title: Site specific phosphorylation of insulin-like growth factor binding protein-1 (IGFBP-1) for evaluating clinical relevancy in fetal growth restriction.
abstract: Fetal growth restriction (FGR) is a leading cause of fetal and neonatal morbidity and mortality. Insulin-like growth factor binding protein-1 (IGFBP-1) is one of the major insulin-like growth factor (IGF) binding proteins involved in fetal growth and development. Our recent data shows that phosphorylation of IGFBP-1 carries both functional and biological relevance in FGR. Considering that IGFBP-1 phosphorylation can be valuable in diagnostics, we examined strategies to enrich IGFBP-1 so that its phosphorylation sites could be assessed by mass spectrometry (MS). Using <1 mL of human amniotic fluid, widely employed immunoprecipitation with IGFBP-1 monoclonal antibody (Mab 6303) coenriched IgGs that interfered with MS. Covalent coupling of Mab 6303 with Seize immunoprecipitation resin (Pierce) mitigated this drawback. However, LC-MS/MS analysis with the titanium dioxide (TiO(2)) enriched IGFBP-1 phosphopeptides in the immunoprecipitated samples revealed pSer101 and pSer119, but not pSer169 nor pSer98 of the previously identified phosphorylation sites. The alternative, ZOOM isoelectric focusing (IEF) (Invitrogen) rendered low-IGFBP-1 recovery with overlapping albumin. Subsequently, depletion of albumin using Affi-GelBlue gel (Bio-Rad) maximized IGFBP-1 yield. ELISA estimation showed approximately 8.5% residual albumin (3.73 x 10(5) +/- 2.35 x 10(5) ng/mL), whereas up to approximately 68% IGFBP-1 was recovered (1.36 x 10(3) +/- 0.174 x 10(3) microg/L, IEMA). LC-MS/MS analysis with the albumin depleted samples detected all four expected phosphorylation sites. Additionally, LC-MS analysis semiquantitatively indicated much reduced phosphopeptide peak intensities, approximately 20-fold with pSer169 and approximately 10-fold lower with pSer98 sites as compared to pSer101. With the use of our depletion strategy, this study offers a novel simple proteomic approach to enrich IGFBP-1 for identification of site-specific changes in IGFBP-1 phosphorylation. This strategy will be vital in performing differential IGFBP-1 phosphorylation profiling clinically, to help establish its link with FGR and develop diagnostic assays, as well as elucidating novel mechanisms potentially involved in regulation of fetal growth.
authors.affiliation: Department of Pediatrics, University of Western Ontario, London, Ontario, Canada.
authors.forename: Majida
authors.initials: M
authors.lastname: Abu Shehab
authors.forename: Shinobu
authors.initials: S
authors.lastname: Inoue
authors.forename: Victor K M
authors.initials: VK
authors.lastname: Han
authors.forename: Madhulika B
authors.initials: MB
authors.lastname: Gupta
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods; D005260:Female; D005317:Fetal Growth Retardation / Q000175:diagnosis / Q000378:metabolism; D006801:Humans; D018970:Insulin-Like Growth Factor Binding Protein 1 / Q000737:chemistry / Q000378:metabolism*; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011247:Pregnancy; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D018970:Insulin-Like Growth Factor Binding Protein 1; D010748:Phosphopeptides; D020033:Protein Isoforms