Mapping the 5-50-kDa fraction of human amniotic fluid proteins by 2-DE and ESI-MS

This report presents a proteomic analysis and provides a reference map of the 5-50-kDa components of normal amniotic fluid collected in gestational weeks 16-18. Early amniocentesis samples were pooled and proteins with molecular mass lower than albumin were separated by gel filtration chromatography. The 2-DE protocol was optimized for the separation of the small proteins and peptides in the fraction of interest. A total of 132 Coomassie blue-stained protein spots were analyzed, following in-gel tryptic digestion, by ESI-MS/MS and 49 different gene products were identified. The treatment with alkaline phosphatase caused the shift of the phosphoisoforms of insulin-like growth factor-binding protein-1 and of the N-terminal osteopontin fragment. Of the 33 full-length proteins identified in the 2-DE profile, 23 had not been previously detected in the amniotic fluid and, of these, 22 are not present in the human plasma proteome under physiological conditions. Fragments of 16 larger proteins were identified and the sequence coverage data revealed that several correspond to autonomous domains that may have biological roles on their own. Several of the detected proteins and peptides appear to be involved in critical regulatory processes associated with placentation and early development, thus representing potential markers of various physiological or pathological conditions.