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Quantitative Proteomics Analysis of Chondrogenic Differentiation of C3H10T1/2 Mesenchymal Stem Cells by iTRAQ Labeling Coupled with On-line Two-dimensional LC/MS/MS
The chondrogenic potential of multipotent mesenchymal stem cells (MSCs) makes them a promising source for cell-based therapy of cartilage defects; however, the exact intracellular molecular mechanisms of chondrogenesis as well as self-renewal of MSCs remain largely unknown. To gain more insight into...
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| Published in: | Molecular & cellular proteomics 2010-03, Vol.9 (3), p.550-564 |
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| container_title | Molecular & cellular proteomics |
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| creator | Ji, Yu-hua Ji, Ju-ling Sun, Fen-yong Zeng, Yao-ying He, Xian-hui Zhao, Jing-xian Yu, Yu Yu, Shou-he Wu, Wei |
| description | The chondrogenic potential of multipotent mesenchymal stem cells (MSCs) makes them a promising source for cell-based therapy of cartilage defects; however, the exact intracellular molecular mechanisms of chondrogenesis as well as self-renewal of MSCs remain largely unknown. To gain more insight into the underlying molecular mechanisms, we applied isobaric tag for relative and absolute quantitation (iTRAQ) labeling coupled with on-line two-dimensional LC/MS/MS technology to identify proteins differentially expressed in an in vitro model for chondrogenesis: chondrogenic differentiation of C3H10T1/2 cells, a murine embryonic mesenchymal cell line, was induced by micromass culture and 100 ng/ml bone morphogenetic protein 2 treatment for 6 days. A total of 1756 proteins were identified with an average false discovery rate |
| doi_str_mv | 10.1074/mcp.M900243-MCP200 |
| format | article |
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To gain more insight into the underlying molecular mechanisms, we applied isobaric tag for relative and absolute quantitation (iTRAQ) labeling coupled with on-line two-dimensional LC/MS/MS technology to identify proteins differentially expressed in an in vitro model for chondrogenesis: chondrogenic differentiation of C3H10T1/2 cells, a murine embryonic mesenchymal cell line, was induced by micromass culture and 100 ng/ml bone morphogenetic protein 2 treatment for 6 days. A total of 1756 proteins were identified with an average false discovery rate <0.21%. Linear regression analysis of the quantitative data gave strong correlation coefficients: 0.948 and 0.923 for two replicate two-dimensional LC/MS/MS analyses and 0.881, 0.869, and 0.927 for three independent iTRAQ experiments, respectively (p < 0.0001). Among 1753 quantified proteins, 100 were significantly altered (95% confidence interval), and six of them were further validated by Western blotting. Functional categorization revealed that the 17 up-regulated proteins mainly comprised hallmarks of mature chondrocytes and enzymes participating in cartilage extracellular matrix synthesis, whereas the 83 down-regulated were predominantly involved in energy metabolism, chromatin organization, transcription, mRNA processing, signaling transduction, and cytoskeleton; except for a number of well documented proteins, the majority of these altered proteins were novel for chondrogenesis. Finally, the biological roles of BTF3l4 and fibulin-5, two novel chondrogenesis-related proteins identified in the present study, were verified in the context of chondrogenic differentiation. These data will provide valuable clues for our better understanding of the underlying mechanisms that modulate these complex biological processes and assist in the application of MSCs in cell-based therapy for cartilage regeneration.</description><identifier>ISSN: 1535-9476</identifier><identifier>ISSN: 1535-9484</identifier><identifier>EISSN: 1535-9484</identifier><identifier>DOI: 10.1074/mcp.M900243-MCP200</identifier><identifier>PMID: 20008835</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Animals ; Bone Morphogenetic Protein 2 - metabolism ; Cartilage - metabolism ; Cell Differentiation ; Cell Line ; Chondrocytes - chemistry ; Chondrocytes - cytology ; Chondrocytes - metabolism ; Chondrogenesis ; Chromatography, Liquid ; Down-Regulation ; Extracellular Matrix - metabolism ; Extracellular Matrix Proteins - genetics ; Internet ; Mesenchymal Stem Cells - chemistry ; Mesenchymal Stem Cells - cytology ; Mesenchymal Stem Cells - metabolism ; Mice ; Proteomics ; Recombinant Proteins - genetics ; RNA, Messenger - genetics ; Tandem Mass Spectrometry ; Transcription Factors - genetics</subject><ispartof>Molecular & cellular proteomics, 2010-03, Vol.9 (3), p.550-564</ispartof><rights>2010 © 2010 ASBMB. Currently published by Elsevier Inc; originally published by American Society for Biochemistry and Molecular Biology.</rights><rights>2010 by The American Society for Biochemistry and Molecular Biology, Inc.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c478t-fe75e4c5a4ca1b66d6af9b2a5e5229af12af478b2558cd19ed0c0b885fb222b3</citedby><cites>FETCH-LOGICAL-c478t-fe75e4c5a4ca1b66d6af9b2a5e5229af12af478b2558cd19ed0c0b885fb222b3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2849707/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/S153594762030832X$$EHTML$$P50$$Gelsevier$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,881,3536,27900,27901,45755,53765,53767</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/20008835$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Ji, Yu-hua</creatorcontrib><creatorcontrib>Ji, Ju-ling</creatorcontrib><creatorcontrib>Sun, Fen-yong</creatorcontrib><creatorcontrib>Zeng, Yao-ying</creatorcontrib><creatorcontrib>He, Xian-hui</creatorcontrib><creatorcontrib>Zhao, Jing-xian</creatorcontrib><creatorcontrib>Yu, Yu</creatorcontrib><creatorcontrib>Yu, Shou-he</creatorcontrib><creatorcontrib>Wu, Wei</creatorcontrib><title>Quantitative Proteomics Analysis of Chondrogenic Differentiation of C3H10T1/2 Mesenchymal Stem Cells by iTRAQ Labeling Coupled with On-line Two-dimensional LC/MS/MS</title><title>Molecular & cellular proteomics</title><addtitle>Mol Cell Proteomics</addtitle><description>The chondrogenic potential of multipotent mesenchymal stem cells (MSCs) makes them a promising source for cell-based therapy of cartilage defects; however, the exact intracellular molecular mechanisms of chondrogenesis as well as self-renewal of MSCs remain largely unknown. 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Functional categorization revealed that the 17 up-regulated proteins mainly comprised hallmarks of mature chondrocytes and enzymes participating in cartilage extracellular matrix synthesis, whereas the 83 down-regulated were predominantly involved in energy metabolism, chromatin organization, transcription, mRNA processing, signaling transduction, and cytoskeleton; except for a number of well documented proteins, the majority of these altered proteins were novel for chondrogenesis. Finally, the biological roles of BTF3l4 and fibulin-5, two novel chondrogenesis-related proteins identified in the present study, were verified in the context of chondrogenic differentiation. These data will provide valuable clues for our better understanding of the underlying mechanisms that modulate these complex biological processes and assist in the application of MSCs in cell-based therapy for cartilage regeneration.</description><subject>Animals</subject><subject>Bone Morphogenetic Protein 2 - metabolism</subject><subject>Cartilage - metabolism</subject><subject>Cell Differentiation</subject><subject>Cell Line</subject><subject>Chondrocytes - chemistry</subject><subject>Chondrocytes - cytology</subject><subject>Chondrocytes - metabolism</subject><subject>Chondrogenesis</subject><subject>Chromatography, Liquid</subject><subject>Down-Regulation</subject><subject>Extracellular Matrix - metabolism</subject><subject>Extracellular Matrix Proteins - genetics</subject><subject>Internet</subject><subject>Mesenchymal Stem Cells - chemistry</subject><subject>Mesenchymal Stem Cells - cytology</subject><subject>Mesenchymal Stem Cells - metabolism</subject><subject>Mice</subject><subject>Proteomics</subject><subject>Recombinant Proteins - genetics</subject><subject>RNA, Messenger - genetics</subject><subject>Tandem Mass Spectrometry</subject><subject>Transcription Factors - genetics</subject><issn>1535-9476</issn><issn>1535-9484</issn><issn>1535-9484</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2010</creationdate><recordtype>article</recordtype><recordid>eNp9kc2O0zAUhSMEYoaBF2AB3rHK1HbiJpEQUhV-BqnVzDDZW7Zz3RgldrGTVn0fHhQPKRVskCzZ8v3O8fU9SfKa4GuCi3wxqN31psKY5lm6qe8oxk-SS8IyllZ5mT89n4vlRfIihO-RxKRgz5OLiOKyzNhl8vN-EnY0oxjNHtCddyO4waiAVlb0x2ACchrVnbOtd1uwRqGPRmvwEEVR4-zvenZDcEMWFG0ggFXdcRA9ehhhQDX0fUDyiEzzbXWP1kJCb-wW1W7a9dCigxk7dGvTeAmoObi0NQPYEI2jw7pebB7iepk806IP8Oq0XyXN509NfZOub798rVfrVOVFOaYaCga5YiJXgsjlsl0KXUkqGDBKK6EJFTqCkjJWqpZU0GKFZVkyLSmlMrtKPsy2u0kO0Kr4Ry96vvNmEP7InTD834o1Hd-6PadlXhW4iAbvTgbe_ZggjHwwQcUJCAtuCrzIMlZGjkSSzqTyLgQP-vwKwfwxXB7D5adw-RxuFL35u7-z5E-aEXg7A53ZdgfjgUvjVAcDr3jGGXu0eD8TEMe4N-B5UCYmBm2k1chbZ_7XwS-xfsKz</recordid><startdate>20100301</startdate><enddate>20100301</enddate><creator>Ji, Yu-hua</creator><creator>Ji, Ju-ling</creator><creator>Sun, Fen-yong</creator><creator>Zeng, Yao-ying</creator><creator>He, Xian-hui</creator><creator>Zhao, Jing-xian</creator><creator>Yu, Yu</creator><creator>Yu, Shou-he</creator><creator>Wu, Wei</creator><general>Elsevier Inc</general><general>American Society for Biochemistry and Molecular Biology</general><general>The American Society for Biochemistry and Molecular Biology</general><scope>6I.</scope><scope>AAFTH</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20100301</creationdate><title>Quantitative Proteomics Analysis of Chondrogenic Differentiation of C3H10T1/2 Mesenchymal Stem Cells by iTRAQ Labeling Coupled with On-line Two-dimensional LC/MS/MS</title><author>Ji, Yu-hua ; 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however, the exact intracellular molecular mechanisms of chondrogenesis as well as self-renewal of MSCs remain largely unknown. To gain more insight into the underlying molecular mechanisms, we applied isobaric tag for relative and absolute quantitation (iTRAQ) labeling coupled with on-line two-dimensional LC/MS/MS technology to identify proteins differentially expressed in an in vitro model for chondrogenesis: chondrogenic differentiation of C3H10T1/2 cells, a murine embryonic mesenchymal cell line, was induced by micromass culture and 100 ng/ml bone morphogenetic protein 2 treatment for 6 days. A total of 1756 proteins were identified with an average false discovery rate <0.21%. Linear regression analysis of the quantitative data gave strong correlation coefficients: 0.948 and 0.923 for two replicate two-dimensional LC/MS/MS analyses and 0.881, 0.869, and 0.927 for three independent iTRAQ experiments, respectively (p < 0.0001). Among 1753 quantified proteins, 100 were significantly altered (95% confidence interval), and six of them were further validated by Western blotting. Functional categorization revealed that the 17 up-regulated proteins mainly comprised hallmarks of mature chondrocytes and enzymes participating in cartilage extracellular matrix synthesis, whereas the 83 down-regulated were predominantly involved in energy metabolism, chromatin organization, transcription, mRNA processing, signaling transduction, and cytoskeleton; except for a number of well documented proteins, the majority of these altered proteins were novel for chondrogenesis. Finally, the biological roles of BTF3l4 and fibulin-5, two novel chondrogenesis-related proteins identified in the present study, were verified in the context of chondrogenic differentiation. These data will provide valuable clues for our better understanding of the underlying mechanisms that modulate these complex biological processes and assist in the application of MSCs in cell-based therapy for cartilage regeneration.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>20008835</pmid><doi>10.1074/mcp.M900243-MCP200</doi><tpages>15</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | Molecular & cellular proteomics, 2010-03, Vol.9 (3), p.550-564 |
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| source | ScienceDirect Journals; PubMed Central |
| subjects | Animals Bone Morphogenetic Protein 2 - metabolism Cartilage - metabolism Cell Differentiation Cell Line Chondrocytes - chemistry Chondrocytes - cytology Chondrocytes - metabolism Chondrogenesis Chromatography, Liquid Down-Regulation Extracellular Matrix - metabolism Extracellular Matrix Proteins - genetics Internet Mesenchymal Stem Cells - chemistry Mesenchymal Stem Cells - cytology Mesenchymal Stem Cells - metabolism Mice Proteomics Recombinant Proteins - genetics RNA, Messenger - genetics Tandem Mass Spectrometry Transcription Factors - genetics |
| title | Quantitative Proteomics Analysis of Chondrogenic Differentiation of C3H10T1/2 Mesenchymal Stem Cells by iTRAQ Labeling Coupled with On-line Two-dimensional LC/MS/MS |
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