Proteomic Profiling for Congenital Diaphragmatic Hernia

Pregnant patients for whom amniocentesis will be performed as part of the prenatal management of CDH confirmed by fetal ultrasound will be approached fot inclusion in the study. The indication of amniocentesis will be indicated according to the usual standards of care. Briefly, the study will not involve any additional invasive procedure. In our study, amniocentesis will be indicated during the conventional management of these patients. The needs of our research will not lead to additional amniocenteses, but simply an increase in the volume of amniotic fluid collected. An additional volume of 5 ml will be sampled for volumes usually taken from 20 to 30 ml. This volume of sampling will have a very limited impact on maternal and fetal well-being. An analysis of the proteom of the amniotic fluid of the patients will be carried out. The relative amounts of proteins and peptides contained in the amniotic fluid will be analyzed to explore variations in proteomic profile that may reflect different clinical gravity in terms of further evolution. The usual management of the patients and their children will not be modified by the procedures related to the research.

The first step will consist in carrying out a depletion of the major proteins, in particular albumin. A protein assay will be carried out from the same amount of protein for each sample (standardization). This depletion step will be followed by a migration step on a gel of 1D electrophoresis. Its purpose is both to eliminate all the contaminants that can hinder mass spectrometry analysis, to separate the proteins according to their molecular weight in order to simplify the protein mixture and to have a visual control of the heterogeneity of the sample. A differential visual analysis after staining will allow to define the highly variable zones between the groups of patients and within the same sample. We will select the zone of interest in identical manner for each sample and for the whole of the gel track. The use of commercial electrophoresis gels of the same batch and the parallel analysis of several samples of different groups and the use of quality controls ensure reproducibility. The proteins contained in these bands / gel zones are then digested according to a standardized protocol under controlled conditions. The simultaneous treatment of the different biological samples makes it possible to considerably reduce the impact of the variability of this step in the final differential statistical analysis. At the end of this step the proteins are analyzed by an LC-ESI MS / MS approach. This LC-ESI MS / MS approach consists of separating the peptides on a chromatographic separation column which is coupled directly to the ESI-MS / M mass spectrometer. This analytical approach can allow the identification of more than 1000 proteins and 5000 peptides. This analysis is repeated 3 times per sample so that analytical variability can be taken into account in differential statistical studies. At the end of this protocol the raw data of each zone are pooled by sample and the differential statistical study can be carried out.