title: Using mass spectrometry to detect, differentiate, and semiquantitate closely related peptide hormones in complex milieu: measurement of IGF-II and vesiculin.
abstract: The search for an islet β-cell growth factor has been a key objective in recent diabetes research, because the ability to regenerate and/or protect the functioning β-cell population in patients could result in a great advancement for diabetes treatment. IGF-I and IGF-II are known to play crucial roles in fetal growth and prenatal development, and there is growing evidence that IGF-II increases β-cell proliferation and survival in vitro and in vivo. A search for the source of IGF-II-like immunoreactivity in isolated β-cell secretory granules from the murine cell line βTC6-F7 revealed a novel 2-chain IGF-II-derived peptide, which we named vesiculin and which has been shown to be a full insulin agonist. Here, we present a liquid chromatography-tandem mass spectrometry method that enables selective detection and semiquantitation of the highly related IGF-II and vesiculin molecules. We have used this method to measure these 2 peptides in conditioned media from 2 β-cell lines, produced under increasing glucose concentrations. This technique detected both IGF-II and vesiculin in media conditioned by MIN6 and βTC6-F7 cells at levels in the range of 0 to 6 μM (total insulin, 80-450 μM) and revealed a glucose-stimulated increase in insulin, IGF-II, and vesiculin. IGF-II was detected in adult human and neonatal mouse serum in high levels, but vesiculin was not present. The methodology we present herein has utility for detecting and differentiating active peptides that are highly related and of low abundance.
authors.affiliation: School of Biological Sciences (K.L.L., M.J.M., G.J.S.C.), School of Chemical Sciences (G.M.W., M.A.B.), and Maurice Wilkins Centre for Molecular BioDiscovery (M.J.M., M.A.B., G.J.S.C.), The University of Auckland, Auckland 1010, New Zealand; and Centre for Advanced Discovery and Experimental Therapeutics (G.J.S.C.), Manchester Biomedical Research Centre, Central Manchester University Hospitals NHS Foundation Trust, and the School of Biomedicine, the Medical School, University of Manchester, Manchester M13 9WL, United Kingdom.
authors.forename: Kate L
authors.initials: KL
authors.lastname: Lee
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Middleditch
authors.forename: Geoffrey M
authors.initials: GM
authors.lastname: Williams
authors.forename: Margaret A
authors.initials: MA
authors.lastname: Brimble
authors.forename: Garth J S
authors.initials: GJ
authors.lastname: Cooper
mesh_terms: D000818:Animals; D002460:Cell Line; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000737:chemistry*; D050417:Insulin-Secreting Cells / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D011994:Recombinant Proteins
chemical_list: D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; C012605:vesiculin; D007335:Insulin-Like Growth Factor II