title: Peptidomic profiling of secreted products from pancreatic islet culture results in a higher yield of full-length peptide hormones than found using cell lysis procedures.
abstract: Peptide Hormone Acquisition through Smart Sampling Technique-Mass Spectrometry (PHASST-MS) is a peptidomics platform that employs high resolution liquid chromatography-mass spectrometry (LC-MS) techniques to identify peptide hormones secreted from in vitro or ex vivo cultures enriched in endocrine cells. Application of the methodology to the study of murine pancreatic islets has permitted evaluation of the strengths and weaknesses of the approach, as well as comparison of our results with published islet studies that employed traditional cellular lysis procedures. We found that, while our PHASST-MS approach identified fewer peptides in total, we had greater representation of intact peptide hormones. The technique was further refined to improve coverage of hydrophilic as well as hydrophobic peptides and subsequently applied to human pancreatic islet cultures derived from normal donors or donors with type 2 diabetes. Interestingly, in addition to the expected islet hormones, we identified alpha-cell-derived bioactive GLP-1, consistent with recent reports of paracrine effects of this hormone on beta-cell function. We also identified many novel peptides derived from neurohormonal precursors and proteins related to the cell secretory system. Taken together, these results suggest the PHASST-MS strategy of focusing on cellular secreted products rather than the total tissue peptidome may improve the probability of discovering novel bioactive peptides and also has the potential to offer important new insights into the secretion and function of known hormones.
authors.affiliation: Amylin Pharmaceuticals, LLC., 9360 Towne Centre Drive, San Diego, California 92121, USA. steven.w.taylor@questdiagnostics.com
authors.forename: Steven W
authors.initials: SW
authors.lastname: Taylor
authors.forename: Svetlana E
authors.initials: SE
authors.lastname: Nikoulina
authors.forename: Nancy L
authors.initials: NL
authors.lastname: Andon
authors.forename: Carolyn
authors.initials: C
authors.lastname: Lowe
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D002853:Chromatography, Liquid; D003924:Diabetes Mellitus, Type 2 / Q000378:metabolism*; D052216:Glucagon-Like Peptide 1 / Q000032:analysis*; D006801:Humans; D007515:Islets of Langerhans / Q000378:metabolism*; D013058:Mass Spectrometry; D051379:Mice; D058977:Molecular Sequence Annotation; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D036361:Peptide Hormones / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D046509:Tissue Culture Techniques
chemical_list: D010446:Peptide Fragments; D036361:Peptide Hormones; D052216:Glucagon-Like Peptide 1