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

@article{citeulike:6084258, abstract = {PMID: 19731965 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 (TiO2) 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 ∼8.5\% residual albumin (3.73 × 105 ± 2.35 × 105 ng/mL), whereas up to ∼68\% IGFBP-1 was recovered (1.36 × 103 ± 0.174 × 103 μg/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, ∼20-fold with pSer169 and ∼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.}, author = {Abu Shehab, Majida and Inoue, Shinobu and Han, Victor K. M. and Gupta, Madhulika B.}, citeulike-article-id = {6084258}, citeulike-linkout-0 = {http://dx.doi.org/10.1021/pr900633x}, citeulike-linkout-1 = {http://pubs.acs.org/doi/abs/10.1021/pr900633x}, day = {6}, doi = {10.1021/pr900633x}, journal = {Journal of Proteome Research}, keywords = {phosphorylation}, month = {November}, number = {11}, pages = {5325--5335}, posted-at = {2009-11-08 14:44:19}, priority = {2}, title = {Site Specific Phosphorylation of Insulin-Like Growth Factor Binding Protein-1 (IGFBP-1) for Evaluating Clinical Relevancy in Fetal Growth Restriction}, url = {http://dx.doi.org/10.1021/pr900633x}, volume = {8}, year = {2009} }

TY - JOUR ID - citeulike:6084258 L3 - citeulike-article-id:6084258 N2 - PMID: 19731965 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 (TiO2) 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 ∼8.5% residual albumin (3.73 × 105 ± 2.35 × 105 ng/mL), whereas up to ∼68% IGFBP-1 was recovered (1.36 × 103 ± 0.174 × 103 μg/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, ∼20-fold with pSer169 and ∼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. IS - 11 JF - Journal of Proteome Research EP - 5335 TI - Site Specific Phosphorylation of Insulin-Like Growth Factor Binding Protein-1 (IGFBP-1) for Evaluating Clinical Relevancy in Fetal Growth Restriction VL - 8 SP - 5325 KW - phosphorylation AU - Abu Shehab, Majida AU - Inoue, Shinobu AU - Han, Victor AU - Gupta, Madhulika PY - 2009/11/06/ UR - http://dx.doi.org/10.1021/pr900633x ER -