Proteins interacting with Amuc_1100 identified by Pull-down binding mass spectrometry
Description
To identify potential interacting proteins of Amuc_1100, GST pull-down assays were conducted using lysates from human trophoblast HTR-8/SVneo cells, as well as mouse placental and small intestinal epithelial tissues. Recombinant GST-Amuc_1100 fusion protein and GST control protein were expressed in Escherichia coli BL21 (DE3) cells and purified using the GST pull-down kit (FI8804-20T, Guangzhou Huijun Biotechnology Co., Ltd., China) according to the manufacturer's instructions. Cell and tissue lysates were prepared using lysis buffer (20 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1% NP-40; 1 mM EDTA) supplemented with protease inhibitors (Beyotime, P1005, China). Equal amounts of lysates were incubated with GST or GST-Amuc_1100 immobilized on glutathione-affinity resin at 4°C overnight with gentle rotation. After extensive washing, bound proteins were eluted, separated by SDS-PAGE, and visualized using Coomassie Brilliant Blue staining. Distinct protein bands were excised, subjected to in-gel trypsin digestion, and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific, USA). Identified proteins were annotated using the UniProt database and analyzed for functional enrichment and pathway involvement using appropriate bioinformatics tools.
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Steps to reproduce
GST-Amuc_1100 fusion protein served as bait, with GST alone as the negative control. After incubation with cell lysates, specific interactors were identified by excluding nonspecific proteins detected in controls. To identify potential interacting proteins of Amuc_1100, GST pull-down assays were conducted using lysates from human trophoblast HTR-8/SVneo cells, as well as mouse placental and small intestinal epithelial tissues. Recombinant GST-Amuc_1100 fusion protein and GST control protein were expressed in Escherichia coli BL21 (DE3) cells and purified using the GST pull-down kit (FI8804-20T, Guangzhou Huijun Biotechnology Co., Ltd., China) according to the manufacturer's instructions. Cell and tissue lysates were prepared using lysis buffer (20 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1% NP-40; 1 mM EDTA) supplemented with protease inhibitors (Beyotime, P1005, China). Equal amounts of lysates were incubated with GST or GST-Amuc_1100 immobilized on glutathione-affinity resin at 4°C overnight with gentle rotation. After extensive washing, bound proteins were eluted, separated by SDS-PAGE, and visualized using Coomassie Brilliant Blue staining. Distinct protein bands were excised, subjected to in-gel trypsin digestion, and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific, USA). Identified proteins were annotated using the UniProt database and analyzed for functional enrichment and pathway involvement using appropriate bioinformatics tools.
Institutions
- Southern Medical University