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

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 <i>in vitro</i> or <i>ex vivo</i> 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

Integrating Molecular Networking and Biological Assays To Target the Isolation of a Cytotoxic Cyclic Octapeptide, Samoamide A, from an American Samoan Marine Cyanobacterium

Integrating LC-MS/MS molecular networking and bioassay-guided fractionation enabled the targeted isolation of a new and bioactive cyclic octapeptide, samoamide A (<b>1</b>), from a sample of cf. <i>Symploca</i> sp. collected in American Samoa. The structure of <b>1</b> was established by detailed 1D and 2D NMR experiments, HRESIMS data, and chemical degradation/chromatographic (e.g., Marfey’s analysis) studies. Pure compound <b>1</b> was shown to have in vitro cytotoxic activity against several human cancer cell lines in both traditional cell culture and zone inhibition bioassays. Although there was no particular selectivity between the cell lines tested for samoamide A, the most potent activity was observed against H460 human non-small-cell lung cancer cells (IC₅₀ = 1.1 μM). Molecular modeling studies suggested that one possible mechanism of action for <b>1</b> is the inhibition of the enzyme dipeptidyl peptidase (CD26, DPP4) at a reported allosteric binding site, which could lead to many downstream pharmacological effects. However, this interaction was moderate when tested in vitro at up to 10 μM and only resulted in about 16% peptidase inhibition. Combining bioassay screening with the cheminformatics strategy of LC-MS/MS molecular networking as a discovery tool expedited the targeted isolation of a natural product possessing both a novel chemical structure and a desired biological activity