Site Specific Phosphorylation of Insulin-Like Growth Factor Binding Protein-1 (IGFBP-1) for Evaluating Clinical Relevancy in Fetal Growth Restriction

摘要

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) bindingproteins involved in fetal growth and development. Our recent data shows that phosphorylation ofIGFBP-1 carries both functional and biological relevance in FGR. Considering that IGFBP-1 phospho-rylation can be valuable in diagnostics, we examined strategies to enrich IGFBP-1 so that itsphosphorylation sites could be assessed by mass spectrometry (MS). Using <1 mL of human amnioticfluid, widely employed immunoprecipitation with IGFBP-1 monoclonal antibody (Mab 6303) coenrichedIgGs 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) enrichedIGFBP-1 phosphopeptides in the immunoprecipitated samples revealed pSer101 and pSer119, but notpSer169 nor pSer98 of the previously identified phosphorylation sites. The alternative, ZOOM isoelectricfocusing (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 estimationshowed —8.5% residual albumin (3.73 x 10~5±2.35x 10~6ng/mL), whereas up to —68% IGFBP-1 wasrecovered (1.36 a 10~3± 0.174 x 10~3μg/L, IEMA). LC-MS/MS analysis with the albumin depleted samplesdetected all four expected phosphorylation sites. Additionally, LC-MS analysis semiquantitativelyindicated much reduced phosphopeptide peak intensities, —20-fold with pSer169 and —10-fold lowerwith pSer98 sites as compared to pSer101. With the use of our depletion strategy, this study offers anovel simple proteomic approach to enrich IGFBP-1 for identification of site-specific changes in IGFBP-1phosphorylation. This strategy will be vital in performing differential IGFBP-1 phosphorylation profilingclinically, to help establish its link with FGR and develop diagnostic assays, as well as elucidating novelmechanisms potentially involved in regulation of fetal growth.