2D‐DIGE analysis revealed reduced cytokeratin signaling in placenta with preeclampsia

Abstract

Preeclampsia represents a severe pregnancy disorder associated with premature delivery and fetal growth retardation which also involves certain imbalances of the placental maturation and the placental integration into the surrounding tissues. To characterize possible differences in the development of normal and preeclamptic placentae, two-dimensional SDS-PAGE of 10 normal placental protein homogenates was performed and compared to the protein pattern of homogenates from 10 preeclamptic placentae. Thus, acidic proteins particularly of about 56 kDa were identified in normal placentae which were nearly undetectable in protein homogenates of preeclamptic placentae. Peptide mass finger printing by MALDI identified cytokeratins, especially cytokeratin-10 as one of the differentially expressed protein. Quantitative data were obtained by two-dimensional difference-gel electrophoresis (2D DIGE). Labeling of normal and preeclamptic placental proteins with the fluorophors Cy5 and Cy3, respectively, and subsequent separation of these mixed samples by 2D DIGE revealed a differentially expressed protein spot at a 12.8-fold higher fluorescence intensity in normal placentae as compared to preeclamptic placentae. MALDI analysis of this differentially expressed protein spot identified cytokeratin. In order to verify these results in individual placenta probes, cytokeratin-10 Western blots were performed in 3 normal and preeclamptic placental homogenates of different gestational ages, respectively. Whereas a marked 56 kDa cytokeratin-10 expression appeared in all normal placentae, there was only little if any detectable cytokeratin-10 present in the preeclamptic placentae, respectively. These findings suggest that preeclampsia is accompanied by a significantly reduced cytokeratin signaling provided by 2D-DIGE-coupled MALDI analysis which represents a suitable technique to identify e. g. disease-related alterations in protein patterns.