Man, DmelCG2043, LCMSMS, DmelCG7776, lcp3, Man (Taxonomy), DMLCP3, Type III Procollagen, l(2)28-28-12, alpha1 Chain, procollagen N-terminal peptidase activity, LCP-3, Blood., anon-35Fa, procollagen aminoterminal protease activity, human, DmelCG5861, aminoterminal procollagen peptidase activity, procollagen N-proteinase activity, Type III Collagen, CP3, anon-35Fc, liquid chromatography-tandem mass spectroscopy, scientific observation, DmelLcp3, E(PC), BG:DS02740.11, anon-48Ac, Collagen alpha1(III), Modern Man, liquid chromatography tandem mass spectrometry, CG7776, assay, CG2043, procollagen N-terminal proteinase activity, Collagen, Serumsliquid chromatography tandem mass spectroscopy, Scientific Bias, apparatus, Collagens, determination, Medical Devices, Blood, immunoassay, procollagen aminopeptidase activity, Type III, Somatomedin-C, Somatomedin, Serum, type I/II procollagen N-proteinase activity, Insulin Like Growth Factor I, Insulin-Like Somatomedin Peptide I, peptide, Polypeptides, Techniques, devices, peptido, Method, Epidemiologic Biase, 3, Ecological, Zyderm, Immunoassays, Inventory, treatment, Somatotropins, IGFI, GH-N, Ecological Biases, Insulin-like growth factor I, appliances, LCMSMS, Man (Taxonomy), peptides, Assays, Microfibril Collagen Hemostat, aminoterminal procollagen peptidase activity, Immunochromatographic Assay, collagen degradation, collagen, Methodological Studies, Statistical Biases, scientific observation, Collagen Fleece, xigf1, Mechano growth factor, disease management, Therapies, Ecological Bias, C730016P09Rik, CG7776, Systematic, Collagen, growth hormone receptor ligand, Epidemiologic Biases, LC-MS2, Therapy, IGF-I, Somatomedin C, Scientific, statistics and numerical data, Devices, Epidemiologic, Modern, blood in capillary, e(Pc), Igf-I, LC-MS/MS, Insulin Like Somatomedin Peptide I, type III procollagen, IBP1, Ecological Fallacies, Blood Serum, Procedure, igf1-A, L[[3]]CP3, Outcome Measurement Errors, Experimental, IGF-1, Growth hormone 1, IGF-I-SmC, Supplies and Equipment, Somatotropin, numerical data, Truncation Bias, Igf-1, Bias, DmelCG7776, procollagen N-terminal peptidase activity, Instruments and Apparatus, Immunochromatographic, Methodological, Outcome Measurement, Methodological Study, Treatments, Wachstumshormon, human, portion of blood in capillary, Experimental Bias, procollagen N-proteinase activity, GH, Outcome Measurement Error, Recombinant Growth Hormone, liquid chromatography-tandem mass spectroscopy, DmelLcp3, E(PC), BG:DS02740.11, anon-48Ac, Collagen Felt, Equipment, Polypeptide, Recombinant Pituitary Growth Hormones, GH05739, collagen catabolism, human being, Procedures, Pituitary growth hormone, Ecological Fallacy, Truncation, alpha-Collagen, instruments, Systematic Bias, Procollagen Type III, IA, LC-MS-MS, Human, collagen breakdown, method, Microfibril, III, GH14582, Homo sapiens, utilization, Dermodress, Collastat, method used in an experiment, LC-MSMS, Studies, Collagen Type III, portion of capillary blood, Medical, Man, Technique, IGF1A, DmelCG2043, Fallacies, Growth Hormone, lcp3, alpha1 Chain, Aggregation, LCP-3, Avitene, igf-1, Supplies, procollagen aminoterminal protease activity, alpha Collagen, Study, Type III Collagen, Growth Hormones, CP3, Collagen alpha1(III), peptidos, Collagenfleece, Biase, Medical Device, Apparatus and Instruments, measuring, Inventories, Fallacy, Immunochromatographic Assays, use, LcpIII, Laboratory., Pituitary Growth Hormone, Assay, Truncation Biases, Recombinant Somatotropin, Peptide, polypeptide, LC/MS/MS, Device, aminoprocollagen peptidase activity, equipment, chemical analysis, Collagen Hemostat, Errors, Recombinant Growth Hormones, LCP3, Growth hormone, Pangen, Type III Procollagen, DMLCP3, l(2)28-28-12, other collagen, Biases, anon-35Fa, Avicon, growth hormone, Recombinant, plan specification, DmelCG5861, Therapeutic, Error, anon-35Fc, concentration, Aggregation Bias, MGF, Modern Man, Recombinant Somatotropins, Statistical Bias, Growth Hormones Pituitary, liquid chromatography tandem mass spectrometry, Treatment, Peptid, CG2043, assay, procollagen N-terminal proteinase activity, Pituitary, growth hormones, Pituitary Growth Hormones, Statistical, SerumsfalseA Novel High-Throughput Immunoaffinity LC-MS/MS Assay for P III-NP and other Fragments of Type III Procollagen in Human SerumThe amino-terminal propeptide of type III procollagen (P-III-NP) is used with IGF-I to detect the illicit use of growth hormone and to monitor growth hormone therapy. However, the only currently available assays for P-III-NP are immunoassays, which are not well harmonized. In addition, other fragments of type III procollagen may better evaluate collagen turnover. We developed a high-throughput assay using immunoaffinity enrichment coupled to ultra high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify peptides belonging to three different regions of type III procollagen in human serum simultaneously.Mon Oct 28 00:00:00 GMT 2024PXD052245960639462787