Differential Proteome Analysis of the Preeclamptic Placenta Using Optimized Protein Extraction

Abstract

The human placenta is a difficult tissue to work with using proteomic technology since it contains large amounts of lipids and glycogen. Both lipids and glycogen are known to interfere with the first step in the two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), the isoelectric focusing. In order to gain the best possible protein separation on 2D-PAGE, an optimized sample preparation protocol for placental proteins was developed. Two different buffers, urea/CHAPS and Hepes, were used for solubilization in combination with six different precipitation methods. The removal of glycogen from the samples by centrifugation was crucial for the final proteome maps. Solubilization with urea/CHAPS in combination with dichloromethane/methanol or acidified acetone proved to be the best precipitation procedures. When applied to clinical placenta samples apolipoprotein A1 was found to be accumulated in the preeclamptic placenta, where it may either have a nutritional effect or act as a modifier of signal transduction.

A placental sub-proteome: The apical plasma membrane of the syncytiotrophoblast

“…Pre-eclamptic placentas expressed significantly less dysferlin, at both the mRNA and protein levels, than did normal placentas [37]. Two recent proteomics studies comparing normal and pre-eclamptic tissue extracts prepared from whole placental lysates separated by 2D-polyacrylamide gel electrophoresis have reported an increase in apolipoprotein A1 expression in the pre-eclamptic placenta [38,39]. Soluble forms of endoglin secreted by the placenta have also been studied as a potential marker for pre-eclampsia [40].…”

An effective placental cotyledons proteins extraction method for 2D gel electrophoresis

“…Therefore, this had encouraged us to employ two other commonly used chemical extraction buffers (high urea and dense sucrose protein lysis buffers) coupled with improved TCA‐acetone precipitation with the aim of improving the protein yield and protein profile of placental cotyledons. This protein extraction strategy was clearly supported by several studies in improving the resolution of protein spots on 2D‐gel .…”

Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

“…Furthermore it was decided to add a precipitation step to eliminate impureties that may disrupt the IEF. In this way, as precipitation method determines the solubility of the proteins 26 and methanol has already been described as the most suitable solvent to remove lipids which are abundant in brain tissue, it brought us to choose the chloroform/methanol precipitation 27 . The utilization of the zwitterionic detergent CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-Propane sulfonate) for resuspending the proteins pellet in the 2D buffer is due to its suitability for facilitating the protein entrance in the first dimension step 28 .…”

“…Another important limitation is the controversy use of the precipitation. On one hand, it is highly recommendable to do the precipitation step with chloroform/methanol that enables to efficiently reduce the lipid content 27 , salts, nucleic acids and charged detergents that might interfere with the IEF parameters and impact the resolution and/or reproducibility of 2D. Moreover, on the other hand it cannot be ruled out that some proteins strongly attached to the membrane may be lost.…”