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. 2013 Apr 29;8(4):e62988.
doi: 10.1371/journal.pone.0062988. Print 2013.

Identification and analysis of multi-protein complexes in placenta

Fuqiang Wang  1 , Ling Wang, Zhiyang Xu, Gaolin Liang
Affiliations

Identification and analysis of multi-protein complexes in placenta

Fuqiang Wang et al. PLoS One. .

Abstract

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Blue native PAGE analysis of the placenta lysates.
(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%).
Figure 2
Figure 2. 2D BN/SDS-PAGE proteomic maps of placental protein complexes solubilized with 1.5% Triton X-100.
1D BN strip (1.5% Triton X-100) in Figure 1B 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.
Figure 3
Figure 3. The functions and subcellular locations of the identified proteins from BN/SDS-PAGE.
(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.
Figure 4
Figure 4. Verification of the validity of clathrin-SK channels protein complex.
(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).
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Grants and funding

This work was supported by the National Natural Science Foundation of China (Grants 21175122 and 91127036) and Technology Development Key Project Fund of Nanjing Medical University (Grants 2011NJMU269 and 2011NJMU270). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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