panoramaProteomicsJess BeckerHomo Sapienshttps://panoramaweb.org/TaMADOR_Glucagon.urlbecker78@uw.eduUniversity of Washington, Department of Laboratory Medicine and PathologyPanoramaPublic<h4>Introduction</h4>Glucagon and oxyntomodulin are peptide hormones differentially released from proglucagon that function in regulating blood glucose. Their overlapping amino acid sequences make the development of specific immunoassays difficult, but the specificity of liquid chromatography-tandem mass spectrometry can be used to distinguish the peptides. We aimed to develop a sensitive and specific mass spectrometric assay that uses non-proprietary reagents and normal-flow liquid chromatography in the simultaneous quantification of both analytes.<h4>Methods</h4>Bulk plasma proteins were precipitated in ethanol/ammonium hydroxide. Analytes were enriched using monoclonal antibodies generated in-house and analyzed using liquid chromatography-tandem mass spectrometry. A glucagon calibration material was sourced commercially and characterized for purity and concentration by high-performance liquid chromatography-ultraviolet detection and amino acid analysis. Single-point calibration was used to minimize between-day variability.<h4>Results</h4>The novel antibodies performed acceptably (peptide recovery 45-59 %). The assay was precise (<13 %CV) and linear over the range of 1.3-14.7 pM and 1.1-13.7 pM for glucagon and oxyntomodulin, respectively. The glucagon calibration material concentration was determined to be 1.596 mg/g. Tube-type studies supported the use of protease inhibitor tubes at the time of blood draw. Patients with type 1 diabetes had lower concentrations of glucagon when maintained on an insulin pump, but not with injectable insulin.<h4>Conclusion</h4>We have validated a method with a highly detailed standard operating procedure. We have characterized calibration materials to help maintain accuracy and achieve between-day and between-laboratory harmonization. The assay will be beneficial in better understanding α-cell health and glycemic control in diabetes and other diseases.Quantification of glucagon and oxyntomodulin by protein precipitation-immunoaffinity enrichment-LC-MS/MS.Becker Jessica O JO, Shijo Sara K SK, Huynh Huu-Hien HH, Forrest Katrina L KL, MacCoss Michael J MJ, Emrick Michelle A MA, Goonatilleke Elisha E, Hoofnagle Andrew N ANSoluble, GRPP, Laboratory, Insulin Dependent Diabetes Mellitus 1, PNT-P1, Blood Glucose Control, L-histidyl-L-seryl-L-glutaminylglycyl-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl-L-alpha-aspartyl-L-tyrosyl-L-seryl-L-lysyl-L-tyrosyl-L-leucyl-L-alpha-aspartyl-L-seryl-L-arginyl-L-arginyl-L-alanyl-L-glutaminyl-L-alpha-aspartyl-L-phenylalanyl-L-valyl-L-glutaminyl-L-tryptophyl-L-leucyl-L-methionyl-L-asparaginyl-L-threonine, A4, insuline humaine, growth and development, EY3-1, Long Term, dmTAF[[II]]230, Glucagon Precursor, Polypeptides, Method, Oxyntomodulin, Protease Inhibitor, High Performance Liquid Chromatography, 1, insulin dependent diabetes, D-ets-2, 3520, blood specimen collection, with unspecified complication, Sudden Onset, post-pharynx, notch, WMS, Immunoassays, Juvenile Onset Diabetes, average, gamma sarcoglycan, Grain, amino acids, TFIID TAF250, cel, polypeptide hormone, Assays, Sudden-Onset Diabetes, procedures, Ax, Structures, SURGICAL AND MEDICAL PROCEDURES, HPLC, allergic reaction, Dehydrated ethanol, Peptidohydrolase Inhibitor, Diabetes mellitus, scientific observation, anatomical tube, co, Chromatography, Homo sapiens disease, Blood Protein, High Speed Liquid, juvenile diabetes, NECD, Type 1 Diabetes Mellitus, SGS, l(1)N, db, spiritus vini, LC-MS2, insulinum humanum, Glucagon, dTAF[[II]]230, Regular, DM, Chp, Hormones, His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr, Co, Monoclonal Antibody, Liquid Chromatography, Longterm Effect, TAF200, GLP-1(7-37), Preproglucagons, Normalcy, Procedure, antibodies, Glucagon Precursors, ACMICD, Absolute Alcohol, Regular Insulin, OB-RGRP, Juvenile-Onset, insulin (recombinant), matrix metalloproteinase pump 1, Monoclonal Antibodies, shd, Insulin-dependent diabetes, Hydrolase Inhibitor, numerical data, sarcoglycan, GLP-1(7-36), Soluble Insulin, Glicentin-related polypeptide, [OEtH], fa, Peptide Hormone, Sensor, type I diabetes mellitus, Blood Sugar, Acid, Autoimmune Diabetes, pntP2, Pointed-P1, Antagonists, matrix metalloproteinase 7 activity, Ets94F, insulina humana, Diabetes mellitus type I [insulin dependent type] [IDDM] [juvenile type], blood harvesting, Methodological, alcohol etilico, Methylcarbinol, Obr, clone 1.12, Taf250, liquid chromatography-tandem mass spectroscopy, 35kD dystrophin-associated glycoprotein, duct, Indicator, PUMP-1 proteinase activity, Acids, Endopeptidase, Peptide Peptidohydrolase Inhibitor, antlia, Hyperglycemic-Glycogenolytic Factor, obl, TAF230, Amino Acid Sequences, 0998/12, Juvenile diabetes, Glucose, Effects, Endopeptidase Inhibitors, etanol, glucagonum, Serum Protein, putative metalloproteinase activity, Normalcies, Hormone, Autoimmune Diabete, DMPOINT1A, sensitive, Insulin, LC-MSMS, Diabetes mellitus (disorder), disease or disorder, Peptide Hydrolase, MAM, gamma-SG, Technique, l(1)Ax, Normalities, Chain, Insulin-Dependent Diabetes Mellitus, dTAF[[II]]250, pointed-RC, 2310061K05Rik, Longterm, interventionDescription, cell, ethanol, obese-like, Long-Term, Antagonist, Structure, High-Performance Liquid Chromatographies, EK3-2, Study, Injectable, Plasma Protein, l(3)07825, dTAF250, C2H5OH, His-ser-glu(nh2)-gly-thr-phe-thr-ser-asp-tyr-ser-lys-tyr-leu-asp-ser-arg-arg-ala-glu(NH2)-asp-phe-val-glu(NH2)-trp-leu-met-asp(NH2)-thr, Insulin recombinant, Protease Antagonists, Diabetes Mellitus, Proglucagon (33-61), putative (or punctuated) metalloproteinase-1 activity, Type 1 Diabetes, Peptide Peptidohydrolase Inhibitors, peptidos, CG17077, UPLC, Hydroxide, CT13012, 16-178, measuring, Individual Health, 16-55, Immunochromatographic Assays, Ets, use, T1D, Diabete, 35 kDa dystrophin-associated glycoprotein, pumping organ, Assay, Proteins, disorders, function, Primary, anon-EST:Liang-1.12, BG:DS00004.13, Cell, SGCG, dTAF230, Sequences, polypeptide, adult pharynx, Preproglucagon, chemical analysis, spl, Long Term Effects, Postpharynx, Insulin A Chain, alcool ethylique, TAF[[II]]250/230, condition, MFS1, 1.1, Sodium, Novolin, nd, Protein Structures, pnt-P1, pnt-P2, dJ142L7.2, Mellitus, Intervention, Incretin hormone, Taf[[II]]250, LEPROT, diabetes mellitis type I, DMDA1, postnatal growth, Specificity, Peptidohydrolase Inhibitors, Understanding, diabetes mellitis type 1, HG-Factor, Sugar, Longterm Effects, Ketosis-Prone, plan specification, type 1 diabetes, Peptidase, High Pressure, Antiprotease, Calibration, Pharynx, liquid chromatography tandem mass spectrometry, Peptide Hydrolase Inhibitors, POINT, CG8705, Insulin Dependent, liquid chromatography tandem mass spectroscopy, Modb1, MGC130048, Diabetes Type 1, Insulin B Chain, Endopeptidase Inhibitor, Grain Alcohol, single-organism developmental process, determination, Blood, anterior pharynx, postnatal development, Aminosaeure, DmelCG17077, Glicentin (33-69), Hyperglycemic Glycogenolytic Factor, Peptide Hydrolase Inhibitor, Pro Glucagon, Pnt, Serum, Model Calibration, Insulin Dependent Diabetes, 1-hydroxyethane, alcohol, peptide, Techniques, Ethanol, Insulin dependent diabetes mellitus, Readability, Blood sugar, peptido, png, diseases, human insulin, Alkohol, Juvenile onset diabetes mellitus, Inhibitors, diseases and disorders, not stated as uncontrolled, Hydrolase Inhibitors, Leprb, Effect, Autoimmune, Injectables, DM - Diabetes mellitus, Pnt-P1, insulin, human disease, LCMSMS, peptides, Pumporgan, metalloproteinase pump-1, PUMP, Ethyl, Dietary Fiber, Aethanol, [CH2Me(OH)], pump, Immunochromatographic Assay, Proglucagon (33-69), insulin human, High-Performance, Methodological Studies, gamma-sarcoglycan, High-Performance Liquid, Reagents, GPHYSD2, Long-Term Effects, EG:140G11.1, matrin activity, swb, Primary Protein Structure, Tube, Juvenile Onset, Alcohol, Peptidase Inhibitor, statistics and numerical data, Aminokarbonsaeure, LC-MS/MS, SG-gamma, Brittle, D-Ets-2, uncontrolled, ets94F, 0123/09, Ethyl alcohol, TAFII-250, Peptide Peptidohydrolase, TAF250/230, PUMP-1 activity, TUBE, Diabetes mellitus type I, TAFII250, MMP 7, Diseases, MMP, NOS, Glucagon (1-29), immune mediated diabetes, laboratory, Individual, ETHANOL, Amino Acid, Sudden-Onset, IDDM, EtOH, posterior pharynx, IDDM - Insulin-dependent diabetes mellitus, Glicentin, Diabetes mellitus type 1, Sudden-Onset Diabetes Mellitus, Control, Immunochromatographic, Glucose Control, MASS, Juvenile Diabetes, gamma (35kDa dystrophin-associated glycoprotein), CG17603, Methodological Study, TAF[[II]], Glucagon-like peptide 2, Glucagon-like peptide 1, Type 1, Diabetes NOS, ptd, GLP-1, PntP2, disease, Health, DMDA, Peptidase Inhibitors, Blood Glucose, Specificity and Sensitivity, Patient, SR3-5, Sensor Calibration, PntP1, E(E2F)3D, Antiproteases, Sensor Calibrations, Liquid, Insulin-Dependent Diabetes Mellitus 1, medical condition., PNTP2, Polypeptide, HSQGTFTSDYSKYLDSRRAQDFVQWLMNT, Polypeptide Hormone, Polypeptide Hormones, n[fah], Inhibitor, PNTP1, GLP-2, Juvenile-Onset Diabetes Mellitus, DMI UNSPF NT ST UNCNTRLD, Ammonium, Ketosis-Prone Diabetes Mellitus, other disease, d230, Glucagon-like peptide 1(7-37), SGCG_HUMAN, Procedures, Type I Diabetes, Reagent, ETS2, Ets2, Aminocarbonsaeure, EG:163A10.2, FBN, Protease, dTAFII250, Diabetes mellitus type 1 (disorder), Insulin B, DIABETES MELLITUS TYPE 01, Praepharynx, EfW1, TYPE, LC-MS-MS, Intervention or Procedure, DAGA4, Aethylalkohol, method, Antibody, pntegfr, 0608/07, utilization, dmTAF1, Glucagon-like peptide 1(7-36), ECTOL1, Injection, Taf230, Glukagon, Insulin-Dependent, method used in an experiment, 35DAG, Studies, Protease Antagonist, l(1)3Cb, Type I, glucagon, SCG3, sensitivity, Reagents and Indicators, TAF250, Taf200, Absolute, DmelCG3936, Type I diabetes, Interventional, Taf1p, Serum Proteins, dNotch, Plasma Proteins, Indicators, OCTD, non-neoplastic, Brittle Diabetes Mellitus, Iletin, Pre-Proglucagons, Protein Structure, Clients, pharyngeal valve, Ultra Performance Liquid Chromatography, Sensitivity, disorder, Long-Term Effect, Primary Protein, Model, NICD, TAF, Diabetes, l(3)j1B7, diabetes, High-Performance Liquid Chromatography, T1DM, Intervention Strategies, OXM, TAF[[II]]250, Pro-Glucagon, OXY, muscular dilation of the oesophagus, l(3)84Ab, Monoclonal, DMI UNSPF UNCNTRLD, Client, Ketosis Prone, Peptide, type I diabetes, LGMD2C, Glycemic, diabetes mellitus (disease), development, insulin-dependent diabetes mellitus, Glucagone, LC/MS/MS, Sodium Insulin, Sequence, Model Calibrations, p230, Protein, AI551343, TFIID, peptide hormones, techniques, l(3)s118306, WMS2, Ethyl Alcohol, Plasma, HG Factor, Exubera, TAF[[II]]230, diabetes mellitus, Normality, uterine metalloendopeptidase activity, TAF[II]250, Amino, hydroxyethane, Ets58AB, DmelCG17603, Juvenile-Onset Diabetes, concentration, SSKS, SCARMD2, Calibrations, Peptid, High Performance Liquid, assay, Primary Protein Structures, CG3936, growth, methodology, TAF1Soluble, GRPP, Laboratory, Insulin Dependent Diabetes Mellitus 1, PNT-P1, Blood Glucose Control, L-histidyl-L-seryl-L-glutaminylglycyl-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl-L-alpha-aspartyl-L-tyrosyl-L-seryl-L-lysyl-L-tyrosyl-L-leucyl-L-alpha-aspartyl-L-seryl-L-arginyl-L-arginyl-L-alanyl-L-glutaminyl-L-alpha-aspartyl-L-phenylalanyl-L-valyl-L-glutaminyl-L-tryptophyl-L-leucyl-L-methionyl-L-asparaginyl-L-threonine, A4, insuline humaine, growth and development, EY3-1, Long Term, dmTAF[[II]]230, Glucagon Precursor, Polypeptides, Method, Oxyntomodulin, Protease Inhibitor, High Performance Liquid Chromatography, 1, insulin dependent diabetes, D-ets-2, 3520, blood specimen collection, with unspecified complication, Sudden Onset, post-pharynx, notch, WMS, Immunoassays, Juvenile Onset Diabetes, average, gamma sarcoglycan, Fresh Frozen, Grain, amino acids, TFIID TAF250, cel, polypeptide hormone, Assays, Sudden-Onset Diabetes, procedures, Ax, Structures, SURGICAL AND MEDICAL PROCEDURES, HPLC, allergic reaction, Dehydrated ethanol, Peptidohydrolase Inhibitor, Diabetes mellitus, scientific observation, anatomical tube, co, Chromatography, Homo sapiens disease, Blood Protein, High Speed Liquid, juvenile diabetes, NECD, Type 1 Diabetes Mellitus, plasma, SGS, l(1)N, db, spiritus vini, LC-MS2, insulinum humanum, Glucagon, dTAF[[II]]230, Regular, DM, Chp, Hormones, His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr, Co, Monoclonal Antibody, Liquid Chromatography, Longterm Effect, TAF200, GLP-1(7-37), Preproglucagons, Normalcy, Procedure, antibodies, Glucagon Precursors, ACMICD, Absolute Alcohol, Regular Insulin, OB-RGRP, Juvenile-Onset, insulin (recombinant), matrix metalloproteinase pump 1, Monoclonal Antibodies, shd, Insulin-dependent diabetes, Hydrolase Inhibitor, numerical data, sarcoglycan, GLP-1(7-36), Soluble Insulin, Glicentin-related polypeptide, [OEtH], fa, Peptide Hormone, Sensor, type I diabetes mellitus, Blood Sugar, Acid, Autoimmune Diabetes, pntP2, Pointed-P1, Antagonists, matrix metalloproteinase 7 activity, Ets94F, insulina humana, Diabetes mellitus type I [insulin dependent type] [IDDM] [juvenile type], blood harvesting, Methodological, alcohol etilico, Methylcarbinol, Obr, clone 1.12, Taf250, liquid chromatography-tandem mass spectroscopy, 35kD dystrophin-associated glycoprotein, duct, Indicator, PUMP-1 proteinase activity, Acids, Endopeptidase, Peptide Peptidohydrolase Inhibitor, antlia, Hyperglycemic-Glycogenolytic Factor, obl, TAF230, Amino Acid Sequences, Fresh, 0998/12, Juvenile diabetes, Fresh Frozen Plasma, Glucose, Effects, Endopeptidase Inhibitors, etanol, glucagonum, Serum Protein, putative metalloproteinase activity, Normalcies, Hormone, Autoimmune Diabete, DMPOINT1A, sensitive, Insulin, LC-MSMS, Diabetes mellitus (disorder), disease or disorder, Peptide Hydrolase, MAM, gamma-SG, Technique, l(1)Ax, Normalities, Chain, Insulin-Dependent Diabetes Mellitus, dTAF[[II]]250, pointed-RC, 2310061K05Rik, Longterm, interventionDescription, cell, ethanol, obese-like, Long-Term, Antagonist, Structure, High-Performance Liquid Chromatographies, EK3-2, Study, Injectable, Plasma Protein, l(3)07825, dTAF250, C2H5OH, His-ser-glu(nh2)-gly-thr-phe-thr-ser-asp-tyr-ser-lys-tyr-leu-asp-ser-arg-arg-ala-glu(NH2)-asp-phe-val-glu(NH2)-trp-leu-met-asp(NH2)-thr, Insulin recombinant, Protease Antagonists, Diabetes Mellitus, Proglucagon (33-61), putative (or punctuated) metalloproteinase-1 activity, Type 1 Diabetes, Peptide Peptidohydrolase Inhibitors, peptidos, CG17077, UPLC, Hydroxide, CT13012, 16-178, measuring, Individual Health, 16-55, Immunochromatographic Assays, Ets, use, T1D, Diabete, 35 kDa dystrophin-associated glycoprotein, pumping organ, Assay, Proteins, disorders, function, Primary, anon-EST:Liang-1.12, BG:DS00004.13, Cell, SGCG, dTAF230, Sequences, polypeptide, Frozen Plasmas, adult pharynx, Preproglucagon, chemical analysis, spl, Long Term Effects, Postpharynx, Insulin A Chain, alcool ethylique, TAF[[II]]250/230, condition, MFS1, 1.1, Sodium, Novolin, nd, Protein Structures, pnt-P1, pnt-P2, dJ142L7.2, Mellitus, Intervention, Incretin hormone, Taf[[II]]250, LEPROT, diabetes mellitis type I, DMDA1, postnatal growth, Specificity, Peptidohydrolase Inhibitors, Understanding, diabetes mellitis type 1, HG-Factor, Sugar, Longterm Effects, Ketosis-Prone, plan specification, type 1 diabetes, Peptidase, High Pressure, Antiprotease, Calibration, Pharynx, liquid chromatography tandem mass spectrometry, Peptide Hydrolase Inhibitors, POINT, CG8705, Insulin Dependent, liquid chromatography tandem mass spectroscopy, Modb1, MGC130048, Diabetes Type 1, Insulin B Chain, Endopeptidase Inhibitor, Grain Alcohol, single-organism developmental process, determination, Blood, anterior pharynx, postnatal development, Aminosaeure, DmelCG17077, Glicentin (33-69), Hyperglycemic Glycogenolytic Factor, Peptide Hydrolase Inhibitor, Pro Glucagon, Pnt, Serum, Model Calibration, Insulin Dependent Diabetes, 1-hydroxyethane, alcohol, peptide, Techniques, Ethanol, Insulin dependent diabetes mellitus, Readability, Blood sugar, peptido, png, diseases, human insulin, Alkohol, Juvenile onset diabetes mellitus, Inhibitors, diseases and disorders, not stated as uncontrolled, Hydrolase Inhibitors, Leprb, Effect, Autoimmune, Injectables, DM - Diabetes mellitus, Pnt-P1, Fresh Frozen Plasmas, insulin, human disease, LCMSMS, peptides, Pumporgan, metalloproteinase pump-1, PUMP, Ethyl, Dietary Fiber, Aethanol, [CH2Me(OH)], pump, Immunochromatographic Assay, Proglucagon (33-69), insulin human, High-Performance, Methodological Studies, gamma-sarcoglycan, High-Performance Liquid, Reagents, GPHYSD2, Long-Term Effects, EG:140G11.1, matrin activity, swb, Primary Protein Structure, Tube, Juvenile Onset, Frozen Plasma, Alcohol, Peptidase Inhibitor, statistics and numerical data, Aminokarbonsaeure, LC-MS/MS, SG-gamma, Brittle, D-Ets-2, uncontrolled, ets94F, 0123/09, Ethyl alcohol, TAFII-250, Peptide Peptidohydrolase, TAF250/230, Plasmas, PUMP-1 activity, TUBE, Diabetes mellitus type I, TAFII250, MMP 7, Diseases, MMP, NOS, Glucagon (1-29), immune mediated diabetes, laboratory, Individual, ETHANOL, Amino Acid, Sudden-Onset, IDDM, EtOH, posterior pharynx, IDDM - Insulin-dependent diabetes mellitus, Glicentin, Diabetes mellitus type 1, Sudden-Onset Diabetes Mellitus, Control, Immunochromatographic, Glucose Control, MASS, Juvenile Diabetes, gamma (35kDa dystrophin-associated glycoprotein), CG17603, Methodological Study, TAF[[II]], Glucagon-like peptide 2, Glucagon-like peptide 1, Type 1, Diabetes NOS, ptd, GLP-1, PntP2, disease, Health, DMDA, Peptidase Inhibitors, Blood Glucose, Specificity and Sensitivity, Patient, SR3-5, Sensor Calibration, PntP1, E(E2F)3D, Antiproteases, Sensor Calibrations, Liquid, Insulin-Dependent Diabetes Mellitus 1, medical condition., PNTP2, Polypeptide, HSQGTFTSDYSKYLDSRRAQDFVQWLMNT, Polypeptide Hormone, Polypeptide Hormones, n[fah], Inhibitor, PNTP1, GLP-2, Juvenile-Onset Diabetes Mellitus, DMI UNSPF NT ST UNCNTRLD, Ammonium, Ketosis-Prone Diabetes Mellitus, other disease, d230, Glucagon-like peptide 1(7-37), SGCG_HUMAN, Procedures, Type I Diabetes, Reagent, ETS2, Ets2, Aminocarbonsaeure, EG:163A10.2, FBN, Protease, dTAFII250, Diabetes mellitus type 1 (disorder), Insulin B, DIABETES MELLITUS TYPE 01, Praepharynx, EfW1, TYPE, LC-MS-MS, Intervention or Procedure, DAGA4, Aethylalkohol, method, Antibody, pntegfr, 0608/07, utilization, dmTAF1, Glucagon-like peptide 1(7-36), ECTOL1, Injection, Taf230, Glukagon, Insulin-Dependent, method used in an experiment, 35DAG, Studies, Protease Antagonist, l(1)3Cb, Type I, glucagon, SCG3, sensitivity, Reagents and Indicators, TAF250, Blood Plasma, Taf200, Absolute, DmelCG3936, Type I diabetes, Interventional, Taf1p, Serum Proteins, dNotch, Plasma Proteins, Indicators, OCTD, non-neoplastic, Brittle Diabetes Mellitus, Iletin, Pre-Proglucagons, Protein Structure, Clients, Blood Plasmas, pharyngeal valve, Ultra Performance Liquid Chromatography, Sensitivity, disorder, Long-Term Effect, Primary Protein, Model, NICD, TAF, Diabetes, l(3)j1B7, diabetes, High-Performance Liquid Chromatography, T1DM, Intervention Strategies, OXM, TAF[[II]]250, Pro-Glucagon, OXY, muscular dilation of the oesophagus, l(3)84Ab, Monoclonal, DMI UNSPF UNCNTRLD, Client, Ketosis Prone, Peptide, type I diabetes, LGMD2C, Glycemic, diabetes mellitus (disease), development, insulin-dependent diabetes mellitus, Glucagone, LC/MS/MS, Sodium Insulin, Sequence, Model Calibrations, p230, Protein, AI551343, TFIID, peptide hormones, techniques, l(3)s118306, WMS2, Ethyl Alcohol, Plasma, HG Factor, Exubera, TAF[[II]]230, diabetes mellitus, Normality, uterine metalloendopeptidase activity, portion of plasma, TAF[II]250, Amino, hydroxyethane, Ets58AB, DmelCG17603, Juvenile-Onset Diabetes, concentration, SSKS, SCARMD2, Calibrations, Peptid, High Performance Liquid, assay, Primary Protein Structures, CG3936, growth, methodology, TAF1liquid chromatography tandem mass spectroscopy, LC-MS2, Glucagon, OXM, Glucagon-like peptide 1(7-37), GRPP, His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr, protein complex, Glicentin (33-69), Proteins, L-histidyl-L-seryl-L-glutaminylglycyl-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl-L-alpha-aspartyl-L-tyrosyl-L-seryl-L-lysyl-L-tyrosyl-L-leucyl-L-alpha-aspartyl-L-seryl-L-arginyl-L-arginyl-L-alanyl-L-glutaminyl-L-alpha-aspartyl-L-phenylalanyl-L-valyl-L-glutaminyl-L-tryptophyl-L-leucyl-L-methionyl-L-asparaginyl-L-threonine, OXY, Hyperglycemic Glycogenolytic Factor, LC-MS/MS, glucagonum, GLP-1(7-37), Gene, protein, protein-containing complex, Glucagone, LC/MS/MS, native protein, Glucagon-like peptide 1(7-36), Oxyntomodulin, Glukagon, Protein, LC-MSMS, Gene Products, GLP-1(7-36), Glucagon (1-29), protein aggregate, glucagon, Glicentin-related polypeptide, Incretin hormone, HG Factor, LCMSMS, Glicentin, Glucagon-like peptide 2, Glucagon-like peptide 1, HG-Factor, Protein Gene Products, Gene Proteins, GLP-1, Proglucagon (33-69), His-ser-glu(nh2)-gly-thr-phe-thr-ser-asp-tyr-ser-lys-tyr-leu-asp-ser-arg-arg-ala-glu(NH2)-asp-phe-val-glu(NH2)-trp-leu-met-asp(NH2)-thr, liquid chromatography-tandem mass spectroscopy, Proglucagon (33-61), liquid chromatography tandem mass spectrometry, LC-MS-MS., Hyperglycemic-Glycogenolytic Factor, HSQGTFTSDYSKYLDSRRAQDFVQWLMNT, GLP-2falseEvaluation of Linearity, Lower Limit of Measurement Interval and ImprecisionIntroduction Glucagon and oxyntomodulin are peptide hormones differentially released from proglucagon that function in regulating blood glucose concentrations. Their overlapping amino acid sequences make the development of specific immunoassays difficult, but the specificity of liquid chromatography-tandem mass spectrometry can be used to distinguish the peptides. We aimed to develop a sensitive and specific mass spectrometric assay that uses non-proprietary reagents and normal-flow liquid chromatography in the simultaneous quantification of both analytes. Methods Bulk plasma proteins were precipitated in ethanol/ammonium hydroxide. Analytes were enriched using monoclonal antibodies generated in-house and analyzed using liquid chromatography-tandem mass spectrometry. A purified primary glucagon calibration material was sourced commercially and characterized for purity and concentration by HPLC-UV and amino acid analysis. Single point calibration was used to minimize between-day variability. Results The novel antibodies performed acceptably (peptide recovery 45-59%). The assay was precise (<13 %CV) and linear over the range of 1.3-14.7 pM and 1.1-13.7 pM for glucagon and oxyntomodulin, respectively. The primary calibrator concentration was determined to be 1.596 mg/g. Tube-type studies supported the use of protease inhibitor tubes at the time of blood draw. Patients with type 1 diabetes had lower concentrations of plasma glucagon when maintained on an insulin pump, but not with injectable insulin. Conclusion We have validated a method with a highly detailed standard operating procedure. We have characterized a reference material to help maintain accuracy and achieve between-assay and between-laboratory harmonization. The assay will be beneficial in better understanding α-cell health and glycemic control in diabetes and other diseases.Tue May 20 00:00:00 GMT+01:00 2025PXD041410960640385697