title: Degradation and Stabilization of Peptide Hormones in Human Blood Specimens.
abstract: Plasma hormone peptides, including GLP-1, GIP, Glucagon, and OXM, possess multiple physiological roles and potential therapeutic and diagnostic utility as biomarkers in the research of metabolic disorders. These peptides are subject to proteolytic degradation causing preanalytical variations. Stabilization for accurate quantitation of these active peptides in ex vivo blood specimens is essential for drug and biomarker development. We investigated the protease-driven instability of these peptides in conventional serum, plasma, anticoagulated whole blood, as well as whole blood and plasma stabilized with protease inhibitors. The peptide was monitored by both time-course Matrix-Assisted Laser Desorption Ionization Time-to-Flight Mass Spectrometry (MALDI -TOF MS) and Ab-based assay (ELISA or RIA). MS enabled the identification of proteolytic fragments. In non-stabilized blood samples, the results clearly indicated that dipeptidyl peptidase-IV (DPP-IV) removed the N-terminal two amino acid residues from GLP-1, GIP and OXM(1-37) and not-yet identified peptidase(s) cleave(s) the full-length OXM(1-37) and its fragments. DPP-IV also continued to remove two additional N-terminal residues of processed OXM(3-37) to yield OXM(5-37). Importantly, both DPP-IV and other peptidase(s) activities were inhibited efficiently by the protease inhibitors included in the BD P800* tube. There was preservation of GLP-1, GIP, OXM and glucagon in the P800 plasma samples with half-lives > 96, 96, 72, and 45 hours at room temperature (RT), respectively. In the BD P700* plasma samples, the stabilization of GLP-1 was also achieved with half-life > 96 hours at RT. The stabilization of these variable peptides increased their utility in drug and/or biomarker development. While stability results of GLP-1 obtained with Ab-based assay were consistent with those obtained by MS analysis, the Ab-based results of GIP, Glucagon, and OXM did not reflect the time-dependent degradations revealed by MS analysis. Therefore, we recommended characterizing the degradation of the peptide using the MS-based method when investigating the stability of a specific peptide.
authors.affiliation: BD Diagnostics, One Becton Drive, Franklin Lakes, NJ, United States of America.
authors.forename: Jizu
authors.initials: J
authors.lastname: Yi
authors.affiliation: BD Diagnostics, One Becton Drive, Franklin Lakes, NJ, United States of America.
authors.forename: David
authors.initials: D
authors.lastname: Warunek
authors.affiliation: BD Diagnostics, One Becton Drive, Franklin Lakes, NJ, United States of America.
authors.forename: David
authors.initials: D
authors.lastname: Craft
mesh_terms: D000595:Amino Acid Sequence; D018819:Dipeptidyl Peptidase 4 / Q000097:blood; D005749:Gastric Inhibitory Polypeptide / Q000097:blood / Q000235:genetics; D005934:Glucagon / Q000097:blood / Q000235:genetics; D052216:Glucagon-Like Peptide 1 / Q000097:blood / Q000235:genetics; D006801:Humans; D054795:Incretins / Q000097:blood; D008969:Molecular Sequence Data; D053772:Oxyntomodulin / Q000097:blood / Q000235:genetics; D010446:Peptide Fragments / Q000097:blood / Q000235:genetics; D036361:Peptide Hormones / Q000097:blood* / Q000235:genetics; D055550:Protein Stability; D059748:Proteolysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D054795:Incretins; D053772:Oxyntomodulin; D010446:Peptide Fragments; D036361:Peptide Hormones; D005749:Gastric Inhibitory Polypeptide; D052216:Glucagon-Like Peptide 1; D005934:Glucagon; C042807:DPP4 protein, human; D018819:Dipeptidyl Peptidase 4