3/22/2023 0 Comments Photosenstive protein scaffold![]() ( D) The LFQ intensities of PDGFB and PDGFC from breast cancer cell line conditioned medium (CM). (Scale bar, 20 µm.) ( C) Quantification of IHC score for PDGFRB staining in breast sections from wild-type and MMTV-PyMT mice at different ages ( n = 4 or 7). ( B) IHC staining for PDGFRB in mammary gland sections and tumors from wild-type and MMTV-PyMT mice at different ages. ( A) WB analysis of PDGFRB expression in breast fibroblasts and normal and tumor cells. PDGFRB is a potential therapy target for breast cancer. The thickness of the edge indicates the STRING confidence for protein interaction prediction. ![]() ( F) Protein–protein interaction map of the identified proteins. LFQ intensities of the identified proteins are indicated. The pathway map was adapted from a previous report (21). ( E) Pathway map of EGFR and ERBB2 signaling-related proteins. Red and green dots indicate significantly enriched proteins (FDR = 0.01, s0 = 2, two-sample t test, n = 3). ( D) Volcano plots of the enriched proteins from unstimulated human breast cancer cells (MDA-MB-231 vs. ( C) Volcano plots of the enriched proteins from unstimulated human breast cancer cells (MDA-MB-468 vs. ( B) WB validation of native EGFR capture by N-PI3K and Src superbinder Photo-pTyr-scaffold in unstimulated HeLa cells. ( A) Volcano plots of the enriched proteins upon EGF stimulation of HeLa cells (red, EGF-treated FDR = 0.05, s0 = 2, two-sample t test, n = 3). Src superbinder Photo-pTyr-scaffold approach is superior for profiling native pTyr protein complexes. The Photo-pTyr-scaffold approach may become a generic tool for readily profiling dynamic pTyr signaling complexes in clinically relevant samples.Ĭancer phosphotyrosine signaling protein complex protein labeling proteomics. Among more than 1,000 identified pTyr proteins, receptor tyrosine kinase PDGFRB expressed on cancer-associated fibroblasts was validated as an important intercellular signaling regulator with poor expression correlation to ERBB2, and blockade of PDGFRB signaling could efficiently suppress tumor growth. This approach was successfully used to profile native pTyr protein complexes from breast cancer tissue samples on a proteome scale with high selectivity, achieving about 100 times higher sensitivity for detecting pTyr signaling proteins than that afforded by traditional immunohistochemical methods. Dynamic SH2 domain-scaffolding protein complexes were efficiently cross-linked under mild UV light, captured by biotin tag, and identified by mass spectrometry. We developed a hybrid chemical proteomics approach, termed Photo-pTyr-scaffold, by engineering Src homology 2 (SH2) domains, which specifically bind pTyr proteins, with both trifunctional chemical probes and genetic mutations to overcome these challenges. The systematic characterization of these protein complexes in tumor samples remains a challenge due to their limited access and the transient nature of pTyr-mediated interactions. Phosphotyrosine (pTyr)-regulated protein complexes play critical roles in cancer signaling. 10 Department of Chemistry, Southern University of Science and Technology, Shenzhen 518055, China 11 Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037.8 Department of Biology, Southern University of Science and Technology, Shenzhen 518055, China 9 Guangdong Provincial Key Laboratory of Cell Microenvironment and Disease Research, Southern University of Science and Technology, Shenzhen 518055, China.7 Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, ON N6A 5C1, Canada.6 Division of Life Science, State Key Laboratory of Molecular Neuroscience, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China.5 Department of Pathology, The Second Clinical Medical College, Shenzhen People's Hospital, Jinan University, Shenzhen 518020, China.4 Department of Oncology, The Second Clinical Medical College, Shenzhen People's Hospital, Jinan University, Shenzhen 518020, China.3 Department of Biology, Southern University of Science and Technology, Shenzhen 518055, China.2 Department of Chemistry, Fudan University, Shanghai 200433, China.1 Department of Chemistry, Southern University of Science and Technology, Shenzhen 518055, China.
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