title: Multiplexed Quantification of Proglucagon-Derived Peptides by Immunoaffinity Enrichment and Tandem Mass Spectrometry after a Meal Tolerance Test.
abstract: BACKGROUND
  Proglucagon-derived peptides (PGDPs), which include glucagon-like peptide (GLP)-1, glucagon, and oxyntomodulin, are key regulators of glucose homeostasis and satiety. These peptide hormones are typically measured with immuno-based assays (e.g., ELISA, RIA), which often suffer from issues of selectivity.
  
  
  METHODS
  We developed a multiplexed assay for measuring PGDPs including GLP-1 (7-36) amide, GLP-1 (9-36) amide, glucagon, and oxyntomodulin by mass spectrometry and used this assay to examine the effect of a meal tolerance test on circulating concentrations of these hormones. Participants fasted overnight and were either given a meal (n = 8) or continued to fast (n = 4), with multiple blood collections over the course of 3 h. Plasma samples were analyzed by microflow immunoaffinity (IA)-LC-MS/MS with an isotope dilution strategy.
  
  
  RESULTS
  Assay performance characteristics were examined and established during analytical validation for all peptides. Intra- and interassay imprecision were found to be 2.2%-10.7% and 6.8%-22.5%, respectively. Spike recovery was >76%, and dilution linearity was established up to a 16-fold dilution. Immediately after the meal tolerance test, GLP-1 and oxyntomodulin concentrations increased and had an almost identical temporal relationship, and glucagon concentrations increased with a slight delay.
  
  
  CONCLUSIONS
  IA-LC-MS/MS was used for the simultaneous and selective measurement of PGDPs. This work includes the first indication of the physiological concentrations and modulation of oxyntomodulin after a meal.
authors.affiliation: Translational Biomarkers, Merck Research Laboratories, Rahway, NJ;
authors.forename: Anita Y H
authors.initials: AY
authors.lastname: Lee
authors.affiliation: Translational Biomarkers, Merck Research Laboratories, Rahway, NJ;
authors.forename: Derek L
authors.initials: DL
authors.lastname: Chappell
authors.affiliation: NNF Center for Basic Metabolic Research, Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark;
authors.forename: Monika J
authors.initials: MJ
authors.lastname: Bak
authors.affiliation: Molecular Discovery, Antibody Generation Group and Assay Development, Immunoassay Group, Merck Research Laboratories, Palo Alto, CA;
authors.forename: Michael
authors.initials: M
authors.lastname: Judo
authors.affiliation: Molecular Discovery, Antibody Generation Group and Assay Development, Immunoassay Group, Merck Research Laboratories, Palo Alto, CA;
authors.forename: Linda
authors.initials: L
authors.lastname: Liang
authors.affiliation: Molecular Discovery, Antibody Generation Group and Assay Development, Immunoassay Group, Merck Research Laboratories, Palo Alto, CA;
authors.forename: Tatyana
authors.initials: T
authors.lastname: Churakova
authors.affiliation: Molecular Discovery, Antibody Generation Group and Assay Development, Immunoassay Group, Merck Research Laboratories, Palo Alto, CA;
authors.forename: Gulesi
authors.initials: G
authors.lastname: Ayanoglu
authors.affiliation: Cardio-metabolic Disease Biomarkers, Merck Research Laboratories, Kenilworth, NJ.
authors.forename: Jose
authors.initials: J
authors.lastname: Castro-Perez
authors.affiliation: Cardio-metabolic Disease Biomarkers, Merck Research Laboratories, Kenilworth, NJ.
authors.forename: Haihong
authors.initials: H
authors.lastname: Zhou
authors.affiliation: Cardio-metabolic Disease Biomarkers, Merck Research Laboratories, Kenilworth, NJ.
authors.forename: Stephen
authors.initials: S
authors.lastname: Previs
authors.affiliation: Cardio-metabolic Disease Biomarkers, Merck Research Laboratories, Kenilworth, NJ.
authors.forename: Sandra C
authors.initials: SC
authors.lastname: Souza
authors.affiliation: Translational Biomarkers, Merck Research Laboratories, Rahway, NJ; michael_lassman@merck.com.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Lassman
authors.affiliation: Translational Biomarkers, Merck Research Laboratories, Rahway, NJ;
authors.forename: Omar F
authors.initials: OF
authors.lastname: Laterza
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid; D005215:Fasting; D005934:Glucagon / Q000097:blood* / Q000276:immunology; D052216:Glucagon-Like Peptide 1 / Q000097:blood* / Q000276:immunology; D006801:Humans; D007118:Immunoassay; D051379:Mice; D008807:Mice, Inbred BALB C; D053772:Oxyntomodulin / Q000097:blood* / Q000276:immunology; D053719:Tandem Mass Spectrometry
chemical_list: D053772:Oxyntomodulin; D052216:Glucagon-Like Peptide 1; D005934:Glucagon