Cellular Proteome Dynamics during Differentiation of Human Primary Myoblasts
Muscle stem cells, or satellite cells, play an important role in the maintenance and repair of muscle tissue and have the capacity to proliferate and differentiate in response to physiological or environmental changes. Although they have been extensively studied, the key regulatory steps and the com...
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Veröffentlicht in: | Journal of proteome research 2015-08, Vol.14 (8), p.3348-3361 |
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creator | Le Bihan, Marie-Catherine Barrio-Hernandez, Inigo Mortensen, Tenna Pavia Henningsen, Jeanette Jensen, Søren Skov Bigot, Anne Blagoev, Blagoy Butler-Browne, Gillian Kratchmarova, Irina |
description | Muscle stem cells, or satellite cells, play an important role in the maintenance and repair of muscle tissue and have the capacity to proliferate and differentiate in response to physiological or environmental changes. Although they have been extensively studied, the key regulatory steps and the complex temporal protein dynamics accompanying the differentiation of primary human muscle cells remain poorly understood. Here, we demonstrate the advantages of applying a MS-based quantitative approach, stable isotope labeling by amino acids in cell culture (SILAC), for studying human myogenesis in vitro and characterize the fine-tuned changes in protein expression underlying the dramatic phenotypic conversion of primary mononucleated human muscle cells during in vitro differentiation to form multinucleated myotubes. Using an exclusively optimized triple encoding SILAC procedure, we generated dynamic expression profiles during the course of myogenic differentiation and quantified 2240 proteins, 243 of which were regulated. These changes in protein expression occurred in sequential waves and underlined vast reprogramming in key processes governing cell fate decisions, i.e., cell cycle withdrawal, RNA metabolism, cell adhesion, proteolysis, and cytoskeletal organization. In silico transcription factor target analysis demonstrated that the observed dynamic changes in the proteome could be attributed to a cascade of transcriptional events involving key myogenic regulatory factors as well as additional regulators not yet known to act on muscle differentiation. In addition, we created of a dynamic map of the developing myofibril, providing valuable insights into the formation and maturation of the contractile apparatus in vitro. Finally, our SILAC-based quantitative approach offered the possibility to follow the expression profiles of several muscle disease-associated proteins simultaneously and therefore could be a valuable resource for future studies investigating pathogenesis of degenerative muscle disorders as well as assessing new therapeutic strategies. |
doi_str_mv | 10.1021/acs.jproteome.5b00397 |
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Although they have been extensively studied, the key regulatory steps and the complex temporal protein dynamics accompanying the differentiation of primary human muscle cells remain poorly understood. Here, we demonstrate the advantages of applying a MS-based quantitative approach, stable isotope labeling by amino acids in cell culture (SILAC), for studying human myogenesis in vitro and characterize the fine-tuned changes in protein expression underlying the dramatic phenotypic conversion of primary mononucleated human muscle cells during in vitro differentiation to form multinucleated myotubes. Using an exclusively optimized triple encoding SILAC procedure, we generated dynamic expression profiles during the course of myogenic differentiation and quantified 2240 proteins, 243 of which were regulated. These changes in protein expression occurred in sequential waves and underlined vast reprogramming in key processes governing cell fate decisions, i.e., cell cycle withdrawal, RNA metabolism, cell adhesion, proteolysis, and cytoskeletal organization. In silico transcription factor target analysis demonstrated that the observed dynamic changes in the proteome could be attributed to a cascade of transcriptional events involving key myogenic regulatory factors as well as additional regulators not yet known to act on muscle differentiation. In addition, we created of a dynamic map of the developing myofibril, providing valuable insights into the formation and maturation of the contractile apparatus in vitro. Finally, our SILAC-based quantitative approach offered the possibility to follow the expression profiles of several muscle disease-associated proteins simultaneously and therefore could be a valuable resource for future studies investigating pathogenesis of degenerative muscle disorders as well as assessing new therapeutic strategies.</description><identifier>ISSN: 1535-3893</identifier><identifier>EISSN: 1535-3907</identifier><identifier>DOI: 10.1021/acs.jproteome.5b00397</identifier><identifier>PMID: 26074025</identifier><language>eng</language><publisher>United States: American Chemical Society</publisher><subject>Amino Acids - metabolism ; Blotting, Western ; Cell Differentiation ; Cells, Cultured ; Chromatography, Liquid ; Cluster Analysis ; Humans ; Immunohistochemistry ; Infant, Newborn ; Isotope Labeling - methods ; Kinetics ; Muscle Fibers, Skeletal - cytology ; Muscle Fibers, Skeletal - metabolism ; Proteome - classification ; Proteome - metabolism ; Proteomics - methods ; Satellite Cells, Skeletal Muscle - cytology ; Satellite Cells, Skeletal Muscle - metabolism ; Spectrometry, Mass, Electrospray Ionization ; Tandem Mass Spectrometry ; Time Factors</subject><ispartof>Journal of proteome research, 2015-08, Vol.14 (8), p.3348-3361</ispartof><rights>Copyright © American Chemical Society</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a417t-c7ffaa52523c76f0260b16111bf87d19c1c9e039b6e8d7fa0da617e0fc4de5263</citedby><cites>FETCH-LOGICAL-a417t-c7ffaa52523c76f0260b16111bf87d19c1c9e039b6e8d7fa0da617e0fc4de5263</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://pubs.acs.org/doi/pdf/10.1021/acs.jproteome.5b00397$$EPDF$$P50$$Gacs$$H</linktopdf><linktohtml>$$Uhttps://pubs.acs.org/doi/10.1021/acs.jproteome.5b00397$$EHTML$$P50$$Gacs$$H</linktohtml><link.rule.ids>314,780,784,2765,27076,27924,27925,56738,56788</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/26074025$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Le Bihan, Marie-Catherine</creatorcontrib><creatorcontrib>Barrio-Hernandez, Inigo</creatorcontrib><creatorcontrib>Mortensen, Tenna Pavia</creatorcontrib><creatorcontrib>Henningsen, Jeanette</creatorcontrib><creatorcontrib>Jensen, Søren Skov</creatorcontrib><creatorcontrib>Bigot, Anne</creatorcontrib><creatorcontrib>Blagoev, Blagoy</creatorcontrib><creatorcontrib>Butler-Browne, Gillian</creatorcontrib><creatorcontrib>Kratchmarova, Irina</creatorcontrib><title>Cellular Proteome Dynamics during Differentiation of Human Primary Myoblasts</title><title>Journal of proteome research</title><addtitle>J. Proteome Res</addtitle><description>Muscle stem cells, or satellite cells, play an important role in the maintenance and repair of muscle tissue and have the capacity to proliferate and differentiate in response to physiological or environmental changes. Although they have been extensively studied, the key regulatory steps and the complex temporal protein dynamics accompanying the differentiation of primary human muscle cells remain poorly understood. Here, we demonstrate the advantages of applying a MS-based quantitative approach, stable isotope labeling by amino acids in cell culture (SILAC), for studying human myogenesis in vitro and characterize the fine-tuned changes in protein expression underlying the dramatic phenotypic conversion of primary mononucleated human muscle cells during in vitro differentiation to form multinucleated myotubes. Using an exclusively optimized triple encoding SILAC procedure, we generated dynamic expression profiles during the course of myogenic differentiation and quantified 2240 proteins, 243 of which were regulated. These changes in protein expression occurred in sequential waves and underlined vast reprogramming in key processes governing cell fate decisions, i.e., cell cycle withdrawal, RNA metabolism, cell adhesion, proteolysis, and cytoskeletal organization. In silico transcription factor target analysis demonstrated that the observed dynamic changes in the proteome could be attributed to a cascade of transcriptional events involving key myogenic regulatory factors as well as additional regulators not yet known to act on muscle differentiation. In addition, we created of a dynamic map of the developing myofibril, providing valuable insights into the formation and maturation of the contractile apparatus in vitro. Finally, our SILAC-based quantitative approach offered the possibility to follow the expression profiles of several muscle disease-associated proteins simultaneously and therefore could be a valuable resource for future studies investigating pathogenesis of degenerative muscle disorders as well as assessing new therapeutic strategies.</description><subject>Amino Acids - metabolism</subject><subject>Blotting, Western</subject><subject>Cell Differentiation</subject><subject>Cells, Cultured</subject><subject>Chromatography, Liquid</subject><subject>Cluster Analysis</subject><subject>Humans</subject><subject>Immunohistochemistry</subject><subject>Infant, Newborn</subject><subject>Isotope Labeling - methods</subject><subject>Kinetics</subject><subject>Muscle Fibers, Skeletal - cytology</subject><subject>Muscle Fibers, Skeletal - metabolism</subject><subject>Proteome - classification</subject><subject>Proteome - metabolism</subject><subject>Proteomics - methods</subject><subject>Satellite Cells, Skeletal Muscle - cytology</subject><subject>Satellite Cells, Skeletal Muscle - metabolism</subject><subject>Spectrometry, Mass, Electrospray Ionization</subject><subject>Tandem Mass Spectrometry</subject><subject>Time Factors</subject><issn>1535-3893</issn><issn>1535-3907</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2015</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkDFPwzAQhS0EoqXwE0AZWVLukjhuRtQWilQEA8yW49goVRIXOx767zEk7cp0N7z37t5HyC3CHCHBByHdfLe3plemVXNaAqQFOyNTpCmN0wLY-XFfFOmEXDm3A0DKIL0kkyQHlkFCp2S7VE3jG2Gj9zErWh060dbSRZW3dfcVrWqtlVVdX4u-Nl1kdLTxreiCo26FPUSvB1M2wvXumlxo0Th1M84Z-Xxafyw38fbt-WX5uI1FhqyPJdNaCJrQJJUs1xC-KTFHxFIvWIWFRFmoUKfM1aJiWkAlcmQKtMwqRZM8nZH7ITf0__bK9bytnQxFRKeMdxxZyKQJgyxI6SCV1jhnleb74WuOwH9B8gCSn0DyEWTw3Y0nfNmq6uQ6kgsCHAR_fuNtFxr_E_oDDnaEkw</recordid><startdate>20150807</startdate><enddate>20150807</enddate><creator>Le Bihan, Marie-Catherine</creator><creator>Barrio-Hernandez, Inigo</creator><creator>Mortensen, Tenna Pavia</creator><creator>Henningsen, Jeanette</creator><creator>Jensen, Søren Skov</creator><creator>Bigot, Anne</creator><creator>Blagoev, Blagoy</creator><creator>Butler-Browne, Gillian</creator><creator>Kratchmarova, Irina</creator><general>American Chemical Society</general><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></search><sort><creationdate>20150807</creationdate><title>Cellular Proteome Dynamics during Differentiation of Human Primary Myoblasts</title><author>Le Bihan, Marie-Catherine ; Barrio-Hernandez, Inigo ; Mortensen, Tenna Pavia ; Henningsen, Jeanette ; Jensen, Søren Skov ; Bigot, Anne ; Blagoev, Blagoy ; Butler-Browne, Gillian ; Kratchmarova, Irina</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a417t-c7ffaa52523c76f0260b16111bf87d19c1c9e039b6e8d7fa0da617e0fc4de5263</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2015</creationdate><topic>Amino Acids - metabolism</topic><topic>Blotting, Western</topic><topic>Cell Differentiation</topic><topic>Cells, Cultured</topic><topic>Chromatography, Liquid</topic><topic>Cluster Analysis</topic><topic>Humans</topic><topic>Immunohistochemistry</topic><topic>Infant, Newborn</topic><topic>Isotope Labeling - methods</topic><topic>Kinetics</topic><topic>Muscle Fibers, Skeletal - cytology</topic><topic>Muscle Fibers, Skeletal - metabolism</topic><topic>Proteome - classification</topic><topic>Proteome - metabolism</topic><topic>Proteomics - methods</topic><topic>Satellite Cells, Skeletal Muscle - cytology</topic><topic>Satellite Cells, Skeletal Muscle - metabolism</topic><topic>Spectrometry, Mass, Electrospray Ionization</topic><topic>Tandem Mass Spectrometry</topic><topic>Time Factors</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Le Bihan, Marie-Catherine</creatorcontrib><creatorcontrib>Barrio-Hernandez, Inigo</creatorcontrib><creatorcontrib>Mortensen, Tenna Pavia</creatorcontrib><creatorcontrib>Henningsen, Jeanette</creatorcontrib><creatorcontrib>Jensen, Søren Skov</creatorcontrib><creatorcontrib>Bigot, Anne</creatorcontrib><creatorcontrib>Blagoev, Blagoy</creatorcontrib><creatorcontrib>Butler-Browne, Gillian</creatorcontrib><creatorcontrib>Kratchmarova, Irina</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of proteome research</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Le Bihan, Marie-Catherine</au><au>Barrio-Hernandez, Inigo</au><au>Mortensen, Tenna Pavia</au><au>Henningsen, Jeanette</au><au>Jensen, Søren Skov</au><au>Bigot, Anne</au><au>Blagoev, Blagoy</au><au>Butler-Browne, Gillian</au><au>Kratchmarova, Irina</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Cellular Proteome Dynamics during Differentiation of Human Primary Myoblasts</atitle><jtitle>Journal of proteome research</jtitle><addtitle>J. Proteome Res</addtitle><date>2015-08-07</date><risdate>2015</risdate><volume>14</volume><issue>8</issue><spage>3348</spage><epage>3361</epage><pages>3348-3361</pages><issn>1535-3893</issn><eissn>1535-3907</eissn><abstract>Muscle stem cells, or satellite cells, play an important role in the maintenance and repair of muscle tissue and have the capacity to proliferate and differentiate in response to physiological or environmental changes. Although they have been extensively studied, the key regulatory steps and the complex temporal protein dynamics accompanying the differentiation of primary human muscle cells remain poorly understood. Here, we demonstrate the advantages of applying a MS-based quantitative approach, stable isotope labeling by amino acids in cell culture (SILAC), for studying human myogenesis in vitro and characterize the fine-tuned changes in protein expression underlying the dramatic phenotypic conversion of primary mononucleated human muscle cells during in vitro differentiation to form multinucleated myotubes. Using an exclusively optimized triple encoding SILAC procedure, we generated dynamic expression profiles during the course of myogenic differentiation and quantified 2240 proteins, 243 of which were regulated. These changes in protein expression occurred in sequential waves and underlined vast reprogramming in key processes governing cell fate decisions, i.e., cell cycle withdrawal, RNA metabolism, cell adhesion, proteolysis, and cytoskeletal organization. In silico transcription factor target analysis demonstrated that the observed dynamic changes in the proteome could be attributed to a cascade of transcriptional events involving key myogenic regulatory factors as well as additional regulators not yet known to act on muscle differentiation. In addition, we created of a dynamic map of the developing myofibril, providing valuable insights into the formation and maturation of the contractile apparatus in vitro. Finally, our SILAC-based quantitative approach offered the possibility to follow the expression profiles of several muscle disease-associated proteins simultaneously and therefore could be a valuable resource for future studies investigating pathogenesis of degenerative muscle disorders as well as assessing new therapeutic strategies.</abstract><cop>United States</cop><pub>American Chemical Society</pub><pmid>26074025</pmid><doi>10.1021/acs.jproteome.5b00397</doi><tpages>14</tpages></addata></record> |
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subjects | Amino Acids - metabolism Blotting, Western Cell Differentiation Cells, Cultured Chromatography, Liquid Cluster Analysis Humans Immunohistochemistry Infant, Newborn Isotope Labeling - methods Kinetics Muscle Fibers, Skeletal - cytology Muscle Fibers, Skeletal - metabolism Proteome - classification Proteome - metabolism Proteomics - methods Satellite Cells, Skeletal Muscle - cytology Satellite Cells, Skeletal Muscle - metabolism Spectrometry, Mass, Electrospray Ionization Tandem Mass Spectrometry Time Factors |
title | Cellular Proteome Dynamics during Differentiation of Human Primary Myoblasts |
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