Identification and Analysis of Multi-Protein Complexes in Placenta

<div><p>Placental malfunction induces pregnancy disorders which contribute to life-threatening complications for both the mother and the fetus. Identification and characterization of placental multi-protein complexes is an important step to integratedly understand the protein-protein interaction networks in placenta which determine placental function. In this study, blue native/sodium dodecyl sulfate polyacrylamide gel electrophoresis (BN/SDS-PAGE) and Liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to screen the multi-protein complexes in placenta. 733 unique proteins and 34 known and novel heterooligomeric multi-protein complexes including mitochondrial respiratory chain complexes, integrin complexes, proteasome complexes, histone complex, and heat shock protein complexes were identified. A novel protein complex, which involves clathrin and small conductance calcium-activated potassium (SK) channel protein 2, was identified and validated by antibody based gel shift assay, co-immunoprecipitation and immunofluorescence staining. These results suggest that BN/SDS-PAGE, when integrated with LC-MS/MS, is a very powerful and versatile tool for the investigation of placental protein complexes. This work paves the way for deeper functional characterization of the placental protein complexes associated with pregnancy disorders.</p></div

Blue native PAGE analysis of the placenta lysates.

<p>(A) The solubility of placenta proteins in native sample buffer was tested. After solubilization in varying concentrations of Triton X-100 (0.25%–3%), placenta lysates were subjected to SDS-PAGE. (B) Blue native PAGE analysis of the placenta lysates solubilized with Triton X-100 solutions at different concentrations (0.25%–3%).</p

Comparison of the number of protein-coding genes in chromosomes with those of identified glycoproteins (A) and phosphoproteins (B) in chromosomes with LC-MS/MS in this work.

<p>Comparison of the number of identified glycoproteins with that of phosphoproteins (C) in chromosomes.</p

Go analysis for the differentially expressed N-glyco- and phospho- proteins.

<p>Go analysis for the differentially expressed N-glyco- and phospho- proteins.</p

2D BN/SDS-PAGE proteomic maps of placental protein complexes solubilized with 1.5% Triton X-100.

<p>1D BN strip (1.5% Triton X-100) in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062988#pone-0062988-g001" target="_blank">Figure 1B</a> was loaded and separated on 11.5% acrylamide SDS-PAGE. The 1D BN strip was oriented with top to the left and bottom to the right. The proteomic map presented here is a representative coomassie brilliant blue (CBB)-stained protein gel. The gel spots for mass spectrometry identification were labeled with numbers.</p

Immunohistochemical analyses of ENG expression in the placentas from preeclamptic and normal pregnancies.

<p>ENG protein was localized in the syncytiotrophoblast layer of both control (right, white arrow) and preeclamptic placentas (left, red arrow). ENG staining is increased in placentas from preeclampsia. Scale bar: 100 µm.</p

Representative significant biological pathways in which detected placental N-glyco- and phospho- proteins are predicted to be involved.

<p>Representative significant biological pathways in which detected placental N-glyco- and phospho- proteins are predicted to be involved.</p

The list of differentailly expressed N-glyco- proteins in human placenta plasma membrane from control and preeclamptic pregnancies.

<p>The list of differentailly expressed N-glyco- proteins in human placenta plasma membrane from control and preeclamptic pregnancies.</p

The functions and subcellular locations of the identified proteins from BN/SDS-PAGE.

<p>(A) The functions of the identified proteins according to the GO annotations and literatures. (B) The subcellular locations of the identified proteins according to the GO annotations and literatures.</p

Verification of the validity of clathrin-SK channels protein complex.

<p>(A) Verification of the validity of the protein complex by BN-PAGE supershift assays. Placental lysates were incubated with SK channel protein 2 antibody (Ab) and resolved by BN-PAGE. A supershift band was indicated by arrow; no supershift was observed when lysates were incubated with normal IgG. (B) Copurification of clathrin and SK channel protein 2 protein complex from placental by co-immunoprecipitation. Placental protein lysates were either incubated with a clathrin-specific antibody (anti-clathrin) or a preimmunisation IgG pool (as negative control), then subject to SDS PAGE and western blot analyses. (C) Immunohistochemical analysis of the colocalization of clathrin and SK channel protein 2 in trophoblastic layer of placental. Immunohistochemical localization of SK channel protein 2 (left), clathrin (middle), and colocalization of clathrin and SK channel protein 2 (right).</p