Site specific phosphorylation of insulin-like growth factor binding protein-1 (IGFBP-1) for evaluating clinical relevancy in fetal growth restriction.

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.